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The Journal of Biological Chemistry May 2024The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein...
The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein quality control network. Among inclusion bodies, aggresomes are particularly intriguing due to their association with cellular survival, drug resistance, and cancer-aggressive behavior. Aggresomes are molecular condensates formed when collapsed vimentin cages encircle misfolded proteins before final removal by autophagy. Yet significant gaps persist in the mechanisms governing aggresome formation and elimination in cancer cells. Understanding these mechanisms is crucial, especially considering the involvement of LC3A, a member of the MAP1LC3 family, which plays a unique role in autophagy regulation and has been reported to be epigenetically silenced in many cancers. Herein, we utilized tetracycline-inducible expression of LC3A to investigate its role in choroid plexus carcinoma cells, which inherently exhibit the presence of aggresomes. Live cell imaging was employed to demonstrate the effect of LC3A expression on aggresome-positive cells, while SILAC-based proteomics identified LC3A-induced protein and pathway alterations. Our findings demonstrate that extended expression of LC3A is associated with cellular senescence. However, the obstruction of lysosomal degradation in this context has a deleterious effect on cellular viability. In response to LC3A-induced autophagy, we observed significant alterations in mitochondrial morphology, reflected by mitochondrial dysfunction and increased ROS production. Furthermore, LC3A expression elicited the activation of the PERK-eIF2α-ATF4 axis of the UPR, underscoring a significant change in protein quality control network. In conclusion, our results elucidate that LC3A-mediated autophagy alters the protein quality control network, exposing a vulnerability in aggresome-positive cancer cells.
PubMed: 38777145
DOI: 10.1016/j.jbc.2024.107398 -
The Journal of Cell Biology Jun 2024Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription...
Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.
Topics: Animals; Humans; Mice; Autophagy; Lysosomes; NF-E2-Related Factor 1; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteostasis; Proteotoxic Stress; Stress, Physiological
PubMed: 38656405
DOI: 10.1083/jcb.202306150 -
Scientific Reports Feb 2024Neuroblastoma (NB) is the most common pediatric tumor and is currently treated by several types of therapies including chemotherapies, such as bortezomib treatment....
Neuroblastoma (NB) is the most common pediatric tumor and is currently treated by several types of therapies including chemotherapies, such as bortezomib treatment. However, resistance to bortezomib is frequently observed by mechanisms that remain to be deciphered. Bortezomib treatment leads to caspase activation and aggresome formation. Using models of patients-derived NB cell lines with different levels of sensitivity to bortezomib, we show that the activated form of caspase 3 accumulates within aggresomes of NB resistant cells leading to an impairment of bortezomib-induced apoptosis and increased cell survival. Our findings unveil a new mechanism of resistance to chemotherapy based on an altered subcellular distribution of the executioner caspase 3. This mechanism could explain the resistance developed in NB patients treated with bortezomib, emphasizing the potential of drugs targeting aggresomes.
Topics: Child; Humans; Bortezomib; Caspase 3; Cell Line, Tumor; Apoptosis; Neuroblastoma; Antineoplastic Agents
PubMed: 38355966
DOI: 10.1038/s41598-024-54140-7 -
Nature Communications Feb 2024Aggresomes are the product of misfolded protein aggregation, and the presence of aggresomes has been correlated with poor prognosis in cancer patients. However, the...
Aggresomes are the product of misfolded protein aggregation, and the presence of aggresomes has been correlated with poor prognosis in cancer patients. However, the exact role of aggresomes in tumorigenesis and cancer progression remains largely unknown. Herein, the multiomics screening reveal that OTUD1 protein plays an important role in retaining ovarian cancer stem cell (OCSC) properties. Mechanistically, the elevated OTUD1 protein levels lead to the formation of OTUD1-based cytoplasmic aggresomes, which is mediated by a short peptide located in the intrinsically disordered OTUD1 N-terminal region. Furthermore, OTUD1-based aggresomes recruit ASK1 via protein-protein interactions, which in turn stabilize ASK1 in a deubiquitinase-independent manner and activate the downstream JNK signaling pathway for OCSC maintenance. Notably, the disruption of OTUD1-based aggresomes or treatment with ASK1/JNK inhibitors, including ibrutinib, an FDA-approved drug that was recently identified as an MKK7 inhibitor, effectively reduced OCSC stemness (OSCS) of OTUD1 ovarian cancer cells. In summary, our work suggests that aggresome formation in tumor cells could function as a signaling hub and that aggresome-based therapy has translational potential for patients with OTUD1 ovarian cancer.
Topics: Humans; Female; MAP Kinase Signaling System; Proteins; Ovarian Neoplasms; Peptides; Protein Processing, Post-Translational; Ubiquitin-Specific Proteases
PubMed: 38351029
DOI: 10.1038/s41467-024-45698-x -
Cell Death Discovery Feb 2024Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) triggers a noncanonical form of programmed cell death (PCD) called parthanatos, yet the mechanisms of its...
Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) triggers a noncanonical form of programmed cell death (PCD) called parthanatos, yet the mechanisms of its induction are not fully understood. We have recently demonstrated that the aggresome-like induced structures (ALIS) composed of the autophagy receptor SQSTM1/p62 and K48-linked polyubiquitinated proteins (p62-based ALIS) mediate parthanatos. In this study, we identified the D1 dopamine receptor agonist YM435 as a unique parthanatos inhibitor that acts as the disaggregating agent for the p62-based ALIS. We found that YM435 structurally reduces aggregability of the ALIS, and then increases its hydrophilicity and liquidity, which prevents parthanatos. Moreover, dopamine and L-DOPA, a dopamine precursor, also prevented parthanatos by reducing the aggregability of the ALIS. Together, these observations suggest that aggregability of the p62-based ALIS determines the sensitivity to parthanatos, and the pharmacological properties of YM435 that reduces the aggregability may be suitable for therapeutic drugs for parthanatos-related diseases such as neurodegenerative diseases.
PubMed: 38346947
DOI: 10.1038/s41420-024-01838-2 -
PLoS Genetics Feb 2024The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly...
The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.
Topics: Humans; Intermediate Filaments; Clustered Regularly Interspaced Short Palindromic Repeats; Cytoskeleton; Inclusion Bodies
PubMed: 38315730
DOI: 10.1371/journal.pgen.1011138 -
The Journal of Biological Chemistry Feb 2024The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend...
The inflammasome is a large multiprotein complex that assembles in the cell cytoplasm in response to stress or pathogenic infection. Its primary function is to defend the cell and promote the secretion of pro-inflammatory cytokines, including IL-1β and IL-18. Previous research has shown that in immortalized bone marrow-derived macrophages (iBMDMs) inflammasome assembly is dependent on the deacetylase HDAC6 and the aggresome processing pathway (APP), a cellular pathway involved in the disposal of misfolded proteins. Here we used primary BMDMs from mice in which HDAC6 is ablated or impaired and found that inflammasome activation was largely normal. We also used human peripheral blood mononuclear cells and monocyte cell lines expressing a synthetic protein blocking the HDAC6-ubiquitin interaction and impairing the APP and found that inflammasome activation was moderately affected. Finally, we used a novel HDAC6 degrader and showed that inflammasome activation was partially impaired in human macrophage cell lines with depleted HDAC6. Our results therefore show that HDAC6 importance in inflammasome activation is context-dependent.
Topics: Animals; Humans; Mice; Cell Line; Histone Deacetylase 6; Inflammasomes; Interleukin-1beta; Leukocytes, Mononuclear; Macrophages; NLR Family, Pyrin Domain-Containing 3 Protein; Protein Transport
PubMed: 38199570
DOI: 10.1016/j.jbc.2024.105638 -
Molecular Biology of the Cell Mar 2024Immune cells employ diverse mechanisms for host defense. Macrophages, in response to TLR activation, assemble aggresome-like induced structures (ALIS). Our group has...
Immune cells employ diverse mechanisms for host defense. Macrophages, in response to TLR activation, assemble aggresome-like induced structures (ALIS). Our group has shown TLR4-signaling transcriptionally upregulates p62/sequestome1, which assembles ALIS. We have demonstrated that TLR4-mediated autophagy is, in fact, selective-autophagy of ALIS. We hypothesize that TLR-mediated autophagy and ALIS contribute to host-defense. Here we show that ALIS are assembled in macrophages upon exposure to different bacteria. These structures are associated with pathogen-containing phagosomes. Importantly, we present evidence of increased bacterial burden, where ALIS assembly is prevented with p62-specific siRNA. We have employed 3D-super-resolution structured illumination microscopy (3D-SR-SIM) and mass-spectrometric (MS) analyses to gain insight into the assembly of ALIS. Ultra-structural analyses of known constituents of ALIS (p62, ubiquitin, LC3) reveal that ALIS are organized structures with distinct patterns of alignment. Furthermore, MS-analyses of ALIS identified, among others, several proteins of known antimicrobial properties. We have validated MS data by testing the association of some of these molecules (Bst2, IFITM2, IFITM3) with ALIS and the phagocytosed-bacteria. We surmise that AMPs enrichment in ALIS leads to their delivery to bacteria-containing phagosomes and restricts the bacteria. Our findings in this paper support hitherto unknown functions of ALIS in host-defense.
Topics: Toll-Like Receptor 4; Antimicrobial Peptides; Macrophages; Ubiquitin; Autophagy
PubMed: 38170582
DOI: 10.1091/mbc.E23-09-0347 -
Antimicrobial Agents and Chemotherapy Feb 2024Entering a dormant state is a prevailing mechanism used by bacterial cells to transiently evade antibiotic attacks and become persisters. The dynamic progression of...
Entering a dormant state is a prevailing mechanism used by bacterial cells to transiently evade antibiotic attacks and become persisters. The dynamic progression of bacterial dormancy depths driven by protein aggregation has been found to be critical for antibiotic persistence in recent years. However, our current understanding of the endogenous genes that affects dormancy depth remains limited. Here, we discovered a novel role of phage shock protein A () gene in modulating bacterial dormancy depth. Deletion of of resulted in increased bacterial dormancy depths and prolonged lag times for resuscitation during the stationary phase. exhibited a higher persister ratio compared to the wild type when challenged with various antibiotics. Microscopic images revealed that showed accelerated formation of protein aggresomes, which were collections of endogenous protein aggregates. Time-lapse imaging established the positive correlation between protein aggregation and antibiotic persistence of at the single-cell level. To investigate the molecular mechanism underlying accelerated protein aggregation, we performed transcriptome profiling and found the increased abundance of chaperons and a general metabolic slowdown in the absence of . Consistent with the transcriptomic results, the strain showed a decreased cellular ATP level, which could be rescued by glucose supplementation. Then, we verified that replenishment of cellular ATP levels by adding glucose could inhibit protein aggregation and reduce persister formation in . This study highlights the novel role of in maintaining proteostasis, regulating dormancy depth, and affecting antibiotic persistence during stationary phase.
Topics: Anti-Bacterial Agents; Protein Aggregates; Escherichia coli; Adenosine Triphosphate; Glucose
PubMed: 38169282
DOI: 10.1128/aac.00937-23 -
Biophysical Journal Nov 2023Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2, and UBQLN4 differ in both their specific protein quality control functions and their propensities to...
Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2, and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates, and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2, and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2, and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs and the need to apply caution when using epitope tags to prevent experimental artifacts.
PubMed: 38041404
DOI: 10.1016/j.bpj.2023.11.3401