Mechanism Of Protein Degradation Research Articles

Mimicking Lysosomal Degradation of α-Synuclein

Pathological accumulation of α-synuclein (α-syn) is a feature of Parkinson's disease. This accumulation may be counteracted by mechanisms of protein degradation that involve the proteasome and lysosome. Specifically, the lysosomal protease, Cathepsin D (CatD), has been suggested to be the main enzyme involved in the degradation of α-syn in vivo. In vitro, only C-terminal truncated species are generated, arguing that other mechanisms are needed to fully explain α-syn degradation. Here, we show that N-terminally acetylated α-syn also generates C-terminal as well as N-terminal truncated variants in the presence of CatD. These species are shown to be more aggregation prone. Since α-syn associates with membranes, we have investigated the effects of various glycosphingolipids such as glucosylceramide (GlcCer), on CatD degradation of α-syn. It is known that GlcCer buildup is a hallmark of the lysosomal storage disorder Gaucher disease and that these patients have an increased risk of developing PD. Our data clearly shows that in the presence of GlcCer and CatD, α-syn is completely proteolyzed. These data offer new mechanistic insight into α-syn degradation in the lysosome.

A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation.

The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of ∼45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus.

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.

PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.

ESCRT regulates surface expression of the Kir2.1 potassium channel.

Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2.1 potassium channel because altered Kir2.1 levels lead to human disease and Kir2.1 restores growth on low-potassium medium in yeast mutated for endogenous potassium channels. Using this system, first we find that Kir2.1 is targeted for endoplasmic reticulum-associated degradation (ERAD). Next a synthetic gene array identifies nonessential genes that negatively regulate Kir2.1. The most prominent gene family that emerges from this effort encodes members of endosomal sorting complex required for transport (ESCRT). ERAD and ESCRT also mediate Kir2.1 degradation in human cells, with ESCRT playing a more prominent role. Thus multiple proteolytic pathways control Kir2.1 levels at the plasma membrane.

BC-box protein domain-related mechanism for VHL protein degradation

The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.

Coordinate Regulation of Mature Dopaminergic Axon Morphology by Macroautophagy and the PTEN Signaling Pathway

Macroautophagy is a conserved mechanism for the bulk degradation of proteins and organelles. Pathological studies have implicated defective macroautophagy in neurodegeneration, but physiological functions of macroautophagy in adult neurons remain unclear. Here we show that Atg7, an essential macroautophagy component, regulates dopaminergic axon terminal morphology. Mature Atg7-deficient midbrain dopamine (DA) neurons harbored selectively enlarged axonal terminals. This contrasted with the phenotype of DA neurons deficient in Pten – a key negative regulator of the mTOR kinase signaling pathway and neuron size – that displayed enlarged soma but unaltered axon terminals. Surprisingly, concomitant deficiency of both Atg7 and Pten led to a dramatic enhancement of axon terminal enlargement relative to Atg7 deletion alone. Similar genetic interactions between Atg7 and Pten were observed in the context of DA turnover and DA-dependent locomotor behaviors. These data suggest a model for morphological regulation of mature dopaminergic axon terminals whereby the impact of mTOR pathway is suppressed by macroautophagy.

Abstract PR08: Autophagic degradation of Δ133p53 during replicative cellular senescence: An isoform-specific protein degradation mechanism for p53

Abstract Δ133p53, a p53 isoform that dominant-negatively inhibits full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. We here demonstrate that, in contrast to proteasomal degradation of full-length p53, Δ133p53 is degraded by autophagy. Pharmacological inhibition of autophagy restores the reduced expression of Δ133p53 in replicatively senescent fibroblasts. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also results in restored expression of Δ133p53. The degradation of Δ133p53 via selective autophagy is indicated by its ubiquitination at the C-terminal lysine residues, its colocalization in the cytoplasm with a ubiquitin-binding protein p62/SQSTM1 and an autophagosome component LC3-II upon autophagy induction, and its stabilization by siRNA knockdown of p62/SQSTM1. A chaperone-associated E3 ubiquitin ligase STUB1, rather than MDM2, physically interacts with Δ133p53 and, consistent with the downregulation of endogenous STUB1 at replicative senescence, siRNA knockdown of STUB1 induces autophagic degradation of Δ133p53 and thereby senescence. Our data reveal that Δ133p53 degradation is controlled specifically by STUB1, rather than overall activity of autophagy. This study has identified for the first time a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence and supports the hypothesis that Δ133p53 functions at the crossroads of senescence and autophagy. This abstract is also presented as poster B19. Citation Format: Izumi Horikawa, Kaori Fujita, Lisa Miller Jenkins, Yukiharu Hiyoshi, Abdul M. Mondal, Borivoj Vojtesek, David P. Lane, Ettore Appella, Curtis C. Harris. Autophagic degradation of Δ133p53 during replicative cellular senescence: An isoform-specific protein degradation mechanism for p53. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR08.

Transcriptomic indices of fast and slow disease progression in two mouse models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is heterogeneous with high variability in the speed of progression even in cases with a defined genetic cause such as superoxide dismutase 1 (SOD1) mutations. We reported that SOD1(G93A) mice on distinct genetic backgrounds (C57 and 129Sv) show consistent phenotypic differences in speed of disease progression and life-span that are not explained by differences in human SOD1 transgene copy number or the burden of mutant SOD1 protein within the nervous system. We aimed to compare the gene expression profiles of motor neurons from these two SOD1(G93A) mouse strains to discover the molecular mechanisms contributing to the distinct phenotypes and to identify factors underlying fast and slow disease progression. Lumbar spinal motor neurons from the two SOD1(G93A) mouse strains were isolated by laser capture microdissection and transcriptome analysis was conducted at four stages of disease. We identified marked differences in the motor neuron transcriptome between the two mice strains at disease onset, with a dramatic reduction of gene expression in the rapidly progressive (129Sv-SOD1(G93A)) compared with the slowly progressing mutant SOD1 mice (C57-SOD1(G93A)) (1276 versus 346; Q-value ≤ 0.01). Gene ontology pathway analysis of the transcriptional profile from 129Sv-SOD1(G93A) mice showed marked downregulation of specific pathways involved in mitochondrial function, as well as predicted deficiencies in protein degradation and axonal transport mechanisms. In contrast, the transcriptional profile from C57-SOD1(G93A) mice with the more benign disease course, revealed strong gene enrichment relating to immune system processes compared with 129Sv-SOD1(G93A) mice. Motor neurons from the more benign mutant strain demonstrated striking complement activation, over-expressing genes normally involved in immune cell function. We validated through immunohistochemistry increased expression of the C3 complement subunit and major histocompatibility complex I within motor neurons. In addition, we demonstrated that motor neurons from the slowly progressing mice activate a series of genes with neuroprotective properties such as angiogenin and the nuclear factor (erythroid-derived 2)-like 2 transcriptional regulator. In contrast, the faster progressing mice show dramatically reduced expression at disease onset of cell pathways involved in neuroprotection. This study highlights a set of key gene and molecular pathway indices of fast or slow disease progression which may prove useful in identifying potential disease modifiers responsible for the heterogeneity of human amyotrophic lateral sclerosis and which may represent valid therapeutic targets for ameliorating the disease course in humans.

Effects of corticosterone and amyloid-beta on proteins essential for synaptic function: Implications for depression and Alzheimer's disease

The relationship between Alzheimer's disease (AD) and depression has been well established in terms of epidemiological and clinical observations. Depression has been considered to be both a symptom and risk factor of AD. Several genetic and neurobiological mechanisms have been described to underlie these two disorders. Despite the accumulating knowledge on this topic, the precise neuropathological mechanisms remain to be elucidated. In this study, we propose that synaptic degeneration plays an important role in the disease progression of depression and AD. Using primary culture of hippocampal neurons treated with oligomeric Aβ and corticosterone as model agents for AD and depression, respectively, we found significant changes in the pre-synaptic vesicle proteins synaptophysin and synaptotagmin. We further investigated whether the observed protein changes affected synaptic functions. By using FM®4-64 fluorescent probe, we showed that synaptic functions were compromised in treated neurons. Our findings led us to investigate the involvement of protein degradation mechanisms in mediating the observed synaptic protein abnormalities, namely, the ubiquitin–proteasome system and autophagy. We found up-regulation of ubiquitin-mediated protein degradation, and the preferential signaling for the autophagic–lysosomal degradation pathway. Lastly, we investigated the neuroprotective role of different classes of antidepressants. Our findings demonstrated that the antidepressants Imipramine and Escitalopram were able to rescue the observed synaptic protein damage. In conclusion, our study shows that synaptic degeneration is an important common denominator underlying depression and AD, and alleviation of this pathology by antidepressants may be therapeutically beneficial.

Downregulation of Beclin1 and Impairment of Autophagy in a Small Population of Colorectal Cancer

BackgroundAutophagy is a highly conserved mechanism for degradation and recycling of long-lived proteins and damaged organelle to maintain cell homeostasis. Deregulation of autophagy has been associated with tumorigenesis. Beclin 1 is an essential autophagy protein and its upregulation has been observed in most colorectal cancer tissues. However, there is a small population of colorectal cancers with downregulation of Beclin 1.AimThe purpose of this study was to investigate the role autophagy plays in colorectal cancers with downregulaion of Beclin 1.MethodsLC3 protein, an autophagosome marker, was assessed by ICH and WB in colorectal cancers tissues. An anti-tumor effect of Beclin 1 was examined by introducing exogenous Beclin 1 in vitro. Colony formation assay, growth curves and mouse xenograft were analysed.ResultsOur results showed that LC3 was suppressed in the colorectal cancers (9.86 %) with downregulation of Beclin 1. Moreover, overexpression of Beclin 1 inhibited colorectal cancer cell growth and enhanced the rapamycin-induced antitumor effect in vitro.ConclusionDownregulation of Beclin 1 and autophagy inhibition play an important role in a part of colorectal cancers. Activating autophagy or overexperssion of Beclin 1 may be an effective treatment for some colorectal cancers. Detection of expression profile of Beclin 1 in colorectal cancers could be a strategy for new diagnostic and therapeutic methods.Electronic supplementary materialThe online version of this article (doi:10.1007/s10620-013-2732-8) contains supplementary material, which is available to authorized users.

PKG Primes the Proteasome

Proteostasis, the precisely orchestrated balance between protein synthesis and degradation, plays a critical role in the maintenance of cell function. This is particularly the case for postmitotic cells such as cardiomyocytes and neurons, which rarely re-enter the cell cycle to divide and hence survive for decades. Disease-related stress can promote dysregulation of proteostasis, leading to accumulation of damaged or misfolded proteins as toxic aggregates capable of triggering cell death.1,2 Indeed, in both dilated cardiomyopathy and ischemic heart disease, perturbations in proteostasis are evidenced by accumulation of polyubiquitinated proteins.3 Additionally, genetic disorders that lead to aggregate formation can trigger cardiomyopathy.4 For example, specific mutations in the coding region of the molecular chaperone αB-crystallin (CryAB) disrupt its chaperone function, leading to a distinctive myofibrillar myopathy characterized by accumulation of toxic aggregates in cardiac myocytes reminiscent of Alzheimer disease or polyglutamine expansion disorders. Article see p 365 Cells can be protected from accumulation of abnormal protein aggregates by either a decrease in their formation or an increase in their clearance. Protein aggregate clearance is accomplished by 2 major catabolic processes: the ubiquitin proteasome pathway (UPP) or the autophagy-lysosomal pathway (Figure 1). Inhibition of either pathway provokes increases in toxic, intracellular aggregates that can promote disease pathogenesis. Figure 1. Damaged or misfolded proteins are targeted primarily for degradation by the ubiquitin proteasomal pathway (UPP). When an excess of these proteins accumulates, they aggregate and are degraded by autophagic pathways. If misfolded proteins continue to accumulate, they can be ultimately sequestered by the cell into ubiquitin-rich cytoplasmic inclusions called aggresomes. Autophagy is a highly conserved catabolic process underlying bulk removal of cytoplasmic proteins and organelles. Autophagy acts as an adaptive response both under conditions of starvation, where recycling of intracellular contents is required to replenish life-sustaining nutrients, and in the setting of …

New horizon in autophagy research

New horizon in autophagy research

GSK-3β signaling determines autophagy activation in the breast tumor cell line MCF7 and inclusion formation in the non-tumor cell line MCF10A in response to proteasome inhibition

