A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.

The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.

DNAJB6, a Key Factor in Neuronal Sensitivity to Amyloidogenesis.

CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.

Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity.

G3BP1-dependent mechanism suppressing protein aggregation in Huntington's models and its demise upon stress granule assembly.

Stress granules are membrane-less ribonucleoprotein organelles that assemble upon exposure to stress conditions, but rapidly disassemble upon removal of stress. However, chronic stress can lead to persistent stress granules, a feature of distinct age-related neurodegenerative disorders. Among them, Huntington's disease (HD), which is caused by mutant expansion of the polyglutamine (polyQ) repeats of huntingtin protein (HTT), leading to its aggregation. To identify modulators of mutant HTT aggregation, we define its interactome in striatal neurons differentiated from patient-derived induced pluripotent stem cells (HD-iPSCs). We find that HTT interacts with G3BP1, a characteristic component of stress granules. Knockdown of G3BP1 increases mutant HTT protein levels and abolishes the ability of iPSCs as well as their differentiated neural counterparts to suppress mutant HTT aggregation. Moreover, loss of G3BP1 hastens polyQ-expanded aggregation and toxicity in the neurons of HD C. elegans models. Likewise, the assembly of G3BP1 into stress granules upon distinct stress conditions also reduces its interaction with HTT in human cells, promoting mutant HTT aggregation. Notably, enhancing the levels of G3BP1 is sufficient to induce proteasomal degradation of mutant HTT and prevent its aggregation, whereas the formation of stress granules blocks these ameliorative effects. In contrast, a mutant G3BP1 variant that cannot accumulate into granules retains its capacity to prevent mutant HTT aggregation even when the cells assemble stress granules. Thus, our findings indicate a direct role of G3BP1 and stress granule assembly in mutant HTT aggregation that may have implications for HD.

Kinesin-5 inhibition improves neural regeneration in experimental autoimmune neuritis.

BACKGROUND: Autoimmune neuropathies can result in long-term disability and incomplete recovery, despite adequate first-line therapy. Kinesin-5 inhibition was shown to accelerate neurite outgrowth in different preclinical studies. Here, we evaluated the potential neuro-regenerative effects of the small molecule kinesin-5 inhibitor monastrol in a rodent model of acute autoimmune neuropathies, experimental autoimmune neuritis. METHODS: Experimental autoimmune neuritis was induced in Lewis rats with the neurogenic P2-peptide. At the beginning of the recovery phase at day 18, the animals were treated with 1 mg/kg monastrol or sham and observed until day 30 post-immunisation. Electrophysiological and histological analysis for markers of inflammation and remyelination of the sciatic nerve were performed. Neuromuscular junctions of the tibialis anterior muscles were analysed for reinnervation. We further treated human induced pluripotent stem cells-derived secondary motor neurons with monastrol in different concentrations and performed a neurite outgrowth assay. RESULTS: Treatment with monastrol enhanced functional and histological recovery in experimental autoimmune neuritis. Motor nerve conduction velocity at day 30 in the treated animals was comparable to pre-neuritis values. Monastrol-treated animals showed partially reinnervated or intact neuromuscular junctions. A significant and dose-dependent accelerated neurite outgrowth was observed after kinesin-5 inhibition as a possible mode of action. CONCLUSION: Pharmacological kinesin-5 inhibition improves the functional outcome in experimental autoimmune neuritis through accelerated motor neurite outgrowth and histological recovery. This approach could be of interest to improve the outcome of autoimmune neuropathy patients.

Protein clearance strategies for disease intervention.

Protein homeostasis, or proteostasis, is essential for cell function and viability. Unwanted, damaged, misfolded and aggregated proteins are degraded by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. Growing evidence indicates that alterations in these major proteolytic mechanisms lead to a demise in proteostasis, contributing to the onset and development of distinct diseases. Indeed, dysregulation of the UPS or autophagy is linked to several neurodegenerative, infectious and inflammatory disorders as well as cancer. Thus, modulation of protein clearance pathways is a promising approach for therapeutics. In this review, we discuss recent findings and open questions on how targeting proteolytic mechanisms could be applied for disease intervention.

Mechanism suppressing H3K9 trimethylation in pluripotent stem cells and its demise by polyQ-expanded huntingtin mutations.

Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.

Mechanisms of protein homeostasis (proteostasis) maintain stem cell identity in mammalian pluripotent stem cells.

Protein homeostasis, or proteostasis, is essential for cell function, development, and organismal viability. The composition of the proteome is adjusted to the specific requirements of a particular cell type and status. Moreover, multiple metabolic and environmental conditions challenge the integrity of the proteome. To maintain the quality of the proteome, the proteostasis network monitors proteins from their synthesis through their degradation. Whereas somatic stem cells lose their ability to maintain proteostasis with age, immortal pluripotent stem cells exhibit a stringent proteostasis network associated with their biological function and intrinsic characteristics. Moreover, growing evidence indicates that enhanced proteostasis mechanisms play a central role in immortality and cell fate decisions of pluripotent stem cells. Here, we will review new insights into the melding fields of proteostasis and pluripotency and their implications for the understanding of organismal development and survival.

The ubiquitin ligase Ubr4 controls stability of podocin/MEC-2 supercomplexes.

The PHB-domain protein podocin maintains the renal filtration barrier and its mutation is an important cause of hereditary nephrotic syndrome. Podocin and its Caenorhabditis elegans orthologue MEC-2 have emerged as key components of mechanosensitive membrane protein signalling complexes. Whereas podocin resides at a specialized cell junction at the podocyte slit diaphragm, MEC-2 is found in neurons required for touch sensitivity. Here, we show that the ubiquitin ligase Ubr4 is a key component of the podocin interactome purified both from cultured podocytes and native glomeruli. It colocalizes with podocin and regulates its stability. In C. elegans, this process is conserved. Here, Ubr4 is responsible for the degradation of mislocalized MEC-2 multimers. Ubiquitylomic analysis of mouse glomeruli revealed that podocin is ubiquitylated at two lysine residues. These sites were Ubr4-dependent and were conserved across species. Molecular dynamics simulations revealed that ubiquitylation of one site, K301, do not only target podocin/MEC-2 for proteasomal degradation, but may also affect stability and disassembly of the multimeric complex. We suggest that Ubr4 is a key regulator of podocyte foot process proteostasis.

RPN-6 determines C. elegans longevity under proteotoxic stress conditions.

Organisms that protect their germ-cell lineages from damage often do so at considerable cost: limited metabolic resources become partitioned away from maintenance of the soma, leaving the ageing somatic tissues to navigate survival amid an environment containing damaged and poorly functioning proteins. Historically, experimental paradigms that limit reproductive investment result in lifespan extension. We proposed that germline-deficient animals might exhibit heightened protection from proteotoxic stressors in somatic tissues. We find that the forced re-investment of resources from the germ line to the soma in Caenorhabditis elegans results in elevated somatic proteasome activity, clearance of damaged proteins and increased longevity. This activity is associated with increased expression of rpn-6, a subunit of the 19S proteasome, by the FOXO transcription factor DAF-16. Ectopic expression of rpn-6 is sufficient to confer proteotoxic stress resistance and extend lifespan, indicating that rpn-6 is a candidate to correct deficiencies in age-related protein homeostasis disorders.

Increased proteasome activity in human embryonic stem cells is regulated by PSMD11.

Embryonic stem cells can replicate continuously in the absence of senescence and, therefore, are immortal in culture. Although genome stability is essential for the survival of stem cells, proteome stability may have an equally important role in stem-cell identity and function. Furthermore, with the asymmetric divisions invoked by stem cells, the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore, a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4, an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates, regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity.

Proteostasis of Huntingtin in Health and Disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by motor dysfunction, cognitive deficits and psychosis. HD is caused by mutations in the Huntingtin (HTT) gene, resulting in the expansion of polyglutamine (polyQ) repeats in the HTT protein. Mutant HTT is prone to aggregation, and the accumulation of polyQ-expanded fibrils as well as intermediate oligomers formed during the aggregation process contribute to neurodegeneration. Distinct protein homeostasis (proteostasis) nodes such as chaperone-mediated folding and proteolytic systems regulate the aggregation and degradation of HTT. Moreover, polyQ-expanded HTT fibrils and oligomers can lead to a global collapse in neuronal proteostasis, a process that contributes to neurodegeneration. The ability to maintain proteostasis of HTT declines during the aging process. Conversely, mechanisms that preserve proteostasis delay the onset of HD. Here we will review the link between proteostasis, aging and HD-related changes.

The Mechanistic Links Between Proteasome Activity, Aging and Age-related Diseases.

Damaged and misfolded proteins accumulate during the aging process, impairing cell function and tissue homeostasis. These perturbations to protein homeostasis (proteostasis) are hallmarks of age-related neurodegenerative disorders such as Alzheimer's, Parkinson's or Huntington's disease. Damaged proteins are degraded by cellular clearance mechanisms such as the proteasome, a key component of the proteostasis network. Proteasome activity declines during aging, and proteasomal dysfunction is associated with late-onset disorders. Modulation of proteasome activity extends lifespan and protects organisms from symptoms associated with proteostasis disorders. Here we review the links between proteasome activity, aging and neurodegeneration. Additionally, strategies to modulate proteasome activity and delay the onset of diseases associated to proteasomal dysfunction are discussed herein.

ALS-FUS mutations cause abnormal PARylation and histone H1.2 interaction, leading to pathological changes.

The majority of severe early-onset and juvenile cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the FUS gene, resulting in rapid disease progression. Mutant FUS accumulates within stress granules (SGs), thereby affecting the dynamics of these ribonucleoprotein complexes. Here, we define the interactome of the severe mutant FUSP525L variant in human induced pluripotent stem cell (iPSC)-derived motor neurons. We find increased interaction of FUSP525L with the PARP1 enzyme, promoting poly-ADP-ribosylation (PARylation) and binding of FUS to histone H1.2. Inhibiting PARylation or reducing H1.2 levels alleviates mutant FUS aggregation, SG alterations, and apoptosis in human motor neurons. Conversely, elevated H1.2 levels exacerbate FUS-ALS phenotypes, driven by the internally disordered terminal domains of H1.2. In C. elegans models, knockdown of H1.2 and PARP1 orthologs also decreases FUSP525L aggregation and neurodegeneration, whereas H1.2 overexpression worsens ALS-related changes. Our findings indicate a link between PARylation, H1.2, and FUS with potential therapeutic implications.

The anti-leprosy drug clofazimine reduces polyQ toxicity through activation of PPARγ.

BACKGROUND: PolyQ diseases are autosomal dominant neurodegenerative disorders caused by the expansion of CAG repeats. While of slow progression, these diseases are ultimately fatal and lack effective therapies. METHODS: A high-throughput chemical screen was conducted to identify drugs that lower the toxicity of a protein containing the first exon of Huntington's disease (HD) protein huntingtin (HTT) harbouring 94 glutamines (Htt-Q94). Candidate drugs were tested in a wide range of in vitro and in vivo models of polyQ toxicity. FINDINGS: The chemical screen identified the anti-leprosy drug clofazimine as a hit, which was subsequently validated in several in vitro models. Computational analyses of transcriptional signatures revealed that the effect of clofazimine was due to the stimulation of mitochondrial biogenesis by peroxisome proliferator-activated receptor gamma (PPARγ). In agreement with this, clofazimine rescued mitochondrial dysfunction triggered by Htt-Q94 expression. Importantly, clofazimine also limited polyQ toxicity in developing zebrafish and neuron-specific worm models of polyQ disease. INTERPRETATION: Our results support the potential of repurposing the antimicrobial drug clofazimine for the treatment of polyQ diseases. FUNDING: A full list of funding sources can be found in the acknowledgments section.

The emerging role and therapeutic implications of bacterial and parasitic deubiquitinating enzymes.

Deubiquitinating enzymes (DUBs) are emerging as key factors for the infection of human cells by pathogens such as bacteria and parasites. In this review, we discuss the most recent studies on the role of deubiquitinase activity in exploiting and manipulating ubiquitin (Ub)-dependent host processes during infection. The studies discussed here highlight the importance of DUB host-pathogen research and underscore the therapeutic potential of inhibiting pathogen-specific DUB activity to prevent infectious diseases.

A tale of two pathways: Regulation of proteostasis by UPRmt and MDPs.

Mitochondrial fitness is critical to organismal health and its impairment is associated with aging and age-related diseases. As such, numerous quality control mechanisms exist to preserve mitochondrial stability, including the unfolded protein response of the mitochondria (UPRmt). The UPRmt is a conserved mechanism that drives the transcriptional activation of mitochondrial chaperones, proteases, autophagy (mitophagy), and metabolism to promote restoration of mitochondrial function under stress conditions. UPRmt has direct ramifications in aging, and its activation is often ascribed to improve health whereas its dysfunction tends to correlate with disease. This review pairs a description of the most recent findings within the field of UPRmt with a more poorly understood field: mitochondria-derived peptides (MDPs). Similar to UPRmt, MDPs are microproteins derived from the mitochondria that can impact organismal health and longevity. We then highlight a tantalizing interconnection between UPRmt and MDPs wherein both mechanisms may be efficiently coordinated to maintain organismal health.

Cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ-induced proteasomes.

Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective mechanisms to extend longevity in both poikilotherms and homeotherms, a better understanding of cold-induced changes can lead to converging modifiers of pathological protein aggregation. Here, we find that cold temperature (15 °C) selectively induces the trypsin-like activity of the proteasome in Caenorhabditis elegans through PSME-3, the worm orthologue of human PA28γ/PSME3. This proteasome activator is required for cold-induced longevity and ameliorates age-related deficits in protein degradation. Moreover, cold-induced PA28γ/PSME-3 diminishes protein aggregation in C. elegans models of age-related diseases such as Huntington's and amyotrophic lateral sclerosis. Notably, exposure of human cells to moderate cold temperature (36 °C) also activates trypsin-like activity through PA28γ/PSME3, reducing disease-related protein aggregation and neurodegeneration. Together, our findings reveal a beneficial role of cold temperature that crosses evolutionary boundaries with potential implications for multi-disease prevention.