Dithiothreitol-based protein equalisation in the context of multiple myeloma: Enhancing proteomic analysis and therapeutic insights.

In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine.

NatB Protects Procaspase-8 from UBR4-Mediated Degradation and Is Required for Full Induction of the Extrinsic Apoptosis Pathway.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB (Naa20-/-) to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, Naa20-/- MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Sleep maintains excitatory synapse diversity in the cortex and hippocampus.

Insufficient sleep is a global problem with serious consequences for cognition and mental health.1 Synapses play a central role in many aspects of cognition, including the crucial function of memory consolidation during sleep.2 Interference with the normal expression or function of synapse proteins is a cause of cognitive, mood, and other behavioral problems in over 130 brain disorders.3 Sleep deprivation (SD) has also been reported to alter synapse protein composition and synapse number, although with conflicting results.4,5,6,7 In our study, we conducted synaptome mapping of excitatory synapses in 125 regions of the mouse brain and found that sleep deprivation selectively reduces synapse diversity in the cortex and in the CA1 region of the hippocampus. Sleep deprivation targeted specific types and subtypes of excitatory synapses while maintaining total synapse density (synapse number/area). Synapse subtypes with longer protein lifetimes exhibited resilience to sleep deprivation, similar to observations in aging and genetic perturbations. Moreover, the altered synaptome architecture affected the responses to neural oscillations, suggesting that sleep plays a vital role in preserving cognitive function by maintaining the brain's synaptome architecture.

Time-resolved interactome profiling deconvolutes secretory protein quality control dynamics.

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes.

Alzheimer's disease risk gene CD2AP is a dose-sensitive determinant of synaptic structure and plasticity.

CD2-Associated protein (CD2AP) is a candidate susceptibility gene for Alzheimer's disease, but its role in the mammalian central nervous system remains largely unknown. We show that CD2AP protein is broadly expressed in the adult mouse brain, including within cortical and hippocampal neurons, where it is detected at pre-synaptic terminals. Deletion of Cd2ap altered dendritic branching and spine density, and impaired ubiquitin-proteasome system activity. Moreover, in mice harboring either one or two copies of a germline Cd2ap null allele, we noted increased paired-pulse facilitation at hippocampal Schaffer-collateral synapses, consistent with a haploinsufficient requirement for pre-synaptic release. Whereas conditional Cd2ap knockout in the brain revealed no gross behavioral deficits in either 3.5- or 12-month-old mice, Cd2ap heterozygous mice demonstrated subtle impairments in discrimination learning using a touchscreen task. Based on unbiased proteomics, partial or complete loss of Cd2ap triggered perturbation of proteins with roles in protein folding, lipid metabolism, proteostasis, and synaptic function. Overall, our results reveal conserved, dose-sensitive requirements for CD2AP in the maintenance of neuronal structure and function, including synaptic homeostasis and plasticity, and inform our understanding of possible cell-type specific mechanisms in Alzheimer's Disease.

Pharmacological inhibition of USP14 delays proteostasis-associated aging in a proteasome-dependent but foxo-independent manner.

Aging is often accompanied by a decline in proteostasis, manifested as an increased propensity for misfolded protein aggregates, which are prevented by protein quality control systems, such as the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy. Although the role of the UPS and autophagy in slowing age-induced proteostasis decline has been elucidated, limited information is available on how these pathways can be activated in a collaborative manner to delay proteostasis-associated aging. Here, we show that activation of the UPS via the pharmacological inhibition of USP14 (ubiquitin specific peptidase 14) using IU1 improves proteostasis and autophagy decline caused by aging or proteostatic stress in Drosophila and human cells. Treatment with IU1 not only alleviated the aggregation of polyubiquitinated proteins in aging Drosophila flight muscles but also extended the fly lifespan with enhanced locomotive activity via simultaneous activation of the UPS and autophagy. Interestingly, the effect of this drug disappeared when proteasomal activity was inhibited, but was evident upon proteostasis disruption by foxo mutation. Overall, our findings shed light on potential strategies to efficiently ameliorate age-associated pathologies associated with perturbed proteostasis.Abbreviations: AAAs: amino acid analogs; foxo: forkhead box, sub-group O; IFMs: indirect flight muscles; UPS: ubiquitin-proteasome system; USP14: ubiquitin specific peptidase 14.

ProSAAS is preferentially up-regulated during homeostatic scaling and reduces amyloid plaque burden in the 5xFAD mouse hippocampus.

