TEX13B is essential for metabolic reprogramming during germ cell differentiation.

STUDY QUESTION: What is the functional significance of Tex13b in male germ cell development and differentiation? SUMMARY ANSWER: Tex13b regulates male germ cell differentiation by metabolic reprogramming during spermatogenesis. WHAT IS KNOWN ALREADY: Studies in mice and humans suggest that TEX13B is a transcription factor and is exclusively expressed in germ cells. STUDY DESIGN, SIZE, DURATION: We sequenced the coding regions of TEX13B in 628 infertile men and 427 ethnically matched fertile control men. Further, to identify the molecular function of Tex13b, we created a Tex13b knockout and conditional overexpression system in GC-1spg (hereafter, GC-1) cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Our recent exome sequencing study identified novel candidate genes for male infertility. TEX13B was found to be one of the potential candidates, hence we explored the role of TEX13B in male infertility within a large infertile case-control cohort. We performed functional analyses of Tex13b in a GC-1 cell line using CRISPR-Cas9. We differentially labelled the cell proteins by stable isotope labelling of amino acids in cell culture (SILAC) and performed mass spectrometry-based whole-cell proteomics to identify the differential protein regulation in knockout cells compared to wild-type cells. We found that Tex13b knockout leads to downregulation of the OXPHOS complexes and upregulation of glycolysis genes, which was further validated by western blotting. These results were further confirmed by respirometry analysis in Tex13b knockout cells. Further, we also performed a conditional overexpression of TEX13B in GC-1 cells and studied the expression of OXPHOS complex proteins by western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a rare variant, rs775429506 (p.Gly237Glu), exclusively in two non-obstructive-azoospermia (NOA) men, that may genetically predispose these men for infertility. Further, we demonstrated that Tex13b functions in the transcription regulation of OXPHOS complexes. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: We examined the function of Tex13b in GC-1 in vitro by knocking out and conditional overexpression, for understanding the function of Tex13b in germ cells. Unfortunately, this could not be replicated in either an animal model or in patient-derived tissue due to the non-availability of an animal model or patient's testis biopsies. WIDER IMPLICATIONS OF THE FINDINGS: This study identified that Tex13b plays an important role in male germ cell development and differentiation. The findings of this study would be useful for screening infertile males with spermatogenic failure and counselling them before the implementation of assisted reproduction technique(s). STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by the Council of Scientific and Industrial Research (CSIR) under the network project (BSC0101 and MLP0113) and SERB, the Department of Science and Technology, Government of India (J C Bose Fellowship: JCB/2019/000027). The authors do not have any competing interest.

Widespread nuclear lamina injuries defeat proteostatic purposes of α-synuclein amyloid inclusions.

Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids sequester into insoluble protein deposits. Lewy bodies (LBs) are filamentous amyloid inclusions of α-synuclein, but PQC benefits and drawbacks associated with LB-like IBs remain underexplored. Here, we report that crosstalk between filamentous LB-like IBs and aggresome-like IBs of α-synuclein (Syn-aggresomes) buffer the load, aggregation state, and turnover of the amyloidogenic protein in mouse primary neurons and HEK293T cells. Filamentous LB-like IBs possess unorthodox PQC capacities of self-quarantining α-synuclein amyloids and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of filamentous LB-like IBs by facilitating spontaneous degradation of α-synuclein and conditional turnover of disintegrated α-synuclein amyloids. Thus, both types of IB primarily contribute to PQC. Incidentally, the overgrown perinuclear LB-like IBs become degenerative once these are misidentified by BICD2, a cargo-adapter for the cytosolic motor-protein dynein. Microscopy indicates that microtubules surrounding the perinuclear filamentous inclusions are also distorted, misbalancing the cytoskeleton-nucleoskeleton tension leading to widespread lamina injuries. Together, nucleocytoplasmic mixing, DNA damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of the filamentous amyloids of α-synuclein.

Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria.

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

Increased supraorganization of respiratory complexes is a dynamic multistep remodelling in response to proteostasis stress.

