RESUMO
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.
Assuntos
Proteínas Amiloidogênicas/genética , Diferenciação Celular/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Ácido Glutâmico/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Agregados Proteicos/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.
RESUMO
FOXO1, a transcription factor downstream of the insulin/insulin like growth factor axis, has been linked to protein degradation. Elevated expression of FOXO orthologs can also prevent the aggregation of cytosine adenine guanine (CAG)-repeat disease causing polyglutamine (polyQ) proteins but whether FOXO1 targets mutant proteins for degradation is unclear. Here, we show that increased expression of FOXO1 prevents toxic polyQ aggregation in human cells while reducing FOXO1 levels has the opposite effect and accelerates it. Although FOXO1 indeed stimulates autophagy, its effect on polyQ aggregation is independent of autophagy, ubiquitin-proteasome system (UPS) mediated protein degradation and is not due to a change in mutant polyQ protein turnover. Instead, FOXO1 specifically downregulates protein synthesis rates from expanded pathogenic CAG repeat transcripts. FOXO1 orchestrates a change in the composition of proteins that occupy mutant expanded CAG transcripts, including the recruitment of IGF2BP3. This mRNA binding protein enables a FOXO1 driven decrease in pathogenic expanded CAG transcript- and protein levels, thereby reducing the initiation of amyloidogenesis. Our data thus demonstrate that FOXO1 not only preserves protein homeostasis at multiple levels, but also reduces the accumulation of aberrant RNA species that may co-contribute to the toxicity in CAG-repeat diseases.
Assuntos
Proteína Forkhead Box O1/genética , Peptídeos/genética , Agregação Patológica de Proteínas/genética , Proteínas de Ligação a RNA/genética , Adenina/metabolismo , Proteínas Amiloidogênicas , Autofagia/genética , Citosina/metabolismo , Proteína Forkhead Box O1/biossíntese , Regulação da Expressão Gênica/genética , Guanina/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes/genética , Peptídeos/toxicidade , Agregação Patológica de Proteínas/patologia , Biossíntese de Proteínas/genética , Proteólise , RNA Mensageiro/genética , Repetições de Trinucleotídeos/genéticaRESUMO
Disturbances to protein homeostasis (proteostasis) can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. DNAJB proteins are molecular chaperones that have been identified as potent suppressors of disease-related protein aggregation. In this work, a destabilised isoform of firefly luciferase (R188Q/R261Q Fluc; termed FlucDM) was overexpressed in cells to assess the capacity of DNAJBs to inhibit inclusion formation. Co-expression of all DNAJB proteins tested significantly inhibited the intracellular aggregation of FlucDM. Moreover, we show that DNAJB proteins suppress aggregation by supporting the Hsp70 (HSPA)-dependent degradation of FlucDM via the proteasome. The serine-rich stretch in DNAJB6 and DNAJB8, essential for preventing fibrillar aggregation, is not involved in the suppression of FlucDM inclusion formation. Conversely, deletion of the C-terminal TTK-LKS motif in DNAJB6 and DNAJB8, a region not required to suppress polyglutamine aggregation, abolished the ability to inhibit inclusion formation by FlucDM. Thus, our data suggest that DNAJB6 and DNAJB8 possess two distinct regions for binding substrates, one that is responsible for binding ß-hairpins that form during amyloid formation and another that interacts with exposed hydrophobic patches in aggregation-prone clients. This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas de Choque Térmico HSP40 , Agregados Proteicos , Amiloide/metabolismo , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , ProteostaseRESUMO
Heat shock protein 70 (HSP70) chaperones play a central role in protein quality control and are crucial for many cellular processes, including protein folding, degradation, and disaggregation. Human HSP70s compose a family of 13 members that carry out their functions with the aid of even larger families of co-chaperones. A delicate interplay between HSP70s and co-chaperone recruitment is thought to determine substrate fate, yet it has been generally assumed that all Hsp70 paralogs have similar activities and are largely functionally redundant. However, here we found that when expressed in human cells, two highly homologous HSP70s, HSPA1A and HSPA1L, have opposing effects on cellular handling of various substrates. For example, HSPA1A reduced aggregation of the amyotrophic lateral sclerosis-associated protein variant superoxide dismutase 1 (SOD1)-A4V, whereas HSPA1L enhanced its aggregation. Intriguingly, variations in the substrate-binding domain of these HSP70s did not play a role in this difference. Instead, we observed that substrate fate is determined by differential interactions of the HSP70s with co-chaperones. Whereas most co-chaperones bound equally well to these two HSP70s, Hsp70/Hsp90-organizing protein (HOP) preferentially bound to HSPA1L, and the Hsp110 nucleotide-exchange factor HSPH2 preferred HSPA1A. The role of HSPH2 was especially crucial for the HSPA1A-mediated reduction in SOD1-A4V aggregation. These findings reveal a remarkable functional diversity at the level of the cellular HSP70s and indicate that this diversity is defined by their affinities for specific co-chaperones such as HSPH2.
Assuntos
Proteínas de Choque Térmico HSP110/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Homeodomínio/química , Agregação Patológica de Proteínas , Superóxido Dismutase-1/química , Proteínas Supressoras de Tumor/química , Substituição de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Proteínas de Choque Térmico HSP110/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Homeodomínio/genética , Humanos , Mutação de Sentido Incorreto , Superóxido Dismutase-1/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Heat shock protein family B (small) member 7 (HSPB7) is a unique, relatively unexplored member within the family of human small heat shock proteins (HSPBs). Unlike most HSPB family members, HSPB7 does not oligomerize and so far has not been shown to associate with any other member of the HSPB family. Intriguingly, it was found to be the most potent member within the HSPB family to prevent aggregation of proteins with expanded polyglutamine (polyQ) stretches. How HSPB7 suppresses polyQ aggregation has remained elusive so far. Here, using several experimental strategies, including in vitro aggregation assay, immunoblotting and fluorescence approaches, we show that the polyQ aggregation-inhibiting activity of HSPB7 is fully dependent on its flexible N-terminal domain (NTD). We observed that the NTD of HSPB7 is both required for association with and inhibition of polyQ aggregation. Remarkably, replacing the NTD of HSPB1, which itself cannot suppress polyQ aggregation, with the NTD of HSPB7 resulted in a hybrid protein that gained anti-polyQ aggregation activity. The hybrid NTDHSPB7-HSPB1 protein displayed a reduction in oligomer size and, unlike WT HSPB1, associated with polyQ. However, experiments with phospho-mimicking HSPB1 mutants revealed that de-oligomerization of HSPB1 alone does not suffice to gain polyQ aggregation-inhibiting activity. Together, our results reveal that the NTD of HSPB7 is both necessary and sufficient to bind to and suppress the aggregation of polyQ-containing proteins.
Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Peptídeos/química , Agregados Proteicos , Proteínas de Choque Térmico HSP27/química , Humanos , Peptídeos/metabolismo , Ligação Proteica , ProteóliseRESUMO
Impaired neuronal proteostasis is a salient feature of many neurodegenerative diseases, highlighting alterations in the function of the endoplasmic reticulum (ER). We previously reported that targeting the transcription factor XBP1, a key mediator of the ER stress response, delays disease progression and reduces protein aggregation in various models of neurodegeneration. To identify disease modifier genes that may explain the neuroprotective effects of XBP1 deficiency, we performed gene expression profiling of brain cortex and striatum of these animals and uncovered insulin-like growth factor 2 (Igf2) as the major upregulated gene. Here, we studied the impact of IGF2 signaling on protein aggregation in models of Huntington's disease (HD) as proof of concept. Cell culture studies revealed that IGF2 treatment decreases the load of intracellular aggregates of mutant huntingtin and a polyglutamine peptide. These results were validated using induced pluripotent stem cells (iPSC)-derived medium spiny neurons from HD patients and spinocerebellar ataxia cases. The reduction in the levels of mutant huntingtin was associated with a decrease in the half-life of the intracellular protein. The decrease in the levels of abnormal protein aggregation triggered by IGF2 was independent of the activity of autophagy and the proteasome pathways, the two main routes for mutant huntingtin clearance. Conversely, IGF2 signaling enhanced the secretion of soluble mutant huntingtin species through exosomes and microvesicles involving changes in actin dynamics. Administration of IGF2 into the brain of HD mice using gene therapy led to a significant decrease in the levels of mutant huntingtin in three different animal models. Moreover, analysis of human postmortem brain tissue and blood samples from HD patients showed a reduction in IGF2 level. This study identifies IGF2 as a relevant factor deregulated in HD, operating as a disease modifier that buffers the accumulation of abnormal protein species.
Assuntos
Doença de Huntington/metabolismo , Doença de Huntington/patologia , Fator de Crescimento Insulin-Like II/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Humanos , Fator de Crescimento Insulin-Like II/farmacologia , Camundongos , Camundongos Transgênicos , Agregados Proteicos/efeitos dos fármacosRESUMO
Several neurodegenerative diseases like Huntington's, a polyglutamine (PolyQ) disease, are initiated by protein aggregation in neurons. Furthermore, these diseases are also associated with a multitude of responses in non-neuronal cells in the brain, in particular glial cells, like astrocytes. These non-neuronal responses have repeatedly been suggested to play a disease-modulating role, but how these may be exploited to delay the progression of neurodegeneration has remained unclear. Interestingly, one of the molecular changes that astrocytes undergo includes the upregulation of certain Heat Shock Proteins (HSPs) that are classically considered to maintain protein homeostasis, thus resulting in cell autonomous protection. Previously, we discovered DNAJB6, a member of the human DNAJ family, as potent cell autonomous suppressor of PolyQ aggregation and related neurodegeneration. Using cell type specific expression systems in D. melanogaster, we show that exclusive expression of DNAJB6 in astrocytes (that do not express PolyQ protein) can delay neurodegeneration and expands lifespan when the PolyQ protein is exclusively expressed in neurons (that do not co-express DNAJB6 themselves). This provides direct evidence for a non-cell autonomous protective role of astrocytes in PolyQ diseases.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Doença de Huntington/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Proteínas de Choque Térmico HSP40/genética , Proteína Huntingtina/metabolismo , Masculino , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Peptídeos/metabolismoRESUMO
With the discovery in the late 1980s that the DNA-repair gene RAD6 encodes a ubiquitin-conjugating enzyme, it became clear that protein modification by ubiquitin conjugation has a much broader significance than had previously been assumed. Now, two decades later, ubiquitin and its cousin SUMO are implicated in a range of human diseases, including breast cancer and Fanconi anaemia, giving fresh momentum to studies focused on the relationships between ubiquitin, SUMO and DNA-repair pathways.
Assuntos
Reparo do DNA , Proteína SUMO-1/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , UbiquitinaçãoRESUMO
Progressive neurocognitive dysfunction is the leading cause of a reduced quality of life in patients with primary brain tumors. Understanding how the human brain responds to cancer and its treatment is essential to improve the associated cognitive sequelae. In this study, we performed integrated transcriptomic and tissue analysis on postmortem normal-appearing non-tumor brain tissue from glioblastoma (GBM) patients that had received cancer treatments, region-matched brain tissue from unaffected control individuals and Alzheimer's disease (AD) patients. We show that normal-appearing non-tumor brain regions of patients with GBM display hallmarks of accelerated aging, in particular mitochondrial dysfunction, inflammation, and proteostasis deregulation. The extent and spatial pattern of this response decreased with distance from the tumor. Gene set enrichment analyses and a direct comparative analysis with an independent cohort of brain tissue samples from AD patients revealed a significant overlap in differentially expressed genes and a similar biological aging trajectory. Additionally, these responses were validated at the protein level showing the presence of increased lysosomal lipofuscin, phosphorylated microtubule-associated protein Tau, and oxidative DNA damage in normal-appearing brain areas of GBM patients. Overall, our data show that the brain of GBM patients undergoes accelerated aging and shared AD-like features, providing the basis for novel or repurposed therapeutic targets for managing brain tumor-related side effects.
Assuntos
Doença de Alzheimer , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Qualidade de Vida , Encéfalo/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Doença de Alzheimer/patologiaRESUMO
Cooling at 4°C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, γ-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures.
Assuntos
Dano ao DNA , Histonas , Trifosfato de Adenosina/metabolismo , Animais , DNA/metabolismo , Histonas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , SuínosRESUMO
Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , InterfaseRESUMO
A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.
Cells are constantly perceiving and responding to changes in their surroundings, and challenging conditions such as extreme heat or toxic chemicals can put cells under stress. When this happens, protein production can be affected. Proteins are long chains of chemical building blocks called amino acids, and they can only perform their roles if they fold into the right shape. Some proteins fold easily and remain folded, but others can be unstable and often become misfolded. Unfolded proteins can become a problem because they stick to each other, forming large clumps called aggregates that can interfere with the normal activity of cells, causing damage. The causes of stress that have a direct effect on protein folding are called proteotoxic stresses, and include, for example, high temperatures, which make proteins more flexible and unstable, increasing their chances of becoming unfolded. To prevent proteins becoming misfolded, cells can make 'protein chaperones', a type of proteins that help other proteins fold correctly and stay folded. The production of protein chaperones often increases in response to proteotoxic stress. However, there are other types of stress too, such as genotoxic stress, which damages DNA. It is unclear what effect genotoxic stress has on protein folding. Huiting et al. studied protein folding during genotoxic stress in human cells grown in the lab. Stress was induced by either blocking the proteins that repair DNA or by 'trapping' the proteins that release DNA tension, both of which result in DNA damage. The analysis showed that, similar to the effects of proteotoxic stress, genotoxic stress increased the number of proteins that aggregate, although certain proteins formed aggregates even without stress, particularly if they were common and relatively unstable proteins. Huiting et al.'s results suggest that aggregation increases in cells under genotoxic stress because the cells fail to produce enough chaperones to effectively fold all the proteins that need it. Indeed, Huiting et al. showed that aggregates contain many proteins that rely on chaperones, and that increasing the number of chaperones in stressed cells reduced protein aggregation. This work shows that genotoxic stress can affect protein folding by limiting the availability of chaperones, which increases protein aggregation. Remarkably, there is a substantial overlap between proteins that aggregate in diseases that affect the brain such as Alzheimer's disease and proteins that aggregate after genotoxic stress. Therefore, further research could focus on determining whether genotoxic stress is involved in the progression of these neurological diseases.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA Topoisomerases/metabolismo , Chaperonas Moleculares/metabolismo , Dano ao DNA , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Peptídeos/metabolismo , Agregados Proteicos , Dobramento de Proteína , Proteoma/metabolismo , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Genome instability and loss of protein homeostasis are hallmark events of age-related diseases that include neurodegeneration. Several neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis are characterized by protein aggregation, while an impaired DNA damage response (DDR) as in many genetic DNA repair disorders leads to pronounced neuropathological features. It remains unclear to what degree these cellular events interconnect with each other in the development of neurological diseases. This review highlights how the loss of protein homeostasis and genome instability influence one other. We will discuss studies that illustrate this connection. DNA damage contributes to many neurodegenerative diseases, as shown by an increased level of DNA damage in patients, possibly due to the effects of protein aggregates on chromatin, the sequestration of DNA repair proteins and novel putative DNA repair functions. Conversely, genome stability is also important for protein homeostasis. For example, gene copy number variations and the loss of key DDR components can lead to marked proteotoxic stress. An improved understanding of how protein homeostasis and genome stability are mechanistically connected is needed and promises to lead to the development of novel therapeutic interventions.
Assuntos
Suscetibilidade a Doenças , Instabilidade Genômica , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Proteostase , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Animais , Dano ao DNA , Reparo do DNA , Expressão Gênica , Predisposição Genética para Doença , Humanos , Doenças Neurodegenerativas/patologia , Estresse Oxidativo , Fenótipo , Agregação Patológica de Proteínas , Proteostase/genéticaRESUMO
Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5-6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI.
Assuntos
Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Chaperonas Moleculares/genética , Proteostase/genética , Proteostase/fisiologiaRESUMO
Protein ubiquitination is critical for numerous cellular functions, including DNA damage response pathways. Histones are the most abundant monoubiquitin conjugates in mammalian cells; however, the regulation and the function of monoubiquitinated H2A (uH2A) and H2B (uH2B) remain poorly understood. In particular, little is known about mammalian deubiquitinating enzymes (DUBs) that catalyze the removal of ubiquitin from uH2A/uH2B. Here we identify the ubiquitin-specific protease 3 USP3 as a deubiquitinating enzyme for uH2A and uH2B. USP3 dynamically associates with chromatin and deubiquitinates H2A/H2B in vivo. The ZnF-UBP domain of USP3 mediates uH2A-USP3 interaction. Functional ablation of USP3 by RNAi leads to delay of S phase progression and to accumulation of DNA breaks, with ensuing activation of DNA damage checkpoint pathways. In addition, we show that in response to ionizing radiation, (1) uH2A redistributes and colocalizes in gamma-H2AX DNA repair foci and (2) USP3 is required for full deubiquitination of ubiquitin-conjugates/uH2A and gamma-H2AX dephosphorylation. Our studies identify USP3 as a novel regulator of H2A and H2B ubiquitination, highlight its role in preventing replication stress, and suggest its involvement in the response to DNA double-strand breaks. Together, our results implicate USP3 as a novel chromatin modifier in the maintenance of genome integrity.
Assuntos
Cromatina/metabolismo , Endopeptidases/fisiologia , Instabilidade Genômica/fisiologia , Fase S/fisiologia , Células HeLa , Histonas/metabolismo , Humanos , Proteases Específicas de Ubiquitina , Ubiquitinação/fisiologiaRESUMO
The pathophysiology of Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and many others converge at alpha-synuclein (α-Syn) aggregation. Although it is still not entirely clear what precise biophysical processes act as triggers, cumulative evidence points towards a crucial role for protein quality control (PQC) systems in modulating α-Syn aggregation and toxicity. These encompass distinct cellular strategies that tightly balance protein production, stability, and degradation, ultimately regulating α-Syn levels. Here, we review the main aspects of α-Syn biology, focusing on the cellular PQC components that are at the heart of recognizing and disposing toxic, aggregate-prone α-Syn assemblies: molecular chaperones and the ubiquitin-proteasome system and autophagy-lysosome pathway, respectively. A deeper understanding of these basic protein homeostasis mechanisms might contribute to the development of new therapeutic strategies envisioning the prevention and/or enhanced degradation of α-Syn aggregates.
Assuntos
Redes e Vias Metabólicas , Agregação Patológica de Proteínas/metabolismo , Sinucleinopatias/metabolismo , alfa-Sinucleína/metabolismo , Animais , HumanosRESUMO
Maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. Here, we analyzed Polycomb group protein (PcG) complex integrity in response to heat shock (HS). Upon HS, various Polycomb Repressive Complex (PRC)1 and PRC2 subunits, including CBX proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. In parallel, binding of PRC1/2 to target genes is strongly reduced, coinciding with a dramatic loss of H2AK119ub and H3K27me3 marks. Nucleolar-accumulated CBX proteins are immobile, but remarkably both CBX protein accumulation and loss of PRC1/2 epigenetic marks are reversible. This post-heat shock recovery of pan-nuclear CBX protein localization and reinstallation of epigenetic marks is HSP70 dependent. Our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock.
Assuntos
Nucléolo Celular/metabolismo , Epigênese Genética/efeitos da radiação , Resposta ao Choque Térmico , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Linhagem Celular , Proteínas de Choque Térmico HSP70/metabolismo , HumanosRESUMO
Like many other cellular processes, regulation of the DNA damage response (DDR) is regulated at different levels, ranging from transcriptional control to an array of distinct post-translational modifications. Involvement of ubiquitylation and the ubiquitin proteasome system in adjusting DDR are such protein modifications that were receiving increasing attention in the field. In this review we summarize and discuss a few recent key publications addressing the issue of DDR factor ubiquitylation, focusing on UV-induced DDR. We discuss the implications of these modifications to allow swift adaptation and regulation of genome surveillance factors.
Assuntos
Dano ao DNA/efeitos da radiação , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Raios Ultravioleta , Reparo do DNA/efeitos da radiação , Humanos , Complexo de Endopeptidases do Proteassoma , Processamento de Proteína Pós-TraducionalRESUMO
BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery.