Maintain Protein Quality Control Research Articles

Cathepsin D overexpression in the nervous system rescues lethality and Aβ42 accumulation of cathepsin D systemic knockout invivo.

The lysosome is responsible for protein and organelle degradation and homeostasis and the cathepsins play a key role in maintaining protein quality control. Cathepsin D (CTSD), is one such lysosomal protease, which when deficient in humans lead to neurolipofuscinosis (NCL) and is important in removing toxic protein aggregates. Prior studies demonstrated that CTSD germ-line knockout-CtsdKO (CDKO) resulted in accumulation of protein aggregates, decreased proteasomal activities, and postnatal lethality on Day 26±1. Overexpression of wildtype CTSD, but not cathepsin B, L or mutant CTSD, decreased α-synuclein toxicity in worms and mammalian cells. In this study we generated a mouse line expressing human CTSD with a floxed STOP cassette between the ubiquitous CAG promoter and the cDNA. After crossing with Nestin-cre, the STOP cassette is deleted in NESTIN+cells to allow CTSD overexpression-CTSDtg (CDtg). The CDtg mice exhibited normal behavior and similar sensitivity to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced neurodegeneration. By breeding CDtg mice with CDKO mice, we found that over-expression of CTSD extended the lifespan of the CDKO mice, partially rescued proteasomal deficits and the accumulation of Aβ42 in the CDKO. This new transgenic mouse provides supports for the key role of CTSD in protecting against proteotoxicity and offers a new model to study the role of CTSD enhancement invivo.

Chemogenomic Profiling of a Plasmodium falciparum Transposon Mutant Library Reveals Shared Effects of Dihydroartemisinin and Bortezomib on Lipid Metabolism and Exported Proteins.

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance. IMPORTANCE Malaria control is seriously threatened by the emergence and spread of Plasmodium falciparum resistance to the leading antimalarial, artemisinin. The potent killing activity of artemisinin results from oxidative damage unleashed by free heme activation released by hemoglobin digestion. Although the ubiquitin-proteasome system is considered critical for parasite survival of this toxicity, the diverse genetic changes linked to artemisinin resistance are complex and, so far, have not included the ubiquitin-proteasome system. In this study, we use a systematic forward genetic approach by screening a library of P. falciparum random piggyBac mutants to decipher the genetic factors driving malaria parasite responses to the oxidative stress caused by antimalarial drugs. This study compares phenotype-genotype associations influencing dihydroartemisinin responses with the proteasome inhibitor bortezomib to delineate the role of ubiquitin-proteasome system. Our study highlights shared and unique pathways from the complex array of molecular processes critical for P. falciparum survival resulting from the oxidative damage of artemisinin.

Covalent drug discovery targeting G protein-coupled receptors

G protein-coupled receptors (GPCRs) play pivotal roles in converting physicochemical stimuli due to environmental changes to intracellular responses. After ligand stimulation, many GPCRs are desensitized and then recycled or degraded through phosphorylation and β-arrestin-dependent internalization, an important process to maintain protein quality control of GPCRs. However, it is unknown how GPCRs with low β-arrestin sensitivity are controlled. Here we unmasked a β-arrestin-independent GPCR internalization, named Redox-dependent Alternative Internalization (REDAI), focusing on β-arrestin-resistant purinergic P2Y6 receptor (P2Y6R). P2Y6R is highly expressed in macrophage and pathologically contributes to the development of colitis in mice. Natural electrophiles including in functional foods induce REDAI-mediated P2Y6R degradation leading to anti-inflammation in macrophages. Prevention of Cys220 modification on P2Y6R resulted in aggravation of the colitis. These results strongly suggest that targeting REDAI on GPCRs will be a breakthrough strategy for the prevention and treatment of inflammatory diseases.

Abstract 2558: Understanding the shared roles of obesity and genetic mutation in the development of endometrial cancer

Abstract Up to 20% of all cancer deaths in women can be attributed to excess body weight. Endometrial cancer was the first cancer identified with an increased risk among obese populations, and incidence of endometrial cancer is rising steadily due to the obesity epidemic. Obesity is associated with over 60% of endometrial cancer occurring in the United States, but like many cancers, the causes of endometrial cancer are multi-faceted. Increased estrogen abundance in obese women is considered the primary driver, but obesity-linked complications, including hyperinsulinemia, are also known to contribute to endometrial cancer. However, obesity alone is not sufficient for tumorigenesis. Genetic mutations driving endometrial cancer are well characterized, but these mutations alone are not sufficient for EC pathogenesis as they exist in uteri without cancer. In normal cells, the PI3K pathway maintains metabolic homeostasis, but in cancer the PI3K pathway is activated to promote cell survival. There is an urgent need to understand the mechanisms by which obesity and genetic mutation interact to promote EC. Metabolic signaling influences protein production and folding. The unfolded protein response pathway acts to maintain protein quality control, and is activated upon endoplasmic reticulum stress. During endoplasmic reticulum stress, if proteostasis cannot be achieved, the unfolded protein response is activated, resulting in cell death. Obesity results in systemic endoplasmic reticulum stress, with overabundant estrogen and hyperinsulinemia likely as contributing factors. In our preliminary studies, we have identified a role for PI3K pathway mutations in regulating endoplasmic reticulum stress in the endometrial epithelium. By antagonizing endoplasmic reticulum stress, PI3K pathway mutations may promote survival of endometrial epithelium through resistance to unfolded protein response-mediated cell death mechanisms. Through understanding endoplasmic reticulum stress resistance in the endometrial epithelium, we hope to develop preventative interventions for obese women at risk for endometrial cancer. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Understanding the shared roles of obesity and genetic mutation in the development of endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2558.

The Glyoxysomal Protease LON2 Is Involved in Fruiting-Body Development, Ascosporogenesis and Stress Resistance in Sordaria macrospora.

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.

Sorting Nexins in Protein Homeostasis.

Protein homeostasis is maintained by removing misfolded, damaged, or excess proteins and damaged organelles from the cell by three major pathways; the ubiquitin-proteasome system, the autophagy-lysosomal pathway, and the endo-lysosomal pathway. The requirement for ubiquitin provides a link between all three pathways. Sorting nexins are a highly conserved and diverse family of membrane-associated proteins that not only traffic proteins throughout the cells but also provide a second common thread between protein homeostasis pathways. In this review, we will discuss the connections between sorting nexins, ubiquitin, and the interconnected roles they play in maintaining protein quality control mechanisms. Underlying their importance, genetic defects in sorting nexins are linked with a variety of human diseases including neurodegenerative, cardiovascular diseases, viral infections, and cancer. This serves to emphasize the critical roles sorting nexins play in many aspects of cellular function.

78 kDa Glucose-Regulated Protein Attenuates Protein Aggregation and Monocyte Adhesion Induced by Angiotensin II in Vascular Cells.

Investigations of vascular smooth muscle cell (VSMC) phenotypic modulation due to angiotensin II (AngII) stimulation are important for understanding molecular mechanisms contributing to hypertension and associated vascular pathology. AngII induces endoplasmic reticulum (ER) stress in VSMCs, which has been implicated in hypertensive vascular remodeling. Under ER stress, 78 kDa glucose-regulated protein (GRP78) acts as an endogenous chaperone, as well as a master controller of unfolded protein response (UPR) to maintain protein quality control. However, the potential downstream consequences of ER stress induced by AngII on protein quality control and pro-inflammatory phenotype in VSMCs remain elusive. This study aims to identify protein aggregation as evidence of the disruption of protein quality control in VSMCs, and to test the hypothesis that preservation of proteostasis by overexpression of GRP78 can attenuate the AngII-induced pro-inflammatory phenotype in VSMCs. Increases in protein aggregation and enhanced UPR were observed in VSMCs exposed to AngII, which were mitigated by overexpression of GRP78. Moreover, GRP78 overexpression attenuated enhanced monocyte adhesion to VSMCs induced by AngII. Our results thus indicate that the prevention of protein aggregation can potentially mitigate an inflammatory phenotype in VSMCs, which may suggest an alternative therapy for the treatment of AngII-associated vascular disorders.

Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase.

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH4) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH4 triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.