The accumulation of ß-amyloid in Alzheimer's disease greatly impacts neuronal health and synaptic function. To maintain network stability in the face of altered synaptic activity, neurons engage a feedback mechanism termed homeostatic scaling; however, this process is thought to be disrupted during disease progression. Previous proteomics studies have shown that one of the most highly regulated proteins in cell culture models of homeostatic scaling is the small secretory chaperone proSAAS. Our prior work has shown that proSAAS exhibits anti-aggregant behavior against alpha-synuclein and ß-amyloid fibrillation in vitro and is up-regulated in cell models of proteostatic stress. However, the specific role that this protein might play in homeostatic scaling, and its anti-aggregant role in Alzheimer's progression, is not clear. To learn more about the role of proSAAS in maintaining hippocampal proteostasis, we compared its expression in a primary neuron model of homeostatic scaling to other synaptic components using western blotting and qPCR, revealing that proSAAS protein responses to homeostatic up- and down-regulation were significantly higher than those of two other synaptic vesicle components, 7B2 and carboxypeptidase E. However, proSAAS mRNA expression was static, suggesting translational control and/or altered protein degradation. ProSAAS was readily released upon depolarization of differentiated hippocampal cultures, supporting its synaptic localization. Immunohistochemical analysis demonstrated abundant proSAAS within the mossy fiber layer of the hippocampus in both wild-type and 5xFAD mice; in the latter, proSAAS was also concentrated around amyloid plaques. Importantly, overexpression of proSAAS in the CA1 region via stereotaxic injection of proSAAS-encoding AAV2/1 significantly decreased amyloid plaque burden in 5xFAD mice. We hypothesize that dynamic changes in proSAAS expression play a critical role in hippocampal proteostatic processes, both in the context of normal homeostatic plasticity and in the control of protein aggregation during Alzheimer's disease progression.

Protein misfolding releases human Heat Shock Factor 1 (HSF1) from HSP70 latency control.

Heat shock factor 1 (HSF1) responds to stress to mount the heat shock response (HSR), a conserved transcriptional program that allows cells to maintain proteostasis by upregulating heat shock proteins (HSPs). The homeostatic stress regulation of HSF1 plays a key role in human physiology and health but its mechanism has remained difficult to pinpoint. Recent work in the budding yeast model has implicated stress-inducible chaperones of the HSP70 family as direct negative regulators of HSF1 activity. Here, we have investigated the latency control and activation of human HSF1 by HSP70 and misfolded proteins. Purified oligomeric HSF1-HSP70 (HSPA1A) complexes exhibited basal DNA binding activity that was inhibited by increasing the levels of HSP70 and, importantly, misfolded proteins reverted the inhibitory effect. Using site-specific UV photo-crosslinking, we monitored HSP70-HSF1 complexes in HEK293T cells. While HSF1 was bound by the substrate binding domain of HSP70 in unstressed cells, activation of HSF1 by heat shock as well as by inducing the misfolding of newly synthesized proteins resulted in release of HSF1 from the chaperone. Taken our results together, we conclude that latent HSF1 populate dynamic complexes with HSP70, which are sensitive to increased levels of misfolded proteins that compete for binding to the HSP70 substrate binding domain. Thus, human HSF1 is activated by various stress conditions that all titrate available HSP70.

The Thioredoxin Fold Protein (TFP2) from Extreme Acidophilic Leptospirillum sp. CF-1 Is a Chaperedoxin-like Protein That Prevents the Aggregation of Proteins under Oxidative Stress.

Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

Protein folding, cellular stress and cancer.

Proteins are acknowledged as the phenotypical manifestation of the genotype, because protein-coding genes carry the information for the strings of amino acids that constitute the proteins. It is widely accepted that protein function depends on the corresponding "native" structure or folding achieved within the cell, and that native protein folding corresponds to the lowest free energy minimum for a given protein. However, protein folding within the cell is a non-deterministic dissipative process that from the same input may produce different outcomes, thus conformational heterogeneity of folded proteins is the rule and not the exception. Local changes in the intracellular environment promote variation in protein folding. Hence protein folding requires "supervision" by a host of chaperones and co-chaperones that help their client proteins to achieve the folding that is most stable according to the local environment. Such environmental influence on protein folding is continuously transduced with the help of the cellular stress responses (CSRs) and this may lead to changes in the rules of engagement between proteins, so that the corresponding protein interactome could be modified by the environment leading to an alternative cellular phenotype. This allows for a phenotypic plasticity useful for adapting to sudden and/or transient environmental changes at the cellular level. Starting from this perspective, hereunder we develop the argument that the presence of sustained cellular stress coupled to efficient CSRs may lead to the selection of an aberrant phenotype as the resulting adaptation of the cellular proteome (and the corresponding interactome) to such stressful conditions, and this can be a common epigenetic pathway to cancer.

The germline coordinates mitokine signaling.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

The antagonistic role of an E3 ligase pair in regulating plant NLR-mediated autoimmunity and fungal pathogen resistance.

Plant immune homeostasis is achieved through a balanced immune activation and suppression, enabling effective defense while averting autoimmunity. In Arabidopsis, disrupting a mitogen-activated protein (MAP) kinase cascade triggers nucleotide-binding leucine-rich-repeat (NLR) SUPPRESSOR OF mkk1/2 2 (SUMM2)-mediated autoimmunity. Through an RNAi screen, we identify PUB5, a putative plant U-box E3 ligase, as a critical regulator of SUMM2-mediated autoimmunity. In contrast to typical E3 ligases, PUB5 stabilizes CRCK3, a calmodulin-binding receptor-like cytoplasmic kinase involved in SUMM2 activation. A closely related E3 ligase, PUB44, functions oppositely with PUB5 to degrade CRCK3 through monoubiquitylation and internalization. Furthermore, CRCK3, highly expressed in roots and conserved across plant species, confers resistance to Fusarium oxysporum, a devastating soil-borne fungal pathogen, in both Arabidopsis and cotton. These findings demonstrate the antagonistic role of an E3 ligase pair in fine-tuning kinase proteostasis for the regulation of NLR-mediated autoimmunity and highlight the function of autoimmune activators in governing plant root immunity against fungal pathogens.

Dysregulated ribosome quality control in human diseases.

Precise regulation of mRNA translation is of fundamental importance for maintaining homeostasis. Conversely, dysregulated general or transcript-specific translation, as well as abnormal translation events, have been linked to a multitude of diseases. However, driven by the misconception that the transient nature of mRNAs renders their abnormalities inconsequential, the importance of mechanisms that monitor the quality and fidelity of the translation process has been largely overlooked. In recent years, there has been a dramatic shift in this paradigm, evidenced by several seminal discoveries on the role of a key mechanism in monitoring the quality of mRNA translation - namely, Ribosome Quality Control (RQC) - in the maintenance of homeostasis and the prevention of diseases. Here, we will review recent advances in the field and emphasize the biological significance of the RQC mechanism, particularly its implications in human diseases.

Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.

Imaging of Existing and Newly Translated Proteins Elucidates Mechanisms of Sarcomere Turnover.

BACKGROUND: How the sarcomeric complex is continuously turned over in long-living cardiomyocytes is unclear. According to the prevailing model of sarcomere maintenance, sarcomeres are maintained by cytoplasmic soluble protein pools with free recycling between pools and sarcomeres. METHODS: We imaged and quantified the turnover of expressed and endogenous sarcomeric proteins, including the giant protein titin, in cardiomyocytes in culture and in vivo, at the single cell and at the single sarcomere level using pulse-chase labeling of Halo-tagged proteins with covalent ligands. RESULTS: We disprove the prevailing protein pool model and instead show an ordered mechanism in which only newly translated proteins enter the sarcomeric complex while older ones are removed and degraded. We also show that degradation is independent of protein age and that proteolytic extraction is a rate-limiting step in the turnover. We show that replacement of sarcomeric proteins occurs at a similar rate within cells and across the heart and is slower in adult cells. CONCLUSIONS: Our findings establish a unidirectional replacement model for cardiac sarcomeres subunit replacement and identify their turnover principles.

Quantitative proteome analysis of LAP1-deficient human fibroblasts: A pilot approach for predicting the signaling pathways deregulated in LAP1-associated diseases.

Lamina-associated polypeptide 1 (LAP1), a ubiquitously expressed nuclear envelope protein, appears to be essential for the maintenance of cell homeostasis. Although rare, mutations in the human LAP1-encoding TOR1AIP1 gene cause severe diseases and can culminate in the premature death of affected individuals. Despite there is increasing evidence of the pathogenicity of TOR1AIP1 mutations, the current knowledge on LAP1's physiological roles in humans is limited; hence, investigation is required to elucidate the critical functions of this protein, which can be achieved by uncovering the molecular consequences of LAP1 depletion, a topic that remains largely unexplored. In this work, the proteome of patient-derived LAP1-deficient fibroblasts carrying a pathological TOR1AIP1 mutation (LAP1 E482A) was quantitatively analyzed to identify global changes in protein abundance levels relatively to control fibroblasts. An in silico functional enrichment analysis of the mass spectrometry-identified differentially expressed proteins was also performed, along with additional in vitro functional assays, to unveil the biological processes that are potentially dysfunctional in LAP1 E482A fibroblasts. Collectively, our findings suggest that LAP1 deficiency may induce significant alterations in various cellular activities, including DNA repair, messenger RNA degradation/translation, proteostasis and glutathione metabolism/antioxidant response. This study sheds light on possible new functions of human LAP1 and could set the basis for subsequent in-depth mechanistic investigations. Moreover, by identifying deregulated signaling pathways in LAP1-deficient cells, our work may offer valuable molecular targets for future disease-modifying therapies for TOR1AIP1-associated nuclear envelopathies.

Spatial selectivity of ATase inhibition in mouse models of Charcot-Marie-Tooth disease.

The endoplasmic reticulum acetylation machinery has emerged as a new branch of the larger endoplasmic reticulum quality control system. It regulates the selection of correctly folded polypeptides as well as reticulophagy-mediated removal of toxic protein aggregates with the former being a particularly important aspect of the proteostatic functions of endoplasmic reticulum acetylation. Essential to this function is the Nε-lysine acetyltransferase activity of acetyltransferase 1 and acetyltransferase 2, which regulates the induction of endoplasmic reticulum-specific autophagy through the acetylation of the autophagy-related protein 9A. Here, we used three mouse models of Charcot-Marie-Tooth disease, peripheral myelin protein 22/Tr-J, C3-peripheral myelin protein 22 and myelin protein zero/ttrr, to study spatial and translational selectivity of endoplasmic reticulum acetyltransferase inhibitors. The results show that inhibition of the endoplasmic reticulum acetyltransferases selectively targets misfolding/pro-aggregating events occurring in the lumen of the organelle. Therefore, they establish acetyltransferase 1 and acetyltransferase 2 as the first proven targets for disease-causing proteotoxic states that initiate within the lumen of the endoplasmic reticulum/secretory pathway.

Pathophysiological processes underlying hidden hearing loss revealed in Kcnt1/2 double knockout mice.

Presbycusis is a prevalent condition in older adults characterized by the progressive loss of hearing due to age-related changes in the cochlea, the auditory portion of the inner ear. Many adults also struggle with understanding speech in noise despite having normal auditory thresholds, a condition termed "hidden" hearing loss because it evades standard audiological assessments. Examination of animal models and postmortem human tissue suggests that hidden hearing loss is also associated with age-related changes in the cochlea and may, therefore, precede overt age-related hearing loss. Nevertheless, the pathological mechanisms underlying hidden hearing loss are not understood, which hinders the development of diagnostic biomarkers and effective treatments for age-related hearing loss. To fill these gaps in knowledge, we leveraged a combination of tools, including transcriptomic profiling and morphological and functional assessments, to identify these processes and examine the transition from hidden to overt hearing loss. As a novel approach, we took advantage of a recently characterized model of hidden hearing loss: Kcnt1/2 double knockout mice. Using this model, we find that even before observable morphological pathology, hidden hearing loss is associated with significant alteration in several processes, notably proteostasis, in the cochlear sensorineural structures, and increased susceptibility to overt hearing loss in response to noise exposure and aging. Our findings provide the first insight into the pathophysiology associated with the earliest and, therefore, most treatable stages of hearing loss and provide critical insight directing future investigation of pharmaceutical strategies to slow and possibly prevent overt age-related hearing loss.

Cellular turnover and degradation of the most common missense cystathionine beta-synthase variants causing homocystinuria.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU.

O-Sialoglycoprotein Endopeptidase Deficiency Impairs Proteostasis and Induces Autophagy in Human Embryonic Stem Cells.

The OSGEP gene encodes O-sialoglycoprotein endopeptidase, a catalytic unit of the highly conserved KEOPS complex (Kinase, Endopeptidase, and Other Proteins of small Size) that regulates the second biosynthetic step in the formation of N-6-threonylcarbamoyladenosine (t6A). Mutations in KEOPS cause Galloway-Mowat syndrome (GAMOS), whose cellular function in mammals and underlying molecular mechanisms are not well understood. In this study, we utilized lentivirus-mediated OSGEP knockdown to generate OSGEP-deficient human embryonic stem cells (hESCs). OSGEP-knockdown hESCs exhibited reduced stemness factor expression and G2/M phase arrest, indicating a potential role of OSGEP in the regulation of hESC fate. Additionally, OSGEP silencing led to enhanced protein synthesis and increased aggregation of proteins, which further induced inappropriate autophagy, as evidenced by the altered expression of P62 and the conversion of LC3-I to LC3-II. The above findings shed light on the potential involvement of OSGEP in regulating pluripotency and differentiation in hESCs while simultaneously highlighting its crucial role in maintaining proteostasis and autophagy, which may have implications for human disease.