Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/ellerbyLab © 2011 Buck Institute, All Rights Reserved Huntingtons disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin (Htt). Striatal and cortical neuronal loss are prominent features of this disease. No disease modifying treatments have been discovered for HD. To identify new therapeutic targets in HD, we screened a kinase inhibitor library for molecules that block mutant Htt cellular toxicity in a mouse HD striatal cell model. We found that diacylglycerol kinase (DGK) inhibitor II (R59949) decreased caspase3/7 activity after serum withdrawal in striatal Hdh111Q/111Q cells. In addition, R59949 decreased the level of cleaved caspase3, the accumulation of the 513 amino acid Nterminal Htt fragment processed by caspase3, and blocked alterations in lipid metabolism during serum withdrawal. To identify the diacylglyercol kinase mediating this effect, we knocked down all four DGK isoforms expressed in the brain using siRNA (beta, gamma, epsilon and zeta). Only the knockdown of the family member, DGKepsilon (DGK), blocked striatal Hdh111Q/111Qmediated toxicity. We also investigated the significance of these findings in vivo. First, we found that reduced function of the Drosophila DGK homolog significantly improves Httinduced motor dysfunction in a fly model of HD. In addition, we find that the levels of DGK are increased in the striatum of R6/2 HD transgenic mice when compared to controls. Together these findings indicate that increased levels of kinase DGK contribute to HD pathogenesis, and suggest that reducing its levels or activity is a potential therapy for HD. http://www.ncbi.nlm.nih.gov/pubmed/22511757 Sat, 31 Dec 2011 00:00:00 -0800 Huntingtin (Htt) is a protein with a polyglutamine (polyQ) stretch in the Nterminus and expansion of the polyQ stretch causes Huntington disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that posttranslational modifications (PTMs) of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase (HDAC) inhibitors ameliorates HDlike symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1612) in a cell culture model using mass spectrometry (MS). Myctagged Htt23Q (1612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDSPAGE, digested and subjected to HPLC tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys178, Lys236, Lys345 and two previously described sites Lys9 and Lys444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring (MRM) MS assay was developed to compare quantitatively the Lys178 acetylation level between wildtype Htt23Q and mutant Htt148Q (1612). This report represents the first comprehensive mapping of lysine acetylation sites in Nterminal region of Htt. http://www.ncbi.nlm.nih.gov/pubmed/21685499 Fri, 31 Dec 2010 00:00:00 -0800 Huntington's Disease (HD) is characterized bya mutation in the huntingtin (Htt) gene encoding an expansion of glutamine repeats on the N terminus of the Htt protein. Numerous studies have identified Htt proteolysis as a critical pathological event in HD postmortem human tissue and mouse HD models, and proteases known as caspases have emerged as attractive HD therapeutic targets. We report the useof the substrate activity screening method against caspase3 and 6 to identify three novel, pancaspase inhibitors that block proteolysis of Htt at caspase3 and 6 cleavage sites. In HD models these irreversible inhibitors suppressed Hdh(111Q/111Q)mediated toxicity and rescued rat striatal and cortical neurons from cell death. In this study, the identified nonpeptidic caspase inhibitors were used to confirm the role of caspasemediated Htt proteolysis in HD. These results further implicate caspases as promising targets for HD therapeutic development. http://www.ncbi.nlm.nih.gov/pubmed/21095569 Sun, 31 Oct 2010 00:00:00 -0700 Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntington's disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the protease familiescaspases and calpains. Identifying critical proteases involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a highthroughput western blotbased screen to examine the generation of the smallest Nterminal polyglutaminecontaining Htt fragment. We screened 514 siRNAs targeting the repertoire of human protease genes. This screen identified 11 proteases that, when inhibited, reduced Htt fragment accumulation. Three of these belonged to the matrix metalloproteinase (MMP) family. One family member, MMP10, directly cleaves Htt and prevents cell death when knocked down in striatal Hdh(111Q/111Q) cells. Correspondingly, MMPs are activated in HD mouse models, and loss of function of Drosophila homologs of MMPs suppresses Httinduced neuronal dysfunction in vivo. http://www.ncbi.nlm.nih.gov/pubmed/20670829 Wed, 30 Jun 2010 00:00:00 -0700 Recent studies have highlighted the importance of the lysosome in degrading proteins that misfold in neurodegenerative diseases. In this study we explore the role for autophagy in the clearance of an Nterminal caspase7generated fragment of ataxin7, a protein with a pathogenic polyglutamine (polyQ) expansion in the neurodegenerative disease spinocerebellar ataxia 7 (SCA7). Using both cellular and transgenic mouse models of SCA7 we show that the stability of wildtype ataxin7 is modified by macroautophagy, but not by proteasomal, inhibition, whereas both autophagy and proteasomal degradation have little effect on polyQexpanded ataxin7. We also create a posttranslational modificationdeficient ataxin7 mutant that has increased protein turnover of both wildtype and polyQexpanded ataxin7, mediated through the autophagy pathway. Histological analysis reveals that wildtype ataxin7 colocalizes with markers of chaperonemediated autophagy (CMA) and macroautophagy, indicating that both of these mechanisms may play a role in the clearance of ataxin7. Furthermore, there is an increase in LC3, a marker of autophagy initiation, in the cerebellum of SCA7 transgenic mice. Our findings indicate that the ataxin7 fragment may be cleared via autophagy and that this process is altered in SCA7. Identification of the different types of autophagy involved in ataxin7 turnover and the influence of posttranslational modifications on these processes will be pursued in future studies. http://www.ncbi.nlm.nih.gov/pubmed/20104018 Wed, 31 Mar 2010 00:00:00 -0700 Huntingtin proteolysis has been implicated in the molecular pathogenesis of Huntington disease (HD). Despite an intense effort, the identity of the pathogenic smallest Nterminal fragment has not been determined. Using a panel of antihuntingtin antibodies, we employed an unbiased approach to generate proteolytic cleavage maps of mutant and wildtype huntingtin in the HdhQ150 knockin mouse model of HD. We identified 14 prominent Nterminal fragments, which, in addition to the fulllength protein, can be readily detected in cytoplasmic but not nuclear fractions. These fragments were detected at all ages and are not a consequence of the pathogenic process. We demonstrated that the smallest fragment is an exon 1 huntingtin protein, known to contain a potent nuclear export signal. Prior to the onset of behavioral phenotypes, the exon 1 protein, and possibly other small fragments, accumulate in neuronal nuclei in the form of a detergent insoluble complex, visualized as diffuse granular nuclear staining in tissue sections. This methodology can be used to validate the inhibition of specific proteases as therapeutic targets for HD by pharmacological or genetic approaches. http://www.ncbi.nlm.nih.gov/pubmed/20086007 Sun, 28 Feb 2010 00:00:00 -0800 Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first exon of the gene Huntingtin (Htt). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease progresses. A useful advance in HD would be the generation of a humanderived HD model to use for drug screening and understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HDiPS cells into striatal fate required several steps. First, we generated nestin/PAX6/SOX1/OCT4 neural stem cells (NSCs) from HDiPS cells using the method of embryoid body formation. HDNSCs were then subjected to a differentiation condition combining morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained with III tubulin, calbindin and GABA but not DARPP32 (dopamine and cyclic AMPregulated phosphoprotein, Mr = 32,000) were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons resulted in striatal neurons expressing markers like DARPP32. The HDiPS cells derived striatal neurons and neuronal precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. Moreover, the HDNSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs (from iPS or H9 NSCs). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD mechanisms and drug screening. http://www.ncbi.nlm.nih.gov/pubmed/21037797 Thu, 31 Dec 2009 00:00:00 -0800 Polyglutamine (polyQ) expansion within the ataxin7 protein, a member of the STAGA SPT3TAF(II)31GCN5L acetylase and TFTC (GCN5 and TRRAP) chromatin remodeling complexes, causes the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7). Proteolytic processing of ataxin7 by caspase7 generates Nterminal toxic polyQcontaining fragments that accumulate with disease progression and play an important role in SCA7 pathogenesis. To elucidate the basis for the toxicity of these fragments, we evaluated which posttranslational modifications of the Nterminal fragment of ataxin7 modulate turnover and toxicity. Here, we show that mutating lysine 257 (K257), an amino acid adjacent to the caspase7 cleavage site of ataxin7 regulates turnover of the truncation product in a repeatdependent manner. Modification of ataxin7 K257 by acetylation promotes accumulation of the fragment, while unmodified ataxin7 is degraded. The degradation of the caspase7 cleavage product is mediated by macroautophagy in cell culture and primary neuron models of SCA7. Consistent with this, the fragment colocalizes with autophagic vesicle markers, and enhanced fragment accumulation increases in these lysosomal structures. We suggest that the levels of fragment accumulation within the cell is a key event in SCA7 neurodegeneration, and enhancing clearance of polyQcontaining fragments may be an effective target to reduce neurotoxicity in SCA7. http://www.ncbi.nlm.nih.gov/pubmed/19955365 Mon, 30 Nov 2009 00:00:00 -0800 Caspase7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase7 activation by granzyme B and caspase3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase7 is not known. Here, we present that recombinant caspase7 is directly cleaved by calpain1 within the large subunit of caspase7 to produce two novel products, large subunit p18 and p17. This new form of caspase7 has a 6fold increase in V(max) when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase7 product (p17) is 18fold more active than either the caspase3cleaved product (p20) or the larger calpain1 product of caspase7 (p18). Mass spectrometry and sitedirected mutagenesis identified the calpain cleavage sites within the caspase7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase7 p18 and p17 subunits in cortical neurons undergoing Ca(2) dysregulation. Further, cleavage at amino acid 45/47 of caspase7 by calpain results in a reduction in nuclear localization when compared with the caspase3 cleavage product of caspase7 (p20). Our studies suggest the calpainactivated form of caspase7 has unique enzymatic activity, localization, and binding affinity when compared with the caspaseactivated form. http://www.ncbi.nlm.nih.gov/pubmed/19617626 Mon, 31 Aug 2009 00:00:00 -0700 Modulation of testosterone levels is a therapeutic approach for spinal and bulbar muscular atrophy (SBMA), a polyglutamine disorder that affects the motor neurons. The article by Palazzolo et al. in this issue of Neuron provides compelling evidence that the expression of insulin growth hormone is a potential therapeutic for SBMA. http://www.ncbi.nlm.nih.gov/pubmed/19679066 Fri, 31 Jul 2009 00:00:00 -0700 Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by a polyglutamine (polyQ) repeat expansion in the androgen receptor (AR). PolyQAR neurotoxicity may involve generation of an Nterminal truncation fragment, as such peptides occur in SBMA patients and mouse models. To elucidate the basis of SBMA, we expressed Nterminal truncated AR in motor neuronderived cells and primary cortical neurons. Accumulation of polyQAR truncation fragments in the cytosol resulted in neurodegeneration and apoptotic, caspasedependent cell death. Using primary neurons from mice transgenic or deficient for apoptosisrelated genes, we determined that polyQAR apoptotic activation is fully dependent on Bax. Jun Nterminal kinase (JNK) was required for apoptotic pathway activation through phosphorylation of cJun. Expression of polyQAR in DP5/Hrk null neurons yielded significant protection against apoptotic activation, but absence of Bim did not provide protection, apparently due to compensatory upregulation of DP5/Hrk or other BH3only proteins. Misfolded AR protein in the cytosol thus initiates a cascade of events beginning with JNK and culminating in Baxdependent, intrinsic pathway activation, mediated in part by DP5/Hrk. As apoptotic mediators are candidates for toxic fragment generation and other cellular processes linked to neuron dysfunction, delineation of the apoptotic activation pathway induced by polyQexpanded AR may shed light on the pathogenic cascade in SBMA and other motor neuron diseases. http://www.ncbi.nlm.nih.gov/pubmed/19228953 Sat, 31 Jan 2009 00:00:00 -0800 Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine (polyQ) disorder characterized by specific degeneration of cerebellar, brainstem, and retinal neurons. Although they share little sequence homology, proteins implicated in polyQ disorders have common properties beyond their characteristic polyQ tract. These include the production of proteolytic fragments, nuclear accumulation, and processing by caspases. Here we report that ataxin7 is cleaved by caspase7, and we map two putative caspase7 cleavage sites to Asp residues at positions 266 and 344 of the ataxin7 protein. Sitedirected mutagenesis of these two caspase7 cleavage sites in the polyQexpanded form of ataxin7 produces an ataxin7 D266N/D344N protein that is resistant to caspase cleavage. Although ataxin7 displays toxicity, forms nuclear aggregates, and represses transcription in human embryonic kidney 293T cells in a polyQ lengthdependent manner, expression of the noncleavable D266N/D344N form of polyQexpanded ataxin7 attenuated cell death, aggregate formation, and transcriptional interference. Expression of the caspase7 truncation product of ataxin769Q or 92Q, which removes the putative nuclear export signal and nuclear localization signals of ataxin7, showed increased cellular toxicity. We also detected Nterminal polyQexpanded ataxin7 cleavage products in SCA7 transgenic mice similar in size to those generated by caspase7 cleavage. In a SCA7 transgenic mouse model, recruitment of caspase7 into the nucleus by polyQexpanded ataxin7 correlated with its activation. Our results, thus, suggest that proteolytic processing of ataxin7 by caspase7 may contribute to SCA7 disease pathogenesis. http://www.ncbi.nlm.nih.gov/pubmed/17646170 Sun, 30 Sep 2007 00:00:00 -0700 Neuroglobin (Ngb) is a recently discovered vertebrate globin expressed primarily in neurons. Ngb expression is induced by hypoxia and ischemia, and Ngb protects neurons from these insults. However, its normal physiological role and the mechanism underlying its neuroprotective action are uncertain. We report production of a transgenic mouse in which Ngb is overexpressed under the control of the chicken betaactin promoter. This mouse should prove helpful for studying Ngbmediated effects in vitro and in vivo. http://www.ncbi.nlm.nih.gov/pubmed/17537594 Sat, 30 Jun 2007 00:00:00 -0700 Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: highthroughput yeast twohybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 highconfidence Httassociated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This highcontent validation assay showed that 27 of 60 orthologs tested were highconfidence genetic modifiers, as modification was observed with more than one allele. The 45 hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 14 typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast twohybrid and pulldown/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 lossoffunction suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to coimmunoprecipitate with fulllength Htt from mouse brain. These studies demonstrate that highthroughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity. http://www.ncbi.nlm.nih.gov/pubmed/17500595 Mon, 30 Apr 2007 00:00:00 -0700 Mitochondrial dysfunction is believed to participate in Huntington's disease (HD) pathogenesis. Here we compare the bioenergetic behavior of forebrain mitochondria isolated from different transgenic HD mice (R6/2, YAC128 and Hdh150 knockin) and wildtype littermates with the first determination of in situ respiratory parameters in intact HD striatal neurons. We assess the Ca2loading capacity of isolated mitochondria by steady Ca2infusion. Mitochondria from R6/2 mice (1213 weeks) and 12 months YAC128, but not homozygous or heterozygous Hdh150 knockin mice (1517 weeks), exhibit increased Ca2loading capacity when compared with respective wildtype littermates. In situ mitochondria in intact striatal neurons show high respiratory control. Moreover, moderate expression of fulllength mutant huntingtin (in Hdh150 knockin heterozygotes) does not significantly impair mitochondrial respiration in unstimulated neurons. However, when challenged with energydemanding stimuli (NMDAreceptor activation in pyruvatebased media to accentuate the mitochondria role in Ca2handling), Hdh150 neurons are more vulnerable to Ca2deregulation than neurons from their wildtype littermates. These results stress the importance of assessing HD mitochondrial function in the cellular context. http://www.ncbi.nlm.nih.gov/pubmed/17394466 Wed, 28 Feb 2007 00:00:00 -0800 Neuroglobin (Ngb), a protein related to myoglobin and hemoglobin but expressed predominantly in the brain, is induced by neuronal hypoxia and cerebral ischemia and protects against hypoxic or ischemic neuronal injury. We engineered transgenic mice that overexpress murine Ngb under the control of a chicken betaactin promoter, resulting in enhanced Ngb expression in multiple cell types and multiple tissues, including brain and heart. In Ngboverexpressing transgenic mice compared with wildtype littermates, the volume of cerebral infarcts after occlusion of the middle cerebral artery was reduced by approximately 30, and the volume of myocardial infarcts produced by occlusion of the left anterior descending coronary artery was reduced by approximately 25. Ngb overexpression was associated with enhanced expression of endothelial nitric oxide synthase in vascular endothelial cells. These findings extend prior evidence for cytoprotection by Ngb and suggest both direct (parenchymatous) and indirect (vasomotor) protective mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/17098866 Tue, 31 Oct 2006 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/17432184 Tue, 31 Oct 2006 00:00:00 -0800 Evidence suggests that neuronal dysfunction in Huntington's disease (HD) striatum involves deficits in mitochondrial function and in Ca2 handling. However, the relationship between mitochondria and Ca2 handling has been incompletely studied in intact HD striatal cells. Treatment with histone deacetylase (HDAC) inhibitors reduces cell death in HD models, but the effects of this promising therapy on cellular function are mostly unknown. Here, we use realtime functional imaging of intracellular Ca2 and mitochondrial membrane potential to explore the role of in situ HD mitochondria in Ca2 handling. Immortalized striatal (STHdh) cells and striatal neurons from transgenic mice, expressing fulllength mutant huntingtin (Htt), were used to model HD. We show that (1) active glycolysis in STHdh cells occludes the mitochondrial role in Ca2 handling as well as the effects of mitochondrial inhibitors, (2) STHdh cells and striatal neurons in the absence of glycolysis are critically dependent on oxidative phosphorylation for energydependent Ca2 handling, (3) expression of fulllength mutant Htt is associated with deficits in mitochondrialdependent Ca2 handling that can be ameliorated by treatment with HDAC inhibitors (treatment with trichostatin A or sodium butyrate decreases the proportion of STHdh cells losing Ca2 homeostasis after Ca2ionophore challenging, and accelerates the restoration of intracellular Ca2 in striatal neurons challenged with NMDA), and (4) neurons with different response patterns to NMDA receptor activation exhibit different average somatic areas and are differentially affected by treatment with HDAC inhibitors, suggesting subpopulation or functional state specificity. These findings indicate that neuroprotection induced by HDAC inhibitors involves more efficient Ca2 handling, thus improving the neuronal ability to cope with excitotoxic stimuli. http://www.ncbi.nlm.nih.gov/pubmed/17065457 Sat, 30 Sep 2006 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/16900190 Mon, 31 Jul 2006 00:00:00 -0700 Hunterkiller peptides (HKPs) are synthetic peptides that target specific cell types for apoptosis. These studies report functional and structural characteristics of HKP9, an hunterkiller peptide that specifically targets tumor vasculature with a new apoptotic sequence. Vesicle leakage experiments were performed as a model for membrane perturbing activity. Placement of the homing sequence reduces both cell toxicity and vesicle leakage activity. NMR studies elucidate the conformation and orientation of HKP9 in micelles. The positively charged end of the HKP9 killing sequence is solvent exposed however, the central portion of the peptide is helical and buried in dodecylphosphorylcholine micelles. The homing sequence is less solvent exposed than in a previously reported tumorhoming peptide. The results suggest that solvent accessibility of the homing sequence should be considered in design of future peptides. http://www.ncbi.nlm.nih.gov/pubmed/16882316 Mon, 31 Jul 2006 00:00:00 -0700 Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating Nterminal polyQcontaining fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing fulllength myctagged Htt constructs containing 23Q or 148Q repeats. Purified myctagged Htt was subjected to mass spectrometric analysis including matrixassisted laser desorption/ionization mass spectrometry and nanoHPLC tandem mass spectrometry, used in conjunction with ontarget alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in fulllength Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development. http://www.ncbi.nlm.nih.gov/pubmed/16782707 Mon, 31 Jul 2006 00:00:00 -0700 Spinocerebellar ataxia type 7 is a progressive neurodegenerative disorder caused by a CAG DNA triplet repeat expansion leading to an expanded polyglutamine tract in the ataxin7 protein. Ataxin7 appears to be a transcription factor and a component of the STAGA transcription coactivator complex. Here, using live cell imaging and inverted fluorescence recovery after photobleaching, we demonstrate that ataxin7 has the ability to export from the nucleus via the CRM1/exportin pathway and that ataxin7 contains a classic leucinetype nuclear export signal (NES). We have precisely defined the location of this NES in ataxin7 and found it to be fully conserved in all vertebrate species. Polyglutamine expansion was seen to reduce the nuclear export rate of mutant ataxin7 relative to wildtype ataxin7. Subtle point mutation of the NES in polyglutamine expanded ataxin7 increased toxicity in primary cerebellar neurons in a polyglutamine lengthdependent manner in the context of fulllength ataxin7. Our results add ataxin7 to a growing list of polyglutamine disease proteins that are capable of nuclear shuttling, and we define an activity of ataxin7 in the STAGA complex of trafficking between the nucleus and cytoplasm. http://www.ncbi.nlm.nih.gov/pubmed/16314424 Sat, 31 Dec 2005 00:00:00 -0800 Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized behaviorally by chorea, incoordination, and shortened lifespan and neuropathologically by huntingtin inclusions and neuronal degeneration. In order to facilitate studies of pathogenesis and therapeutics, we have generated a new inducible mouse model of HD expressing fulllength huntingtin (Htt) using a tetracyclineregulated promoter. In double transgenic mice Htt was expressed widely in the brain under the control of the tettransactivator (tTA) driven by the prion promoter PrP (in the absence of doxycycline). Mice expressing fulllength mutant Htt, but not fulllength normal Htt, displayed a progressive behavioral phenotype, consisting of slowed and irregular voluntary movements, gait ataxia, tremor and jerky movements, incoordination, and weight loss, with a shortened lifespan. Neuropathology included prominent intranuclear inclusions in cortex and striatum as well as cytoplasmic aggregates. This phenotype is very similar to the phenotypes of previous transgenic mice expressing Nterminal fragments of mutant Htt. The current HDtransgenic mice had nuclear accumulation of Htt, particularly an approximately 60kDa fragment, which appears to represent an Nterminal cleavage product. This fragment is smaller than calpain or caspasederived cleavage products of Htt, but it is comparable to a product, termed cpA, which accumulates in nuclei of cells