Type II Epithelial-Mesenchymal Transition Upregulates Protein N-Glycosylation To Maintain Proteostasis and Extracellular Matrix Production

Type II epithelial-mesenchymal transition (EMT) plays a vital role in airway injury, repair, and remodeling. Triggered by growth factors, such as transforming growth factor beta (TGFβ), EMT induced a biological process that converts epithelial cells into secretory mesenchymal cells with a substantially increased production of extracellular matrix (ECM) proteins. Epithelial cells are not professional secretory cells and produce few ECM proteins under normal conditions. The molecular mechanism underlying the transformation of the protein factory and secretory machinery during EMT is significant because ECM secretion is central to the pathogenesis of airway remodeling. Here we report that type II EMT upregulates the protein N-glycosylation of ECMs. The mechanism study reveals that the substantial increase in synthesis of ECM proteins in EMT activates the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 (XBP1) axis of the unfolded protein response (UPR) coupled to the hexosamine biosynthesis pathway (HBP). These two pathways coordinately up-regulate the protein N-glycosylation of ECM proteins and increase ER folding capacity and ER-associated degradation (ERAD), which improve ER protein homeostasis and protect transitioned cells from proteotoxicity. Inhibition of the alternative splicing of XBP1 or protein N-glycosylation blocks ECM protein secretion, indicating the XBP1-HBP plays a prominent role in regulating the secretion of ECM proteins in the mesenchymal transition. Our data suggest that the activation of XBP1-HBP pathways and elevation of protein N-glycosylation is an adaptive response to maintain protein quality control and facilitate the secretion of ECM proteins during the mesenchymal transition. The components of the XBP1-HBP pathways may be therapeutic targets to prevent airway remodeling.

Erratum to: Early emergence of the FtsH proteases involved in photosystem II repair

The verb ‚make“ was used instead of ‚form“ in the following sentence. Instead of A phylogenetic analysis of over 6000 FtsH protease sequences revealed that there are three major groups of FtsH proteases originating from gene duplication events in the last common ancestor of bacteria, and that the FtsH proteases involved in PSII repair make a distinct clade branching out before the divergence of FtsH proteases found in all groups of anoxygenic phototrophic bacteria. It should read A phylogenetic analysis of over 6000 FtsH protease sequences revealed that there are three major groups of FtsH proteases originating from gene duplication events in the last common ancestor of bacteria, and that the FtsH proteases involved in PSII repair form a distinct clade branching out before the divergence of FtsH proteases found in all groups of anoxygenic phototrophic bacteria. The word ‚photosynthetic“ was omitted in the following sentence: Instead of We conclude that the phylogeny of FtsH proteases is consistent with an early origin of water oxidation chemistry. It should read We conclude that the phylogeny of FtsH proteases is consistent with an early origin of photosynthetic water oxidation chemistry. The words ‚reactions“ and ‚for“ were used mistakenly in the following paragraph: Instead of Oxygenic photosynthetic electron transport from water to NADP+ requires the participation of two functionally distinct reactions centers (RCs) acting in series: photosystem II (PSII, a Type II RC containing quinone electron acceptors) and photosystem I (PSI, a Type I RC containing redox-active iron-sulphur clusters). Early ideas on for the emergence of oxygenic photosynthesis focused on the evolution of PSI and PSII from pre-existing RCs found in anoxygenic photosynthetic bacteria (Nitschke and Rutherford 1991) and the horizontal transfer of genes encoding chlorophyll biosynthetic enzymes and RC proteins (Hohmann-Marriott and Blankenship 2011, Fischer et al. 2016). It should read Oxygenic photosynthetic electron transport from water to NADP+ requires the participation of two functionally distinct reaction centers (RCs) acting in series: photosystem II (PSII, a Type II RC containing quinone electron acceptors) and photosystem I (PSI, a Type I RC containing redox-active iron-sulphur clusters). Early ideas on the emergence of oxygenic photosynthesis focused on the evolution of PSI and PSII from pre-existing RCs found in anoxygenic photosynthetic bacteria (Nitschke and Rutherford 1991) and the horizontal transfer of genes encoding chlorophyll biosynthetic enzymes and RC proteins (Hohmann-Marriott and Blankenship 2011, Fischer et al. 2016). The word ‚other“ was missing in the following sentence: Instead of Possible evolutionary constraints include the need to maintain protein quality control in the multiple membrane compartments found in cyanobacteria, chloroplasts, and mitochondria and the fact that oxygenic photosynthesis is associated with the production of singlet oxygen and ROS (reactive oxygen species), leading to protein damage, particularly in the thylakoid membrane which houses the photosynthetic apparatus. It should read Possible evolutionary constraints include the need to maintain protein quality control in the multiple membrane compartments found in cyanobacteria, chloroplasts and mitochondria and the fact that oxygenic photosynthesis is associated with the production of singlet oxygen and other ROS (reactive oxygen species), leading to protein damage, particularly in the thylakoid membrane which houses the photosynthetic apparatus. The word ‚of“ was mistakenly used instead of ‚to“ in the following sentence: Instead of Recognition of damaged D1 by cyanobacterial FtsH complexes is thought to be mediated by partial disassembly of damaged PSII (Krynicka et al. 2015) and binding of the N-terminal tail of D1 (Komenda et al. 2007). It should read Recognition of damaged D1 by cyanobacterial FtsH complexes is thought to be mediated by partial disassembly of damaged PSII (Krynicka et al. 2015) and binding to the N-terminal tail of D1 (Komenda et al. 2007).

INVESTIGATING THE ROLE OF UBIQUILIN2 IN AGE-RELATED NEURODEGENERATION

Brain accumulation of Ubiquilin2 (UBQLN2) is a shared feature of neurodegenerative diseases including Alzheimer's disease, Lewy Body Dementia, Frontotemporal Dementia (FTD) and polyglutamine expansion disorders such as Huntington disease (HD). UBQLN2's involvement in neurodegeneration reflects the fact that it normally helps maintain protein quality control, principally in protein degradation by the proteasome. Rare mutations in UBQLN2 also directly cause neurodegeneration on the FTD/ALS spectrum. Unfortunately, UBQLN2's normal function in health and its dysfunction in disease remain poorly understood. To explore UBQLN2-mediated disease we employed protein biochemistry, cell-based studies, longitudinal fluorescence microscopy and mouse models of ALS/FTD and HD. Data from in vitro studies of full length UBQLN2, expression studies in cultured neurons, and transgenic mice expressing wildtype (WT) or mutant (MUT) UBQLN2 support the view that the protein is intrinsically prone to form aggregates and that pathogenic mutations can accelerate this process. Studies of UBQLN2 deleted of functional domains implicate a specific domain in driving aggregation of both wild type and mutant UBQLN2. In transgenic mice expressing MUT-UBQLN2, the protein becomes sequestered in large aggregates within neurons throughout the central nervous system including cortex, hippocampus, cerebellum and spinal cord. Mutant mice also exhibit aberrant TDP43 localization, a common pathological hallmark of ALS/FTD generally and of UBQLN2-mediated disease specifically. In contrast, WT-UBQLN2 largely remains diffusely distributed in neurons, though some neurons display spherical puncta enriched for UBQLN2. When overexpressed in a mouse model of HD, WT-UBQLN2 nearly abolishes nuclear aggregates of the HD protein, a characteristic disease feature of HD. In the presence of mutant HD protein, UBQLN2 also moves from its normal cytoplasmic location to the nucleus of affected neurons. Our results support a dominant toxic effect of MUT-UBQLN2 driving disease pathogenesis, associated with aggregation of the protein. Our results further support the view that UBQLN2 normally functions in neurons to help maintain protein homeostasis; whether partial loss of this quality control function contributes to disease pathogenesis remains unknown. Accordingly, ongoing studies are focused in part on evaluating the differences between wild type and mutant forms of UBQLN2 in maintaining neuronal protein homeostasis.

The Impact of the Unfolded Protein Response and the Hexosamine Biosynthetic Pathway on Glycosylation

Glycan changes, in addition to being traditional mediators of protein quality control, cell signaling, and metabolism, are intimately linked to the progression of cancer and can potentially act as novel therapeutic and diagnostic targets. Despite advances in our understanding that glycosylation plays a key role in protein folding, maturation, and function, there is only limited understanding of the interconnected nature of glycan alterations to endoplasmic reticulum stress, the unfolded protein response, and the development of cancer. This review aims to highlight the interdependence of these pathways and the way they feed into each other to maintain protein quality control, regulate key survival mechanisms, and promote cell viability.