The ubiquitin–proteasome system and the autophagy–lysosome pathway are the two main mechanisms for eukaryotic intracellular protein degradation. Proteasome inhibitors are used for the treatment of some types of cancer, whereas autophagy seems to have a dual role in tumor cell survival and death. However, the relationship between both pathways has not been extensively studied in tumor cells. We have investigated both proteolytic systems in the human epithelial breast non-tumor cell line MCF10A and in the human epithelial breast tumor cell line MCF7. In basal condition, tumor cells showed a lower proteasome function but a higher autophagy activity when compared with MCF10A cells. Importantly, proteasome inhibition (PI) leads to different responses in both cell types. Tumor cells showed a dose-dependent glycogen synthase kinase-3 (GSK-3)β inhibition, a huge increase in the expression of the transcription factor CHOP and an active processing of caspase-8. By contrast, MCF10A cells fully activated GSK-3β and showed a lower expression of both CHOP and processed caspase-8. These molecular differences were reflected in a dose-dependent autophagy activation and cell death in tumor cells, while non-tumor cells exhibited the formation of inclusion bodies and a decrease in the cell death rate. Importantly, the behavior of the MCF7 cells can be reproduced in MCF10A cells when GSK-3β and the proteasome were simultaneously inhibited. Under this situation, MCF10A cells strongly activated autophagy, showing minimal inclusion bodies, increased CHOP expression and cell death rate. These findings support GSK-3β signaling as a key mechanism in regulating autophagy activation or inclusion formation in human tumor or non-tumor breast cells, respectively, which may shed new light on breast cancer control.

RGS2 protein degradation is mediated by a novel multiprotein E3 ligase containing Cullin 4b and F box 44

Regulator of G protein signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and selectively regulates Gαq signaling. RGS2 KO mice are hypertensive, show increased responses to angiotensin and α‐adrenergic agonists as well as enhanced cardiomyocyte hypertrophy and death in heart failure models. RGS2 is rapidly degraded through the proteasome, but the exact mechanism for RGS2 protein degradation is not known. We hypothesize that stabilizing RGS2 protein expression could be a novel route in treating cardiovascular disease. RGS proteins are difficult drug targets. Therefore, identifying a more druggable protein within the degradation pathway could be a way to increase RGS2 protein function. The PathHunterTM ProLabel detection assay is a β‐galactosidase complementation assay that efficiently quantifies levels of proteins. Using this assay in a high‐throughput siRNA screen we identified several components of the ubiquitin protein degradation machinery and our results indicate that RGS2 is degraded through a cullin‐RING E3 ligase (CRL) containing Cullin 4B and F box protein 44. Furthermore, our results show that RGS2 and the closely related RGS4 utilize different degradation pathways. Increased understanding of RGS2 protein degradation should help identify new RGS2 modulators for cardiovascular disease. (Supported by NIH‐DA023252 and The Swedish Heart‐and Lung Foundation).

Alterations in ROS Activity and Lysosomal pH Account for Distinct Patterns of Macroautophagy in LINCL and JNCL Fibroblasts

Neuronal Ceroid Lipofuscinoses (NCL) are lysosomal storage disorders characterized by the accumulation of lipofuscin within lysosomes. Late infantile (LINCL) and juvenile (JNCL) are their most common forms and are caused by loss-of-function mutations in tripeptidyl peptidase 1 (TPP1), a lysosomal endopeptidase, and CLN3 protein (CLN3p), whose location and function is still controversial. LINCL patients suffer more severely from NCL consequences than JNCL patients, in spite of having in common an abnormal accumulation of material with a similar composition in the lysosomes. To identify distinctive characteristics that could explain the differences in the severity of LINCL and JNCL pathologies, we compared the protein degradation mechanisms in patientś fibroblasts. Pulse-chase experiments show a significant decrease in protein degradation by macroautophagy in fibroblasts bearing TPP1 (CLN2) and CLN3p (CLN3) mutations. In CLN2 fibroblasts, LC3-II levels and other procedures indicate an impaired formation of autophagosomes, which confirms the pulse-chase experiments. This defect is linked to an accumulation of Reactive Oxygen Species (ROS), an upregulation of the Akt-mTOR signalling pathway and increased activities of the p38α and ERK1/2 MAPKs. In CLN3 fibroblasts, LC3-II analysis indicates impairment in autophagosome maturation and there is also a defect in fluid phase endocytosis, two alterations that can be related to an observed increase of 0.5 units in lysosomal pH. CLN3 fibroblasts also accumulate ROS but to a lower extent than CLN2. TPP1 activity is completely abrogated in CLN2 and partially diminished in CLN3 fibroblasts. TPP1 cleaves small hydrophobic proteins like subunit c of mitochondrial ATP synthase and the lack or a lower activity of this enzyme can contribute to lipofuscin accumulation. These alterations in TPP1 activity lead to an increased ROS production, especially in CLN2 in which it is aggravated by a decrease in catalase activity. This could explain the earlier appearance of the symptoms in the LINCL form.

Analytical methods and formulation factors to enhance protein stability in solution

Development of stable protein formulations needs intimate knowledge of the proteins' physicochemical properties. More specifically, understanding the mechanisms of protein degradation is important in designing and evaluating protein formulations. This review describes briefly the different types of interactions of the major protein degradation pathways. The analytical methods to detect protein degradation are included, along with generalized strategies to suppress protein instability with relevant excipients. With an appreciation of the current practices for stable formulations, the development process will be facilitated in a more efficient way.

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (-96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (-89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.

Enhancement of Ubiquitin Conjugation Activity Reduces Intracellular Aggregation of V76D Mutant γD-Crystallin

The ubiquitin-proteasome pathway (UPP) is an important protein quality control mechanism for selective degradation of abnormal proteins. The objective of this study was to test the hypothesis that enhancement of the UPP capacity could attenuate the accumulation and aggregation of misfolded proteins using V76D-γD-crystallin as a model substrate. Wild type (wt) and V76D mutant γD-crystallin were fused to red fluorescence protein (RFP) and expressed in human lens epithelial cells. The cellular distribution of the expressed proteins was compared by fluorescence microscopy. The solubility of wt- and V76D-γD-crystallin was determined by cellular fractionation and Western blotting. Wt-γD-RFP and V76D-γD-RFP were also cotransfected along with a ubiquitin ligase (CHIP) or a ubiquitin-conjugating enzyme (Ubc5) into cells. Levels of wt- and V76D-γD-crystallin, the percentage of transfected cells with aggregates, and aggregate size were quantified and compared among different groups. Wt-γD-crystallin was evenly distributed in cells, whereas V76D-γD-crystallin formed intracellular aggregates. Eighty percent of wt-γD-crystallin was detected in the soluble fraction, whereas only 7% of V76D-γD-crystallin was soluble. CHIP or Ubc5 coexpression reduced the protein level of V76D-γD and concomitantly its aggregation in transfected cells; these effects could be attenuated by proteasome inhibitor. Mutant CHIP with defect TPR (tetratricopeptide repeat) or U-box domain failed to reduce levels of V76D-γD-crystallin. Enhancing ubiquitin conjugation activity reduces accumulation and aggregation of V76D-γD-crystallin by promoting its degradation. Upregulation of ubiquitin-conjugating activity could be an effective strategy to maintain lens transparency by eliminating other forms of misfolded proteins.

Macroautophagy deficiency mediates age-dependent neurodegeneration through a phospho-tau pathway

BackgroundMacroautophagy is an evolutionarily conserved mechanism for bulk intracellular degradation of proteins and organelles. Pathological studies have implicated macroautophagy defects in human neurodegenerative disorders of aging including Alzheimer’s disease and tauopathies. Neuronal deficiency of macroautophagy throughout mouse embryonic development results in neurodevelopmental defects and early postnatal mortality. However, the role of macroautophagy in mature CNS neurons, and the relationship with human disease neuropathology, remains unclear. Here we describe mice deficient in an essential macroautophagy component, Atg7, specifically within postnatal CNS neurons.ResultsPostnatal forebrain-specific Atg7 conditional knockout (cKO) mice displayed age-dependent neurodegeneration and ubiquitin- and p62-positive inclusions. Phosphorylated tau was significantly accumulated in Atg7 cKO brains, but neurofibrillary tangles that typify end-stage human tauopathy were not apparent. A major tau kinase, glycogen synthase kinase 3β (GSK3β), was also accumulated in Atg7 cKO brains. Chronic pharmacological inhibition of tau phosphorylation, or genetic deletion of tau, significantly rescued Atg7-deficiency-mediated neurodegeneration, but did not suppress inclusion formation.ConclusionsThese data elucidate a role for macroautophagy in the long-term survival and physiological function of adult CNS neurons. Neurodegeneration in the context of macroautophagy deficiency is mediated through a phospho-tau pathway.