in a previously described cell model. This new mouse model may be useful in the future for pathogenic and preclinical therapeutic studies related to HD. The data suggest that proteolytic processing could be a part of the pathogenesis of HD, potentially representing an attractive therapeutic target. http://www.ncbi.nlm.nih.gov/pubmed/16150600 Sat, 31 Dec 2005 00:00:00 -0800 There is no satisfactory treatment for Huntington's disease (HD), a hereditary neurodegenerative disorder that produces chorea, dementia, and death. One potential treatment strategy involves the replacement of dead neurons by stimulating the proliferation of endogenous neuronal precursors (neurogenesis) and their migration into damaged regions of the brain. Because growth factors are neuroprotective in some settings and can also stimulate neurogenesis, we treated HD transgenic R6/2 mice from 8 weeks of age until death by s.c. administration of FGF2. FGF2 increased the number of proliferating cells in the subventricular zone by approximately 30 in wildtype mice, and by approximately 150 in HD transgenic R6/2 mice. FGF2 also induced the recruitment of new neurons from the subventricular zone into the neostriatum and cerebral cortex of HD transgenic R6/2 mice. In the striatum, these neurons were DARPP32expressing medium spiny neurons, consistent with the phenotype of neurons lost in HD. FGF2 was neuroprotective as well, because it blocked cell death induced by mutant expanded Htt in primary striatal cultures. FGF2 also reduced polyglutamine aggregates, improved motor performance, and extended lifespan by approximately 20. We conclude that FGF2 improves neurological deficits and longevity in a transgenic mouse model of HD, and that its neuroprotective and neuroproliferative effects may contribute to this improvement. http://www.ncbi.nlm.nih.gov/pubmed/16326808 Wed, 30 Nov 2005 00:00:00 -0800 CNS neurons are endowed with the ability to recover from cytotoxic insults associated with the accumulation of proteinaceous aggregates in mouse models of polyglutamine disease, but the cellular mechanism underlying this phenomenon is unknown. Here, we show that autophagy is essential for the elimination of aggregated forms of mutant huntingtin and ataxin1 from the cytoplasmic but not nuclear compartments. Human orthologs of yeast autophagy genes, molecular determinants of autophagic vacuole formation, are recruited to cytoplasmic but not nuclear inclusion bodies in vitro and in vivo. These data indicate that autophagy is a critical component of the cellular clearance of toxic protein aggregates and may help to explain why protein aggregates are more toxic when directed to the nucleus. http://www.ncbi.nlm.nih.gov/pubmed/16141322 Wed, 31 Aug 2005 00:00:00 -0700 Spinocerebellar ataxia type3, also known as MachadoJoseph Disease, is one of many inherited neurodegenerative disorders caused by polyglutamineencoding CAG repeat expansions in otherwise unrelated disease genes. Polyglutamine disorders are characterized by disease protein misfolding and aggregation often within the nuclei of affected neurons. Although the precise mechanism of polyglutaminemediated cell death remains elusive, evidence suggests that proteolysis of polyglutamine disease proteins by caspases contributes to pathogenesis. Using cellular models we now show that the endogenous spinocerebellar ataxia type3 disease protein, ataxin3, is proteolyzed in apoptotic paradigms, resulting in the loss of fulllength ataxin3 and the corresponding appearance of an approximately 28kDa fragment containing the glutamine repeat. Broadspectrum caspase inhibitors block ataxin3 proteolysis and studies suggest that caspase1 is a primary mediator of cleavage. Sitedirected mutagenesis experiments eliminating three, six or nine potential caspase cleavage sites in the protein suggest redundancy in the site(s) at which cleavage can occur, as previously described for other disease proteins but also map a major cleavage event to a cluster of aspartate residues within the ubiquitinbinding domain of ataxin3 near the polyglutamine tract. Finally, caspasemediated cleavage of expanded ataxin3 resulted in increased ataxin3 aggregation, suggesting a potential role for caspasemediated proteolysis in spinocerebellar ataxia type3 pathogenesis. http://www.ncbi.nlm.nih.gov/pubmed/15140190 Fri, 30 Apr 2004 00:00:00 -0700 Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain and caspasederived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain1, 5, 7, and 10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain1, 5, 7, and 10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain7 and 10 are found only in the nucleus. These results support the role of calpainderived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis. http://www.ncbi.nlm.nih.gov/pubmed/14981075 Fri, 30 Apr 2004 00:00:00 -0700 Xlinked spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder characterized by lower motor neuron degeneration. SBMA is caused by polyglutamine repeat expansions in the androgen receptor (AR). To determine the basis of AR polyglutamine neurotoxicity, we introduced human AR yeast artificial chromosomes carrying either 20 or 100 CAGs into mouse embryonic stem cells. The AR100 transgenic mice developed a lateonset, gradually progressive neuromuscular phenotype accompanied by motor neuron degeneration, indicating striking recapitulation of the human disease. We then tested the hypothesis that polyglutamineexpanded AR interferes with CREB binding protein (CBP)mediated transcription of vascular endothelial growth factor (VEGF) and observed altered CBPAR binding and VEGF reduction in AR100 mice. We found that mutant ARinduced death of motor neuronlike cells could be rescued by VEGF. Our results suggest that SBMA motor neuronopathy involves altered expression of VEGF, consistent with a role for VEGF as a neurotrophic/survival factor in motor neuron disease. http://www.ncbi.nlm.nih.gov/pubmed/15003169 Sun, 29 Feb 2004 00:00:00 -0800 Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of fulllength mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase2 into an apoptosomelike complex. Binding of caspase2 to Htt is polyglutamine repeatlength dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing fulllength Htt (YAC72). Expression of catalytically inactive (dominantnegative) forms of caspase2, caspase7, and to some extent caspase6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase3, 8, and 9 did not. Histological analysis of postmortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase2 correlates directly with decreased levels of brainderived neurotrophic factor in the cortex and striatum of 3month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase2 in the selective neuronal cell death associated with HD in the striatum and cortex. http://www.ncbi.nlm.nih.gov/pubmed/14713958 Sun, 29 Feb 2004 00:00:00 -0800 Xlinked spinal and bulbar muscular atrophy is a degenerative disease affecting motor neurons that is caused by polyglutamine (polyQ) expansion within the androgen receptor (AR). The polyQexpanded form of AR is cytotoxic to cells, and proteolytic cleavage enhances cell death. The intracellular signaling pathways activated and/or required for cell death induced by the expanded form of AR (AR112) are unknown. We found that AR regulates mitogenactivated protein kinase (MAP kinase) pathways and, therefore, hypothesized that these pathway(s) may be required for AR112induced cell death. The polyQ expansion in AR activates three MAP kinase pathways, causing increasing levels of phosphorylation of p44/42, p38, and SAPK/JNK MAP kinase. Inhibitors of either the JNK or p38 pathways had no effect on AR112induced cell death, suggesting they are not required for polyQinduced cell death. Strikingly, the MEK1/2 inhibitor, U0126, which selectively inhibits the p44/42 MAP kinase pathway, reduces AR112stimulated cell death. The inhibition of the MEK1/2 pathway correlates directly with a change in phosphorylation state of the androgen receptor. Mutation of the MAP kinase consensus phosphorylation site in AR at serine 514 blocked ARinduced cell death and the generation of caspase3derived cleavage products. We propose a mechanism by which phosphorylation at serine 514 of AR enhances the ability of caspase3 to cleave AR and generate cytotoxic polyQ fragments. http://www.ncbi.nlm.nih.gov/pubmed/12824190 Sun, 31 Aug 2003 00:00:00 -0700 Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first nativelike poreforming protein, small globular protein (SGP). We show here that this artificially engineered protein has membranedisrupting properties and antitumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications. http://www.ncbi.nlm.nih.gov/pubmed/12750379 Sun, 31 Aug 2003 00:00:00 -0700 Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder similar to Huntington's disease, with clinical manifestations including chorea, incoordination, ataxia, and dementia. It is caused by an expansion of a CAG trinucleotide repeat encoding polyglutamine in the atrophin1 gene. Both patients and DRPLA transgenic mice have nuclear accumulation of atrophin1, especially an approximately 120kDa fragment, which appears to represent a cleavage product. We now show that this is an Nterminal fragment that does not correspond to the previously described caspase3 fragment, or any other known caspase cleavage product. The atrophin1 sequence contains a putative nuclear localization signal in the N terminus of the protein and a putative nuclear export signal in the C terminus. We have tested the hypothesis that endogenous localization signals are functional in atrophin1, and that nuclear localization and proteolytic cleavage contribute to atrophin1 cell toxicity. In transient cell transfection experiments using a neuroblastoma cell line, fulllength atrophin1 with 26 (normal) or 65 (expanded) glutamines localized to both nucleus and cytoplasm, with no significant difference in toxicity between the normal and mutant proteins. A construct with 65 glutamine repeats encoding an Nterminal fragment (which removes an NES) of atrophin1 similar in size to the truncation product in DRPLA patient tissue, showed increased nuclear labeling, and an increase in cellular toxicity, compared with a similar fragment with 26 glutamines. Fulllength atrophin1 with 65 polyglutamine repeats and mutations inactivating the NES also yielded increased nuclear localization and increased toxicity. These data suggest that truncation enhances cellular toxicity of the mutant protein, and that the NES is a relevant region deleted during truncation. Furthermore, mutating the NLS in the truncated protein shifted atrophin1 more to the cytoplasm and eliminated the increased toxicity, consistent with the idea that nuclear localization enhances toxicity. In none of the experiments were inclusions visible in the nucleus or cytoplasm suggesting that inclusion formation is unrelated to cell death. These data indicate that truncation of atrophin1 may alter its ability to shuttle between the nucleus and cytoplasm, leading to abnormal nuclear interactions and cell toxicity. http://www.ncbi.nlm.nih.gov/pubmed/12464607 Mon, 31 Mar 2003 00:00:00 -0800 Studies on postmortem brains from Parkinson's patients reveal elevated iron in the substantia nigra (SN). Selective cell death in this brain region is associated with oxidative stress, which may be exacerbated by the presence of excess iron. Whether iron plays a causative role in cell death, however, is controversial. Here, we explore the effects of iron chelation via either transgenic expression of the iron binding protein ferritin or oral administration of the bioavailable metal chelator clioquinol (CQ) on susceptibility to the Parkinson'sinducing agent 1methyl4phenyl1,2,3,6tetrapyridine (MPTP). Reduction in reactive iron by either genetic or pharmacological means was found to be well tolerated in animals in our studies and to result in protection against the toxin, suggesting that iron chelation may be an effective therapy for prevention and treatment of the disease. http://www.ncbi.nlm.nih.gov/pubmed/12670420 Fri, 28 Feb 2003 00:00:00 -0800 Huntington's disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspasecleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspasecleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wildtype and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release Nterminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD. http://www.ncbi.nlm.nih.gov/pubmed/12223539 Sat, 31 Aug 2002 00:00:00 -0700 Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG expansion that results in elongation of the polyglutamine tract at the N terminus of huntingtin (Htt). Abnormal proteolytic processing of mutant Htt has been implicated as a critical step in the initiation of HD. The protease(s) involved in this process has not been fully characterized. Here we report that activated calpain was detected in the caudate of human HD tissue but not in agematched controls. In addition, one of the major Nterminal Htt proteolytic fragments found in human HD tissue appears to be derived from calpain cleavage. Htt fragments in HD lysates were similar in size to those produced by exposure of in vitrotranslated Htt to exogenous calpain. Incubation of in vitrotranslated Htt with calpain generated a cascade of cleavage events with an initial intermediate cleavage product at 72 kDa and a final cleavage product at 47 kDa. The rate of cleavage of Htt by calpain was polyglutaminelengthdependent. These results suggest that cleavage of Htt in human HD tissue is mediated in part by the Ca2activated neutral protease, calpain. http://www.ncbi.nlm.nih.gov/pubmed/12077181 Fri, 31 May 2002 00:00:00 -0700 Accumulation of misfolded proteins and alterations in Ca2 homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signaltransducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stressinduced cell death proceeds, we performed studies on Apaf1(/) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldinA, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase12 and caspase9 and by a peptide inhibitor of caspase9 but not by caspase8 inhibitors. Cleavage of caspases and poly(ADPribose) polymerase was observed in cellfree extracts lacking cytochrome c that were isolated from thapsigargin or brefeldintreated cells. To define the molecular requirements for this Apaf1 and cytochrome cindependent apoptosis pathway further, we developed a cellfree system of ER stressinduced apoptosis the addition of microsomes prepared from ER stressinduced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase12 was one of the microsomal components required to activate downstream caspases. Thus, ER stressinduced programmed cell death defines a novel, mitochondrial and Apaf1independent, intrinsic apoptotic pathway. http://www.ncbi.nlm.nih.gov/pubmed/11919205 Fri, 31 May 2002 00:00:00 -0700 Alterations in Ca(2) homeostasis and accumulation of unfolded proteins in the endoplasmic reticulum (ER) lead to an ER stress response. Prolonged ER stress may lead to cell death. Glucoseregulated protein (GRP) 78 (Bip) is an ER lumen protein whose expression is induced during ER stress. GRP78 is involved in polypeptide translocation across the ER membrane, and also acts as an apoptotic regulator by protecting the host cell against ER stressinduced cell death, although the mechanism by which GRP78 exerts its cytoprotective effect is not understood. The present study was carried out to determine whether one of the mechanisms of cell death inhibition by GRP78 involves inhibition of caspase activation. Our studies indicate that treatment of cells with ER stress inducers causes GRP78 to redistribute from the ER lumen with subpopulations existing in the cytosol and as an ER transmembrane protein. GRP78 inhibits cytochrome cmediated caspase activation in a cellfree system, and expression of GRP78 blocks both caspase activation and caspasemediated cell death. GRP78 forms a complex with caspase7 and 12 and prevents release of caspase12 from the ER. Addition of (d)ATP dissociates this complex and may facilitate movement of caspase12 into the cytoplasm to set in motion the cytosolic component of the ER stressinduced apoptotic cascade. These results define a novel protective role for GRP78 in preventing ER stressinduced cell death. http://www.ncbi.nlm.nih.gov/pubmed/11943137 Sun, 31 Mar 2002 00:00:00 -0800 Excitotoxic cell death stimulated by quinolinic acid injection into the striatum has a long history of "mimicking" many aspects of motor, behavioral, and neurochemical changes observed in Huntington's disease patients. In this issue of Neuron, provide insight into the role of NMDA receptors in the cellspecific excitotoxic death observed in Huntington's disease (HD) using a HD mouse model expressing fulllength mutant huntingtin (htt). http://www.ncbi.nlm.nih.gov/pubmed/11906690 Thu, 28 Feb 2002 00:00:00 -0800 Bcl2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT2/D1 (NT2) and SKNMC, overexpressing Bcl2 as compared with vectoronly controls. Bcl2 transfectants of both cell lines were more resistant to H(2)O(2) and showed increases in GSH level and Cu/Znsuperoxide dismutase (SOD1) activity, but not in Mnsuperoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SKNMC cells. Overexpression of Bcl2 did not significantly decrease levels of oxidative DNA damage (measured as 8hydroxyguanine) or lipid peroxidation, but it decreased levels of 3nitrotyrosine in both cell lines and protein carbonyls in SKNMC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl2 overexpression to decrease 3nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species this was confirmed as decreased production of NO(2)()/NO(3)() in the transfected cells and a fall in the level of nNOS protein. http://www.ncbi.nlm.nih.gov/pubmed/11744329 Fri, 30 Nov 2001 00:00:00 -0800 The endoplasmic reticulum (ER) is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. It also regulates cellular responses to stress and intracellular Ca(2) levels. A variety of toxic insults can result in ER stress that ultimately leads to apoptosis. Apoptosis is initiated by the activation of members of the caspase family and serves as a central mechanism in the cell death process. The present study was carried out to determine the role of caspases in triggering ER stressinduced cell death. Treatment of cells with ER stress inducers such as brefeldinA or thapsigargin induces the expression of caspase12 protein and also leads to translocation of cytosolic caspase7 to the ER surface. Caspase12, like most other members of the caspase family, requires cleavage of the prodomain to activate its proapoptotic form. Caspase7 associates with caspase12 and cleaves the prodomain to generate active caspase12, resulting in increased cell death. We propose that any cellular insult that causes prolonged ER stress may induce apoptosis through caspase7mediated caspase12 activation. The data underscore the involvement of ER and caspases associated with it in the ER stressinduced apoptotic process. http://www.ncbi.nlm.nih.gov/pubmed/11448953 Fri, 31 Aug 2001 00:00:00 -0700 Fourteen neurological diseases have been associated with the expansion of trinucleotide repeat regions. These diseases have been categorized into those that give rise to the translation of toxic polyglutamine proteins and those that are untranslated. Thus far, compelling evidence has not surfaced for the inclusion of a model in which a common mechanism may participate in the pathobiology of both translated and untranslated trinucleotide diseases. In these studies we show that a doublestranded RNAbinding protein, PKR, which has previously been linked to virallyinduced and stressmediated apoptosis, preferentially binds mutant huntingtin RNA transcripts immobilized on streptavidin columns that have been incubated with human brain extracts. These studies also show, by immunodetection in tissue slices, that PKR is present in its activated form in both human Huntington autopsy material and brain tissue derived from Huntington yeast artificial chromosome transgenic mice. The increased immunolocalization of the activated kinase is more pronounced in areas most affected by the disease and, coupled with the RNA binding results, suggests a role for PKR activation in the disease process. http://www.ncbi.nlm.nih.gov/pubmed/11468270 Sat, 30 Jun 2001 00:00:00 -0700 We investigated the mechanism of lysosomemediated cell death using purified recombinant proapoptotic proteins, and cellfree extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin Lrelated protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis. http://www.ncbi.nlm.nih.gov/pubmed/11073962 Wed, 31 Jan 2001 00:00:00 -0800 The mechanisms underlying neurotrophin dependence, and cellular dependent states in general, are unknown. We show that a 29 amino acid region in the intracellular domain of the common neurotrophin receptor, p75NTR, is required for the mediation of apoptosis by p75NTR. Furthermore, contrary to results obtained with Fas, monomeric p75NTR is required for apoptosis induction, whereas multimerization inhibits the proapoptotic effect. Within the 29residue domain required for apoptosis induction by p75NTR, a 14residue region is sufficient as a peptide inducer of apoptosis. This 14residue peptide requires the positively charged carboxyterminal residues for its effect on cell death, and these same residues are required by the fulllength p75NTR. These studies define a novel type of domain that mediates neurotrophin dependence, and suggest that other cellular dependent states may be mediated by proteins displaying similar domains. http://www.ncbi.nlm.nih.gov/pubmed/11303785 Thu, 30 Nov 2000 00:00:00 -0800 Both transgenic mouse and cell culture models of familial amyotrophic lateral sclerosis (FALS) support a gainoffunction effect for the mutations in copperzinc superoxide dismutase (CuZnSOD) associated with FALS, but the nature of the function gained remains incompletely characterized. We previously reported an enhanced peroxidase activity for FALSassociated CuZnSOD mutants. Because one of the targets of such activity is CuZnSOD itself, we examined peroxidemediated inactivation of wildtype and mutant CuZnSODs, and found that the mutants are more readily inactivated. Inactivation of the mutants was associated with fragmentation, which did not occur in the wildtype enzyme under these conditions. Furthermore, the reduction of the FALSassociated mutants by ascorbate was enhanced markedly when compared to the wildtype enzyme. The visible spectra of the mutants showed a consistent blue shift of the peak at 680 nm in the wildtype enzyme, suggesting an alteration in coppersite geometry. These results extend previous studies demonstrating enhanced peroxidase activity in the mutants, and suggest that the toxic function that leads to motor neuron degeneration may result from a loss of specificity of the redox reactions catalyzed by CuZnSOD. http://www.ncbi.nlm.nih.gov/pubmed/11220787 Sat, 30 Sep 2000 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/10914037 Sat, 30 Sep 2000 00:00:00 -0700 Huntington's disease is a neurodegenerative disorder caused by CAG expansion that results in expansion of a polyglutamine tract at the extreme N terminus of huntingtin (htt). htt with polyglutamine expansion is proapoptotic in different cell types. Here, we show that caspase inhibitors diminish the toxicity of htt. Additionally, we define htt itself as an important caspase substrate by generating a sitedirected htt mutant that is resistant to caspase3 cleavage at positions 513 and 530 and to caspase6 cleavage at position 586. In contrast to cleavable htt, caspaseresistant htt with an expanded polyglutamine tract has reduced toxicity in apoptotically stressed neuronal and nonneuronal cells and forms aggregates at a much reduced frequency. These results suggest that inhibiting caspase cleavage of htt may therefore be of potential therapeutic benefit in Huntington's disease. http://www.ncbi.nlm.nih.gov/pubmed/10770929 Wed, 31 May 2000 00:00:00 -0700 Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso1 and iso2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a posttranslational modification of lysine 72, Nepsilontrimethylation. This modification was found to abrogate proapoptotic activity of metazoan cytochrome c expressed in yeast. However, iso1 cytochrome c lacking the trimethylation modification also was devoid of proapoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso1 sequence preclude proapoptotic activity. Competition studies suggest that the lack of proapoptotic activity was associated with a low affinity for Apaf1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities. http://www.ncbi.nlm.nih.gov/pubmed/10821864 Sun, 30 Apr 2000 00:00:00 -0700 The amyloid betaprotein precursor gives rise to the amyloid betaprotein, the principal constituent of senile plaques and a cytotoxic fragment involved in the pathogenesis of Alzheimer disease. Here we show that amyloid betaprotein precursor was proteolytically cleaved by caspases in the C terminus to generate a second unrelated peptide, called C31. The resultant C31 peptide was a potent inducer of apoptosis. Both caspasecleaved amyloid betaprotein precursor and activated caspase9 were present in brains of Alzheimer disease patients but not in control brains. These findings indicate the possibility that caspase cleavage of amyloid betaprotein precursor with the generation of C31 may be involved in the neuronal death associated with Alzheimer disease. http://www.ncbi.nlm.nih.gov/pubmed/10742146 Fri, 31 Mar 2000 00:00:00 -0800 We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell deathinducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The proapoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anticancer activity in mice. This approach may yield new therapeutic agents. http://www.ncbi.nlm.nih.gov/pubmed/10470080 Tue, 31 Aug 1999 00:00:00 -0700 Caspase9 is critical for cytochrome c (cytoc)dependent apoptosis and normal brain development. We determined that this apical protease in the cytoc pathway for apoptosis resides inside mitochondria in several types of cells, including cardiomyocytes and many neurons. Caspase9 is released from isolated mitochondria on treatment with Ca2 or Bax, stimuli implicated in ischemic neuronal cell death that are known to induce cytoc release from mitochondria. In neuronal cell culture models, apoptosisinducing agents trigger translocation of caspase9 from mitochondria to the nucleus, which is inhibitable by Bcl2. Similarly, in an animal model of transient global cerebral ischemia, caspase9 release from mitochondria and accumulation in nuclei was observed in hippocampal and other vulnerable neurons exhibiting early postischemic changes preceding apoptosis. Loss of mitochondrial barrier function during neuronal damage from ischemia or other insults therefore may play an important role in making certain caspases available to participate in apoptosis. http://www.ncbi.nlm.nih.gov/pubmed/10318956 Mon, 31 May 1999 00:00:00 -0700 Dentatorubropallidoluysian atrophy (DRPLA) is one of eight autosomal dominant neurodegenerative disorders characterized by an abnormal CAG repeat expansion which results in the expression of a protein with a polyglutamine stretch of excessive length. We have reported recently that four of the gene products (huntingtin, atrophin1 (DRPLA), ataxin3, and androgen receptor) associated with these open reading frame triplet repeat expansions are substrates for the cysteine protease cell death executioners, the caspases. This led us to hypothesize that caspase cleavage of these proteins may represent a common step in the pathogenesis of each of these four neurodegenerative diseases. Here we present evidence that caspase cleavage of atrophin1 modulates cytotoxicity and aggregate formation. Cleavage of atrophin1 at Asp109 by caspases is critical for cytotoxicity because a mutant atrophin1 that is resistant to caspase cleavage is associated with significantly decreased toxicity. Further, the altered cellular localization within the nucleus and aggregate formation associated with the expanded form of atrophin1 are completely suppressed by mutation of the caspase cleavage site at Asp109. These results provide support for the toxic fragment hypothesis whereby cleavage of atrophin1 by caspases may be an important step in the pathogenesis of DRPLA. Therefore, inhibiting caspase cleavage of the polyglutaminecontaining proteins may be a feasible therapeutic strategy to prevent cell death. http://www.ncbi.nlm.nih.gov/pubmed/10085113 Wed, 31 Mar 1999 00:00:00 -0800 Xlinked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the diseaseassociated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions. http://www.ncbi.nlm.nih.gov/pubmed/9886069 Thu, 31 Dec 1998 00:00:00 -0800 The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases8 and 10 directly process the executioner procaspase3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M1 s1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase8 induced activation of procaspase3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, procaspase9, was without consequence. Taken together these data demonstrate that the initiator caspase8 can directly activate procaspase3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors. http://www.ncbi.nlm.nih.gov/pubmed/9765224 Sat, 31 Oct 1998 00:00:00 -0800 The neurodegenerative diseases Huntington disease, dentatorubropallidoluysian atrophy, spinocerebellar atrophy type 3, and spinal bulbar muscular atrophy are caused by expansion of a polyglutamine tract within their respective gene products. There is increasing evidence that generation of truncated proteins containing an expanded polyglutamine tract may be a key step in the pathogenesis of these disorders. We now report that, similar to huntingtin, atrophin1, ataxin3, and the androgen receptor are cleaved in apoptotic extracts. Furthermore, each of these proteins is cleaved by one or more purified caspases, cysteine proteases involved in apoptotic death. The CAG length does not modulate susceptibility to cleavage of any of the fulllength proteins. Our results suggest that by generation of truncated polyglutaminecontaining proteins, caspase cleavage may represent a common step in the pathogenesis of each of these neurodegenerative diseases. http://www.ncbi.nlm.nih.gov/pubmed/9535906 Thu, 30 Apr 1998 00:00:00 -0700 Bax is a proapoptotic member of the Bcl2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Baxtreated mitochondria also induced caspase processing and activation. Recombinant BclXL protein abrogated Baxinduced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broadspecificity caspase inhibitor benzyloxycarbonylvalinylalaninylaspartyl(0methyl) fluoromethylketone (zVADfmk) and the caspaseinhibiting protein XIAP had no effect on Baxinduced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria. http://www.ncbi.nlm.nih.gov/pubmed/9560217 Tue, 31 Mar 1998 00:00:00 -0800 It is unclear how polyglutamine expansion is associated with the pathogenesis of Huntington disease (HD). Here, we provide evidence that polyglutamine expansion leads to the formation of large intracellular aggregates in vitro and in vivo. In vitro these huntingtincontaining aggregates disrupt normal cellular architecture and increase in frequency with polyglutamine length. Huntingtin truncated at nucleotide 1955, close to the caspase3 cleavage site, forms perinuclear aggregates more readily than fulllength huntingtin and increases the susceptibility of cells to death following apoptotic stimuli. Further truncation of huntingtin to nucleotide 436 results in both intranuclear and perinuclear aggregates. For a given protein size, increasing polyglutamine length is associated with increased cellular toxicity. Asymptomatic transgenic mice expressing fulllength huntingtin with 138 polyglutamines form exclusively perinuclear aggregates in neurons. These data support the hypothesis that proteolytic cleavage of mutant huntingtin leads to the development of aggregates which compromise cell viability, and that their localization is influenced by protein length. http://www.ncbi.nlm.nih.gov/pubmed/9462744 Sat, 31 Jan 1998 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/9266721 Sun, 31 Aug 1997 00:00:00 -0700 Apoptosis is a fundamental process required for normal development of the nervous system and is triggered during neurodegenerative disease. To dissect the molecular events leading to neuronal cell death, we have developed a cellfree model of neuronal apoptosis. The model faithfully reproduces key elements of apoptosis, including chromatin condensation, DNA fragmentation, caspase activation/processing, and selective substrate cleavage. We report that cellfree apoptosis is activated in premitochondrial, mitochondrial, and postmitochondrial phases by tamoxifen, mastoparan, and cytochrome c, respectively, allowing a functional ordering of these proapoptotic modulators. Furthermore, this is the first report of mitochondrialmediated activation of cellfree apoptosis in a cell extract. Although Bcl2 blocks activation at the premitochondrial and mitochondrial levels, it does not affect the postmitochondrial level. The cellfree system described here provides a valuable tool to elucidate the molecular events leading to neuronal cell death. http://www.ncbi.nlm.nih.gov/pubmed/9236228 Thu, 31 Jul 1997 00:00:00 -0700 We expressed the human antiapoptotic protein, Bcl2, in Saccharomyces cerevisiae to investigate its effects on antioxidant protection and stationary phase survival. Yeast lacking copperzinc superoxide dismutase (sod1Delta) show a profound defect in entry into and survival during stationary phase even under conditions optimal for survival of wildtype strains (incubation in water after stationary phase is reached). Expression of Bcl2 in the sod1Delta strain caused a large improvement in viability at entry into stationary phase, as well as increased resistance to 100 oxygen and increased catalase activity. In addition, Bcl2 expression reduced mutation frequency in both wildtype and sod1Delta strains. In another set of experiments, wildtype yeast incubated in expired minimal medium instead of water lost viability quickly expression of Bcl2 significantly delayed this stationary phase death. Our results demonstrate that Bcl2 has activities in yeast that are similar to activities it is known to possess in mammalian cells: (a) stimulation of antioxidant protection and (b) delay of processes leading to cell death. http://www.ncbi.nlm.nih.gov/pubmed/9199172 Mon, 30 Jun 1997 00:00:00 -0700 A series of mutant human and yeast copperzinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metalbinding characteristics and their redox reactivities. Replacement of Zn2 ion in the zinc sites of several of these proteins with either Cu2 or Co2 gave metalsubstituted derivatives with spectroscopic properties different from those of the analogous derivative of the wildtype proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALSassociated mutant copperzinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wildtype proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copperzinc superoxide dismutaseassociated familial ALSi.e., how such a diverse set of mutations can result in the same gain of function that causes the disease. http://www.ncbi.nlm.nih.gov/pubmed/8901564 Sat, 30 Nov 1996 00:00:00 -0800 Mutations in copperzinc superoxide dismutase (CuZnSOD) that are associated with familial ALS (FALS) are dominant, gainoffunction mutations, but the nature of the function gained has not been identified. In addition to catalyzing the dismutation of superoxide, copperzinc superoxide dismutase also displays peroxidase activity. Whereas mutants A4V and G93A retained superoxide dismutase activity, they demonstrated a markedly enhanced copperdependent peroxidase activity in comparison with that of the wild type enzyme as detected by the spin trap 5,5'dimethyl1pyrroline Noxide (DMPO) in electron paramagnetic resonance measurements. Two copper chelators, diethyldithiocarbamate and penicillamine, inhibited the mutants' peroxidase activity, but not that of the wild type enzyme, at stoichiometric concentrations furthermore, these copper chelators enhanced neural survival in a cellculture model of ALS but did not alter survival of cells expressing only wild type copperzinc superoxide dismutase. These observations suggest that oxidative reactions catalyzed by mutant copperzinc superoxide dismutases may initiate the neuropathologic changes of FALS. http://www.ncbi.nlm.nih.gov/pubmed/8858049 Thu, 31 Oct 1996 00:00:00 -0800 Expression of the protooncogene bcl2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl2 transfectants in two different neural cell linesrat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT17. Both neural cell lines overexpressing bcl2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT17 cells overexpressing bcl2 were significantly reduced. In addition, the NAD/NADH ratio of bcl2expressing PC12 and GT17 cells was two to threefold less than that of control cell lines. GT17 cells overexpressing bcl2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl2 were similar to those of control cells. These results indicate that the overexpression of bcl2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes. http://www.ncbi.nlm.nih.gov/pubmed/8752134 Mon, 30 Sep 1996 00:00:00 -0700 Ubiquinone (Q) is an essential, lipid soluble, redox component of the mitochondrial respiratory chain. Much evidence suggests that ubiquinol (QH2) functions as an effective antioxidant in a number of membrane and biological systems by preventing peroxidative damage to lipids. It has been proposed that superoxide dismutase (SOD) may protect QH2 form autoxidation by acting either directly as a superoxidesemiquinone oxidoreductase or indirectly by scavenging superoxide. In this study, such an interaction between QH2 and SOD was tested by monitoring the fluorescence of cisparinaric acid (cPN) incorporated phosphatidylcholine (PC) liposomes. Q6H2 was found to prevent both fluorescence decay and generation of lipid peroxides (LOOH) when peroxidation was initiated by the lipidsoluble azo initiator DAMP, dimethyl 2,2'azobis (2methylpropionate), while Q6 or SOD alone had no inhibitory effect. Addition of either SOD or catalase to Q6H2containing liposomes had little effect on the rate of peroxidation even when incubated in 100 O2. Hence, the autoxidation of QH2 is a competing reaction that reduces the effectiveness of QH2 as an antioxidant and was not slowed by either SOD or catalase. The in vivo interaction of SOD and QH2 was also tested by employing yeast mutant strains harboring deletions in either CuZnSOD and/or MnSOD. The sod mutant yeast strains contained the same percent Q6H2 per cell as wildtype cells. These results indicate that the autoxidation of QH2 is independent of SOD. http://www.ncbi.nlm.nih.gov/pubmed/8639607 Sun, 30 Jun 1996 00:00:00 -0700 Copperzinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copperzinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxidegenerating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1deletion strains, indicating that copper metallothioneins substitute for copperzinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from wholecell extracts, exhibits copperdependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants. http://www.ncbi.nlm.nih.gov/pubmed/8367458 Thu, 30 Sep 1993 00:00:00 -0700 Novel solgel synthetic techniques were used to immobilize copperzinc superoxide dismutase (CuZnSOD), cytochrome c, and myoglobin (Mb) by encapsulation in stable, optically transparent, porous silica glass matrices under mild conditions such that the biomolecules retained their characteristic reactivities and spectroscopic properties. The resulting glasses allowed transport of small molecules into and out of the glasses at reasonable rates but nevertheless retained the protein molecules within their pores. Chemical reactions of the immobilized proteins could be monitored by means of changes in their visible absorption spectra. Silica glasses containing the immobilized proteins were observed to have similar reactivities and spectroscopic properties to those found for the proteins in solution. For example, encapsulated CuZnSOD was demetallated and remetallated, encapsulated ferricytochrome c was reduced and then reoxidized, and encapsulated met Mb was reduced to deoxy Mb and then reacted either with dioxygen to make oxy Mb or with carbon monoxide to make carbonyl Mb. http://www.ncbi.nlm.nih.gov/pubmed/1312257 Tue, 31 Mar 1992 00:00:00 -0800 Lys234 has been postulated to participate in betalactamase catalysis by acting as an electrostatic anchor for the C3 carboxylate of penicillins Herzberg, O., Moult, J. (1987) Science 236, 694701. To test this hypothesis, sitedirected mutagenesis was used to convert the Lys234 in Bacillus licheniformis betalactamase into Glu234 or Ala234. The wildtype, Glu234, and Ala234 betalactamases have been expressed in Bacillus subtilis and purified to homogeneity. The wildtype, K234E, and K234A enzymes have virtually identical circular dichroism and fluorescence spectra, similar thermal stabilities at neutral pH, and the same susceptibilities to proteolysis, indicating the lack of significant structural perturbation caused by the mutation. At acidic and basic pH the mutant enzymes have the same native circular dichroism as the wildtype enzyme but the thermal stability is significantly different. The mutations cause perturbations of the pK values of the ionizing groups responsible for the pH dependence of the catalytic reaction in both the free enzyme and the E.S complex. As expected, conversion of Lys234 to Ala or Glu decreased substrate binding (Km) by 12 orders of magnitude for several penicillin and cephalosporin substrates at neutral and higher pH. However, at low pH, Km is essentially the same for the K234E and K234A enzymes as for the wildtype enzyme. Furthermore, decreases of 23 orders of magnitude in kcat were also observed, indicating substantial effects on the transitionstate binding, as well as on groundstate binding. Surprisingly, changing the C3 carboxylate of phenoxymethylpenicillin to a hydroxymethyl group led to little difference in kinetic properties with the K234E or K234A enzyme. The results of this investigation indicate the Lys234 is an important activesite residue involved in both groundstate and transitionstate binding. http://www.ncbi.nlm.nih.gov/pubmed/1974463 Fri, 31 Aug 1990 00:00:00 -0700