The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli.

In eubacteria, the tmRNA system frees ribosomes that stall during protein synthesis and adds an ssrA tag to the incompletely translated polypeptide to target it for degradation. The AAA+ ClpXP protease degrades most ssrA-tagged proteins in the Escherichia coli cytoplasm and was recently shown to degrade an ssrA-tagged protein in the inner membrane. However, we find that tmRNA-mediated tagging of E. coli ProW1-182, a different inner-membrane protein, results in degradation by the membrane-tethered AAA+ FtsH protease. ClpXP played no role in the degradation of ProW1-182 in vivo. These studies suggest that a complex distribution of proteolytic labor maintains protein quality control in the inner membrane.

Molecular Insight on the Relationship Between the Opposing Co‐chaperones CHIP and HOP

Cellular chaperones and co-chaperones maintain protein quality control (PQC) and are vital to cellular stress responses. The TPR-containing co-chaperones CHIP (C-terminus of Hsp70 Interacting Prote...

Crystal structure of the stress-inducible human heat shock protein 70 substrate-binding domain in complex with peptide substrate.

The HSP70 family of molecular chaperones function to maintain protein quality control and homeostasis. The major stress-induced form, HSP70 (also called HSP72 or HSPA1A) is considered an important anti-cancer drug target because it is constitutively overexpressed in a number of human cancers and promotes cancer cell survival. All HSP70 family members contain two functional domains: an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate-binding domain (SBD); the latter is subdivided into SBDα and SBDβ subdomains. The NBD and SBD structures of the bacterial ortholog, DnaK, have been characterized, but only the isolated NBD and SBDα segments of eukaryotic HSP70 proteins have been determined. Here we report the crystal structure of the substrate-bound human HSP70-SBD to 2 angstrom resolution. The overall fold of this SBD is similar to the corresponding domain in the substrate-bound DnaK structures, confirming a similar overall architecture of the orthologous bacterial and human HSP70 proteins. However, conformational differences are observed in the peptide-HSP70-SBD complex, particularly in the loop Lα, β that bridges SBDα to SBDβ, and the loop LL,1 that connects the SBD and NBD. The interaction between the SBDα and SBDβ subdomains and the mode of substrate recognition is also different between DnaK and HSP70. This suggests that differences may exist in how different HSP70 proteins recognize their respective substrates. The high-resolution structure of the substrate-bound-HSP70-SBD complex provides a molecular platform for the rational design of small molecule compounds that preferentially target this C-terminal domain, in order to modulate human HSP70 function.

P300 Is a preferred client protein for nuclear chaperones (800.6)

The KAT3 family of histone acetyl‐transferases, p300 and CBP, are a class of highly versatile epigenetic regulators capable of forming a diverse array of chromatin regulatory complexes that readily adapt to a wide variety of gene promoter contexts to control transcription. These properties require forming multiple conformations through flexible positioning and folding of their various functional domains. Here we show that HSP90 and HSP70 molecular chaperones play a major role in assisting p300/CBP in these molecular functions at gene promoters. Disruption of this chaperone function alters the genome‐wide distribution of p300 and CBP while causing global changes in histone acetylation and gene expression. Major targeted genes include diverse regulators of mammalian protein degradation pathways, thus revealing a previously unrecognized function for molecular chaperones in the epistatic regulation of protein homeostasis through epigenetically regulated transcription of numerous components of protein degradation pathways essential for maintaining protein quality control in the cell.