Proteasome-mediated degradation of misfolded proteins prevents aggregation inside and outside mitochondria. But how do cells safeguard the mitochondrial proteome and mitochondrial functions despite increased aggregation during proteasome inactivation? Here, using a novel two-dimensional complexome profiling strategy, we report increased supraorganization of respiratory complexes (RCs) in proteasome-inhibited cells that occurs simultaneously with increased pelletable aggregation of RC subunits inside mitochondria. Complex II (CII) and complex V (CV) subunits are increasingly incorporated into oligomers. Complex I (CI), complex III (CIII) and complex IV (CIV) subunits are engaged in supercomplex formation. We unravel unique quinary states of supercomplexes during early proteostatic stress that exhibit plasticity and inequivalence of constituent RCs. The core stoichiometry of CI and CIII is preserved, whereas the composition of CIV varies. These partially disintegrated supercomplexes remain functionally competent via conformational optimization. Subsequently, increased stepwise integration of RC subunits into holocomplexes and supercomplexes re-establishes steady-state stoichiometry. Overall, the mechanism of increased supraorganization of RCs mimics the cooperative unfolding and folding pathways for protein folding, but is restricted to RCs and is not observed for any other mitochondrial protein complexes.This article has an associated First Person interview with the first author of the paper.

Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation.

Muscle differentiation is controlled by adhesion and growth factor-dependent signalling through common effectors that regulate muscle-specific transcriptional programs. Here we report that mDiaphanous1, an effector of adhesion-dependent RhoA-signalling, negatively regulates myogenesis at the level of Myogenin expression. In myotubes, over-expression of mDia1ΔN3, a RhoA-independent mutant, suppresses Myogenin promoter activity and expression. We investigated mDia1-interacting proteins that may counteract mDia1 to permit Myogenin expression and timely differentiation. Using yeast two-hybrid and mass-spectrometric analysis, we report that mDia1 has a stage-specific interactome, including Prohibitin2, MyoD, Akt2, and ß-Catenin, along with a number of proteosomal and mitochondrial components. Of these interacting partners, Prohibitin2 colocalises with mDia1 in cytoplasmic punctae in myotubes. We mapped the interacting domains of mDia1 and Phb2, and used interacting (mDia1ΔN3/Phb2 FL or mDia1ΔN3/Phb2-Carboxy) and non-interacting pairs (mDia1H + P/Phb2 FL or mDia1ΔN3/Phb2-Amino) to dissect the functional consequences of this partnership on Myogenin promoter activity. Co-expression of full-length as well as mDia1-interacting domains of Prohibitin2 reverse the anti-myogenic effects of mDia1ΔN3, while non-interacting regions do not. Our results suggest that Prohibitin2 sequesters mDia1, dampens its anti-myogenic activity and fine-tunes RhoA-mDia1 signalling to promote differentiation. Overall, we report that mDia1 is multi-functional signalling effector whose anti-myogenic activity is modulated by a differentiation-dependent interactome. The data have been deposited to the ProteomeXchange with identifier PXD012257.

Aggregation of Respiratory Complex Subunits Marks the Onset of Proteotoxicity in Proteasome Inhibited Cells.

Proteostasis is maintained by optimal expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers protein aggregation and is implicated in many age-related pathologies. In this study, using quantitative proteomics and microscopy, we show that aggregation of many nuclear-encoded mitochondrial proteins is an early protein destabilization event during short-term proteasome inhibition. Among these, respiratory chain complex (RCC) subunits represent a group of functionally related proteins consistently forming aggregates under multiple proteostasis stresses with varying aggregation propensities. Sequence analysis reveals that several RCC subunits, irrespective of the cleavable mitochondrial targeting sequence, contain low-complexity regions at the N-terminus. Using different chimeric and mutant constructs, we show that these low-complexity regions partially contribute to the intrinsic instability of multiple RCC subunits. Taken together, we propose that physicochemically driven aggregation of unassembled RCC subunits destabilizes their functional assembly inside mitochondria. This eventually deregulates the biogenesis of respiratory complexes and marks the onset of mitochondrial dysfunction.

Identification of a splice variant of optineurin which is defective in autophagy and phosphorylation.

Optineurin (Optn) is an autophagy receptor that performs various functions in cargo-selective and non-selective autophagy. Here, we have identified and characterized a splice variant of mouse optineurin mRNA, which produces a truncated protein lacking N-terminal 157 amino acids (d157mOptn). This mRNA and protein are expressed in several tissues and cells. d157mOptn has an intact LC3-interacting region and a serine (S187) in it. However, unlike normal optineurin, the d157mOptn was not phosphorylated at this site when expressed in mammalian cells, and showed reduced interaction with TBK1 (tank binding kinase) that mediates phosphorylation at S187 (S177 in human OPTN). This phosphorylation of Optn required intact N-terminal sequence as well as functional C-terminal ubiquitin-binding domain. Unlike normal optineurin, d157mOptn was unable to promote autophagosome and autolysosome formation upon expression in Optn-deficient cells. d157mOptn was recruited to mutant huntingtin aggregates, but unlike wild type optineurin, it was unable to clear these aggregates by autophagy in neuronal NSC-34 cells. Phospho-TBK1 was seen around mutant Huntingtin aggregates in Optn overexpressing cells but it was reduced in cells overexpressing d157mOptn. Thus, we have identified an isoform of mouse optineurin which is defective in cargo-selective and non-selective autophagy possibly due to loss of phosphorylation and impaired interaction with TBK1. This isoform, which inhibits autophagosome formation in neuronal cells, might be involved in selectively modulating some of the functions of Optn, such as autophagy. Our results provide an insight into the role of N-terminal domain of Optn in various autophagic functions.

Conserved C-terminal nascent peptide binding domain of HYPK facilitates its chaperone-like activity.

Human HYPK (Huntingtin Yeast-two-hybrid Protein K) is an intrinsically unstructured chaperone-like protein with no sequence homology to known chaperones. HYPK is also known to be a part of ribosome-associated protein complex and present in polysomes. The objective of the present study was to investigate the evolutionary influence on HYPK primary structure and its impact on the protein's function. Amino acid sequence analysis revealed 105 orthologs of human HYPK from plants, lower invertebrates to mammals. C-terminal part of HYPK was found to be particularly conserved and to contain nascent polypeptide-associated alpha subunit (NPAA) domain. This region experiences highest selection pressure, signifying its importance in the structural and functional evolution. NPAA domain of human HYPK has unique amino acid composition preferring glutamic acid and happens to be more stable from a conformational point of view having higher content of a-helices than the rest. Cell biology studies indicate that overexpressed C-terminal human HYPK can interact with nascent proteins, co-localizes with huntingtin, increases cell viability and decreases caspase activities in Huntington's disease (HD) cell culture model. This domain is found to be required for the chaperone-like activity of HYPK in vivo. Our study suggested that by virtue of its flexibility and nascent peptide binding activity, HYPK may play an important role in assisting protein (re)folding.

Interplay of acetyltransferase EP300 and the proteasome system in regulating heat shock transcription factor 1.

When exposed to proteotoxic environmental conditions, mammalian cells activate the cytosolic stress response in order to restore protein homeostasis. A key feature of this response is the heat shock transcription factor 1 (HSF1)-dependent expression of molecular chaperones. Here, we describe the results of an RNA interference screen in HeLa cells to identify modulators of stress response induction and attenuation. The modulator proteins are localized in multiple cellular compartments, with chromatin modifiers and nuclear protein quality control playing a central regulatory role. We find that the acetyltransferase, EP300, controls the cellular level of activatable HSF1. This involves acetylation of HSF1 at multiple lysines not required for function and results in stabilization of HSF1 against proteasomal turnover. Acetylation of functionally critical lysines during stress serves to fine-tune HSF1 activation. Finally, the nuclear proteasome system functions in attenuating the stress response by degrading activated HSF1 in a manner linked with the clearance of misfolded proteins.

Identification of HYPK-interacting proteins reveals involvement of HYPK in regulating cell growth, cell cycle, unfolded protein response and cell death.

Huntingtin Yeast Two-Hybrid Protein K (HYPK) is an intrinsically unstructured huntingtin (HTT)-interacting protein with chaperone-like activity. To obtain more information about the function(s) of the protein, we identified 27 novel interacting partners of HYPK by pull-down assay coupled with mass spectrometry and, further, 9 proteins were identified by co-localization and co-immunoprecipitation (co-IP) assays. In neuronal cells, (EEF1A1 and HSPA1A), (HTT and LMNB2) and (TP53 and RELA) were identified in complex with HYPK in different experiments. Various Gene Ontology (GO) terms for biological processes, like protein folding (GO: 0006457), response to unfolded protein (GO: 0006986), cell cycle arrest (GO: 0007050), anti-apoptosis (GO: 0006916) and regulation of transcription (GO: 0006355) were significantly enriched with the HYPK-interacting proteins. Cell growth and the ability to refold heat-denatured reporter luciferase were decreased, but cytotoxicity was increased in neuronal cells where HYPK was knocked-down using HYPK antisense DNA construct. The proportion of cells in different phases of cell cycle was also altered in cells with reduced levels of HYPK. These results show that HYPK is involved in several biological processes, possibly through interaction with its partners.

Firefly luciferase mutants as sensors of proteome stress.

Maintenance of cellular protein homeostasis (proteostasis) depends on a complex network of molecular chaperones, proteases and other regulatory factors. Proteostasis deficiency develops during normal aging and predisposes individuals for many diseases, including neurodegenerative disorders. Here we describe sensor proteins for the comparative measurement of proteostasis capacity in different cell types and model organisms. These sensors are increasingly structurally destabilized versions of firefly luciferase. Imbalances in proteostasis manifest as changes in sensor solubility and luminescence activity. We used EGFP-tagged constructs to monitor the aggregation state of the sensors and the ability of cells to solubilize or degrade the aggregated proteins. A set of three sensor proteins serves as a convenient toolkit to assess the proteostasis status in a wide range of experimental systems, including cell and organism models of stress, neurodegenerative disease and aging.

Protein folding in the cytoplasm and the heat shock response.

Proteins generally must fold into precise three-dimensional conformations to fulfill their biological functions. In the cell, this fundamental process is aided by molecular chaperones, which act in preventing protein misfolding and aggregation. How this machinery assists newly synthesized polypeptide chains in navigating the complex folding energy landscape is now being understood in considerable detail. The mechanisms that ensure the maintenance of a functional proteome under normal and stress conditions are also of great medical relevance, as the aggregation of proteins that escape the cellular quality control underlies a range of debilitating diseases, including many age-of-onset neurodegenerative disorders.

The role of intrinsically unstructured proteins in neurodegenerative diseases.

The number and importance of intrinsically disordered proteins (IUP), known to be involved in various human disorders, are growing rapidly. To test for the generalized implications of intrinsic disorders in proteins involved in Neurodegenerative diseases, disorder prediction tools have been applied to three datasets comprising of proteins involved in Huntington Disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD). Results show, in general, proteins in disease datasets possess significantly enhanced intrinsic unstructuredness. Most of these disordered proteins in the disease datasets are found to be involved in neuronal activities, signal transduction, apoptosis, intracellular traffic, cell differentiation etc. Also these proteins are found to have more number of interactors and hence as the proportion of disorderedness (i.e., the length of the unfolded stretch) increased, the size of the interaction network simultaneously increased. All these observations reflect that, "Moonlighting" i.e. the contextual acquisition of different structural conformations (transient), eventually may allow these disordered proteins to act as network "hubs" and thus they may have crucial influences in the pathogenecity of neurodegenerative diseases.

Huntingtin interacting protein HYPK is intrinsically unstructured.

To characterize HYPK, originally identified as a novel huntingtin (Htt) interacting partner by yeast two hybrid assay, we used various biophysical and biochemical techniques. The molecular weight of the protein, determined by gel electrophoresis, was found to be about 1.3-folds ( approximately 22 kDa) higher than that obtained from mass spectrometric analysis (16.9 kDa). In size exclusion chromatography experiment, HYPK was eluted in three fractions, the hydrodynamic radii for which were calculated to be approximately 1.5-folds (23.06 A) higher than that expected for globular proteins of equivalent mass (17.3 A). The protein exhibited predominantly (63%) random coil characteristics in circular dichroism spectroscopy and was highly sensitive to limited proteolysis by trypsin and papain, indicating absence of any specific domain. Experimental evidences with theoretical analyses of amino acids composition of HYPK and comparison with available published data predicts that HYPK is an intrinsically unstructured protein (IUP) with premolten globule like conformation. In presence of increasing concentration of Ca(2+), HYPK showed conformational alterations as well as concomitant reduction of hydrodynamic radius. Even though any link between the natively unfolded nature of HYPK, its conformational sensitivity towards Ca(2+) and interaction with Htt is yet to be established, its possible involvement in Huntington's disease pathogenesis is discussed.

HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity.

Expansion of polymorphic glutamine (Q) numbers present at the protein Huntingtin (Htt) beyond 36Q results in its misfolding and aggregation, and the aggregates recruit several other proteins. Here we show that HYPK, initially identified as an Htt-interacting partner by yeast two-hybrid assay, physically interacts with N-terminal Htt in Neuro2A cells and alters the numbers and distribution of aggregates formed by N-terminal Htt with 40Q. HYPK also alters the kinetics of mutated N-terminal Htt-mediated aggregate formation. Fluorescence recovery after photobleaching studies reveal that over-expression of HYPK results in the appearance of Htt poly Q aggregates, which upon bleaching recovers approximately 80% of initial fluorescence intensity within 6 min. Fluorescence loss in photobleaching studies indicate loss off fluorescence intensity of the aggregates with time in presence of HYPK. Over-expression of this protein reduces poly Q-mediated caspase-2, caspase-3 and caspase-8 activations, whereas gamma ray-induced activations of these enzymes are not affected. In vitro and in vivo studies demonstrate that HYPK possesses a novel chaperone-like activity. We conclude that HYPK, without having any sequence similarity with known chaperones, plays an effective role in protecting neuronal cells against apoptosis induced by mutated N-terminal Htt by modulating the aggregate formation.

Increased caspase-2, calpain activations and decreased mitochondrial complex II activity in cells expressing exogenous huntingtin exon 1 containing CAG repeat in the pathogenic range.

(1) Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by the expansion of polymorphic CAG repeats beyond 36 at exon 1 of huntingtin gene (htt). To study cellular effects by expressing N-terminal domain of Huntingtin (Htt) in specific cell lines, we expressed exon 1 of htt that codes for 40 glutamines (40Q) and 16Q in Neuro2A and HeLa cells. (2) Aggregates and various apoptotic markers were detected at various time points after transfection. In addition, we checked the alterations of expressions of few apoptotic genes by RT-PCR. (3) Cells expressing exon 1 of htt coding 40Q at a stretch exhibited nuclear and cytoplasmic aggregates, increased caspase-1, caspase-2, caspase-8, caspase-9/6, and calpain activations, release of cytochrome c and AIF from mitochondria in a time-dependent manner. Truncation of Bid was increased, while the activity of mitochondrial complex II was decreased in such cells. These changes were significantly higher in cells expressing N-terminal Htt with 40Q than that obtained in cells expressing N-terminal Htt with 16Q. Expressions of caspase-1, caspase-2, caspase-3, caspase-7, and caspase-8 were increased while expression of Bcl-2 was decreased in cells expressing mutated Htt-exon 1. (4) Results presented in this communication showed that expression of mutated Htt-exon 1 could mimic the cellular phenotypes observed in Huntington's disease and this cell model can be used for screening the agents that would interfere with the apoptotic pathway and aggregate formation.