Cardiac myocyte‐specific deletion of Heat shock protein 10 results in altered cytosolic and mitochondrial protein expression (699.6)

Heat shock proteins (HSPs) are molecular chaperones that prevent protein damage and proteolysis. HSP10 forms mitochondrial (Mito) chaperoning complexes with HSP60 to maintain protein quality control. Studies in vitro suggest that HSP10 plays a role in normal Mito function; however, its physiological function in the heart in vivo remains to be determined. To investigate the function of cardiac HSP10, we generated a floxed allele of the Hspe1 gene, and found that cardiac specific, tamoxifen‐inducible deletion of HSP10 in adult mice resulted in death between 12 and 18 weeks after HSP10 deletion. Increased mortality after HSP10 deletion was associated with dramatically altered protein expression patterns. For instance, cardiac hypertrophic biomarkers atrial natriuretic factor (ANF), β‐Myosin Heavy Chain (βMHC) and skeletal muscle actin were increased in the HSP10 KO mice. In Addition, there was a dramatic reduction in levels of Mito respiratory complex subunits including CI‐NDUFA9, CII‐30, COX I, COX IV, and COX Vb. Furthermore, the apoptotic pathway is activated in HSP10 KO mice as revealed by the cleavage of Caspases 9 and 3. Our results suggest that mitochondrial protein damage may be the primary pathological cause of the death. In conclusion, HSP10 plays a critical and vital role in regulating mitochondrial protein homeostasis.Grant Funding Source: Supported by P. Robert Majumder Charitable Foundation.

Mechanical Protein Unfolding and Translocation by AAA+ Proteases

In all cells, protein degradation regulates key processes and maintains protein quality control. The AAA+ protease ClpXP is one of the best-studied examples of the ATP-dependent machinery responsible for intracellular proteolysis. A ring-shaped hexamer of the ClpX ATPase first recognizes and denatures folded protein substrate, then translocates the unfolded polypeptide into the barrel-shaped ClpP peptidase for degradation. In recent studies, dual-laser optical trapping was used to dissect the molecular events resulting in protein degradation by single molecules of ClpXP (Aubin-Tam et al. 2011, Maillard et al. 2011). This technique allows for the detection of small events (nm scale) with high temporal resolution (sub-ms scale). Here, we adapt our single-molecule assay to study a related AAA+ protease ClpAP and to address whether the similar architecture shared by all AAA+ proteases indicates a common mechanism of protein denaturation and degradation. Unlike ClpX, ClpA contains two ATPase modules per hexamer subunit and represents a major subclass of double-ring protein remodeling enzymes. We find similarities but also differences in mechanochemical activity between single- and double-ring ATPases, which contribute to their distinct activities in protein unfolding and degradation. We anticipate that the methods used to study ClpXP and ClpAP at the single-molecule level can be readily applied to examine the activity of other AAA+ proteases and protein remodeling enzymes.Aubin-Tam ME, Olivares AO, Sauer RT, Baker TA, Lang MJ (2011). Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145: 257-67.Maillard RA, Chistol G, Sen M, Righini M, Tan J, Kaiser CM, Hodges C, Martin A, Bustamante C (2011). ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145: 459-69.

CHIP has a protective role against oxidative stress-induced cell death through specific regulation of Endonuclease G

Oxidative stress is implicated in carcinogenesis, aging, and neurodegenerative diseases. The E3 ligase C terminus of Hsc-70 interacting protein (CHIP) has a protective role against various stresses by targeting damaged proteins for proteasomal degradation, and thus maintains protein quality control. However, the detailed mechanism by which CHIP protects cells from oxidative stress has not been demonstrated. Here, we show that depletion of CHIP led to elevated Endonuclease G (EndoG) levels and enhanced cell death upon oxidative stress. In contrast, CHIP overexpression reduced EndoG levels, and resulted in reduced or no oxidative stress-induced cell death in cancer cells and primary rat cortical neurons. Under normal conditions Hsp70 mediated the interaction between EndoG and CHIP, downregulating EndoG levels in a Hsp70/proteasome-dependent manner. However, under oxidative stress Hsp70 no longer interacted with EndoG, and the stabilized EndoG translocated to the nucleus and degraded chromosomal DNA. Our data suggest that regulation of the level of EndoG by CHIP in normal conditions may determine the sensitivity to cell death upon oxidative stress. Indeed, injection of H2O2 into the rat brain markedly increased cell death in aged mice compared with young mice, which correlated with elevated levels of EndoG and concurrent downregulation of CHIP in aged mice. Taken together, our findings demonstrate a novel protective mechanism of CHIP against oxidative stress through regulation of EndoG, and provide an opportunity to modulate oxidative stress-induced cell death in cancer and aging.

Impaired proteasome function in sporadic amyotrophic lateral sclerosis

Abstract The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS.