Journal of Cellular Biochemistry

The continuous reliance of cancer cells to acquire energy and communicate their nutrient needs makes them resilient and vulnerable. It provides an opportunity to stifle cancer cells by restricting their energy generation and communication ability. Autophagy and exosome biogenesis are two such pathways that are essential in maintaining the robust growth and survival of cancer cells. In this study we observed that inhibition of one pathway altered the expression of genes in other pathway. Exosome biogenesis, when blocked, led to an increase in breast cancer cell proliferation, while inhibition of autophagy did not significantly affect cancer cell proliferation. Therefore, the two pathways, when independently inhibited, did not present any significant effect on restricting cancer cell growth. However, we observed a substantial reduction in cancer cell proliferation upon combined inhibition of two pathways. To evaluate the reciprocal regulation of two pathways, we blocked the autophagy pathway and observed increase in the secretion of exosomes from MDA-MB-231 cells, along with decreased expression of Alix and CD63. On contrary, inhibition of exosome biogenesis led to an increase in the expression of ATG5 and ATG16L1, which caused a significant decrease in expression of GABARAPL2. Interestingly, the knockdown of GABARAPL2 abrogated the decrease in Alix expression upon autophagy inhibition, thus highlighting the essential role of GABARAPL2 in Alix secretion. Thus, our study highlights for the first time the synergistic effects of autophagy and exosome pathway inhibition in restricting cancer cell growth as well as the involvement of GABARAPL2 in the regulation of exosome secretion via modulating Alix expression.

Dysfunctional autophagy is connected to multiple diseases. Withaferin-A, a biologically active withanolide, has been shown to impair autophagy in the breast cancer cell line, MCF-7; however, the underlying mechanism remains unclear. Here while uncovering the mechanism, it was found that treatment of MCF-7 cells with WA declines Beclin1 protein synthesis by restricting association of its mRNA to polysomes with no significant reduction in its mRNA level. Reporter assay established that WA-treatment enhanced the expression level of mature miR-20a which directly interacts with the 3UTR of beclin1 mRNA. RNA affinity chromatography revealed association of mRNA stabilizing protein, HuR with the 3UTR of beclin1 mRNA. Additionally, co-immunoprecipitation assay determined the interactions of GW182, an essential component of GW-body and Ago2, a crucial member of miRNA related silencing complex (RISC) with the 3UTR of beclin1 mRNA. In parallel, it was found that knocking down of HuR eliminates the interaction of GW182 with beclin1 3UTR, while the association of miRNA (Ago2) remains unaffected. These results suggested simultaneous association of HuR, GW182 and microRNA with beclin1 mRNA which could be pivotal for sequestration of beclin1 mRNA in GW-bodies. Thus, our findings culminated in identifying an unconventional mechanism of regulation of Beclin1 expression by the dual interplay between hsa-miR-20a and HuR during WA-induced impaired autophagy in MCF-7 cells.

Many of the molecular mechanisms affected by ubiquitylation are highly conserved from yeast to humans and are associated to a plethora of diseases including cancers. To elucidate the regulatory role of epigenetic factors such as the catalytic subunits of SAGA complex, KAT-Gcn5 and Ub-protease Ubp8, on ubiquitylation of non-histone proteins we have performed a comprehensive analysis of the Ub-proteome in yeast Saccharomyces cerevisiae in strains disrupted in Gcn5, Ubp8 or both respect to wild type. We found significative alteration of ubiquitylation in proteins belonging to different functional categories with a recurrence of identical proteins in absence of Gcn5 or Ubp8 indicating shared targets and their interlaced function. Among the processes involved we noteworthy identified all major enzymes engaged in energy metabolism and glycolysis such as PFK1, PFK2 and others showing increased ubiquitylation respect to WT. We showed that the higher degree of ubiquitylation found is at post-translational level and does not depend on transcription. Noteworthy, we found in vivo severe defects of growth in poor sugar medium and inability to adaptive switch from fermentative to respiratory growth in strains lacking Gcn5 and Ubp8. Our findings data provide a novel, direct link, between metabolism and epigenetic control with a novel role of DUB-Ubp8 and KAT-Gcn5 on the ubiquitylation marking all the main glycolytic enzymes required for an effective execution of the glycolytic flux. Collectively our experimental results and the proposed model can lead to future research and innovative strategies that by targeting epigenetic modulators might be able to lower sugar utilization also in human cells.\n\nAuthor SummaryMolecular mechanisms dissected in simple yeast might be translated to similar circuitries in human cells for new discoveries in human diseases including cancer. Ubiquitylation of proteins is an evolutionary conserved mechanism required for many biological processes. Different post-translational modifications (PTMs) such as ubiquitylation, acetylation, methylation etc. are reciprocally regulated for deposition or removal. Epigenetic factors writing the PTMs code are often components of multiproteic complexes such as SAGA complex that holds the K-acetyltransferase (KAT) Gcn5 and the Ubiquitin-protease (DUB) Ubp8 highly conserved in Evolution. Cells respond to environment and nutrients by changing metabolism and group of enzymes involved in specific pathways are often coregulated by the deposition of selected PTMs. This study analyses the composition and quantitation of Ub-proteins differentially modified in absence of KAT-Gcn5 and DUB-Ubp8 in yeast. Interstingly, we highlighted the role of Gcn5/Ubp8 dependent ubiquitylation in marking major glycolytic enzymes necessary for glucose utilization. Our study suggests a novel regulatory pathway and, considering that lowering glycolysis is a promising strategy to target tumor metabolism, we propose this study as an interesting perspective to lower enhanced glycolysis in tumors.

The co-chaperone BAG3 is a hub for a variety of cellular pathways via its multiple domains and its interaction with HSP70 and HSPB8. Under aging and cellular stress conditions in particular, together with molecular chaperones, BAG3 ensures the sequestration of aggregated or aggregation prone ubiquitinated proteins to the autophagic-lysosomal system via ubiquitin receptors. There are emerging indications that BAG3-mediated selective macroautophagy also copes with non-ubiquitinated cargo. Phylogenetically, BAG3 comprises several highly conserved predicted LIRs, LC3-interacting regions, which might directly target BAG3 including its cargo to ATG8 proteins and directly drive their autophagic degradation. Based on pull-down experiments, peptide arrays and proximity ligation assays, our results provide evidence of an interaction of BAG3 with ATG8 proteins. In addition, we could demonstrate that mutations within the LIRs impair co-localization with ATG8 proteins in immunofluorescence. A BAG3 variant mutated in all LIRs results in a substantial decrease of BAG3 levels within purified native autophagic vesicles compared to wild-type BAG3. These results strongly suggest LC3-mediated sequestration of BAG3. Therefore, we conclude that in addition of being a key co-chaperone to HSP70, BAG3 may also act as cargo receptor for client proteins, which would significantly extend the role of BAG3 in selective macroautophagy and protein quality control. SynopsisBAG3 ensures sequestration of aggregated ubiquitinated proteins to the autophagic-lysosomal degradation. Based on emerging indications this BAG3-mediated macroautophagy may also cope with non-ubiquitinated clients and comprises conserved predicted LC3 interacting regions, we analyzed the interaction with LC3 proteins. We evidenced an interaction of BAG3 with LC3 proteins by various measures including pull-down experiments, peptide arrays, proximity ligation assays, co-localization and native autophagic vesicles analysis. These results suggest BAG3 may additionally act as cargo receptor for client proteins. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/526551v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@ac40d2org.highwire.dtl.DTLVardef@d3bf3dorg.highwire.dtl.DTLVardef@1b06a53org.highwire.dtl.DTLVardef@114a453_HPS_FORMAT_FIGEXP M_FIG C_FIG

The nuclear protein transport between the nucleus and cytosol can be considered a core process of cell regulation. Specially designed proteins in nature such as importins, exportins, and some other transporters facilitate this transport in the cell and control the cellular processes. Transient and weak protein-protein interactions are basis of these various biomolecular processes. Prior to cargo transports, the transport proteins recognize the Nuclear localization signals (NLSs) and Nuclear export signals (NESs) of cargo proteins and, bind to the RanGTP. Also, these proteins bind with other similar protein subunits along with RanGTP to transport cargos. Cell is enormously crowded place where DNA, RNA, proteins, lipids and small molecules cooperatively facilitate numerous cellular processes. In such environment, existence of nonspecific interactions between proteins is quite obvious. Considering this hypothesis, in this study, protein-protein docking approach was applied to determine the binding affinities of 12 human nuclear transport proteins. Results showed that KPNA1, TNPO1 and TNPO3 have greater affinity to bind with other transport proteins. Also, among 78 complexes (12 homodimers and 66 heterodimers), KPNA1-KPNB1, KPNA1-TNPO1 and KPNA1-TNPO3 complexes have the highest stability. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/436462v2_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d81d48org.highwire.dtl.DTLVardef@6a929dorg.highwire.dtl.DTLVardef@bfa16eorg.highwire.dtl.DTLVardef@ff610b_HPS_FORMAT_FIGEXP M_FIG C_FIG Initially, 12 human nuclear transport proteins PDB structures were retrieved from the 1. Protein data bank (PDB). These proteins had some missing terminals and residues thus, we used 2. SWISS-MODEL and 3. MODELLER v.10.1 to model those regions in these proteins. Next, we used widely popular web server, 4. ClusPro v.2.0 for protein-protein docking analysis among 12 proteins. Then, we employed 5. PRODIGY web server to calculate the binding affinities of 78 complexes (12 homodimers & 66 heterodimers). Finally, we utilised three web tools, 6. Arpeggio, 7. PIMA and 8. PDBePISA to analyse top-three complexes (KPNA1-KPNB1, KPNA1-TNPO1 & TNPO3) for in-depth interactions and energetics.

The induction of apoptosis upon USP7 (HAUSP) inhibition is established in cancers that contain a wild-type p53 (p53Wt) through the USP7-Mdm2-p53 axis, but no clear explanation has yet been reported for the same to occur in cancers containing mutant 53 (p53Mut) or even p53 null (p53Null) systems. Instead of this USP7-Mdm2-p53 axis USP7 also works through an alternative new pathway identified in this study. Here in this study, we observed that the magnitude of apoptosis induction in response to USP7 inhibition was remarkably similar between cancer cells showing p53Null or p53Mut and those with p53Wt. Through a proteomics-based approach, we were able to identify XIAP as a novel interacting partner for USP7. XIAP is a potent and well-characterized member of the inhibitor of apoptosis proteins (IAPs), which function through caspase inhibition. We successfully identified USP7 as a positive regulator of XIAP at post-translational but not at its transcriptional level. Using molecular modelling coupled with domain deletion studies, we show that the first three Ubl domains in association with the catalytic domain of USP7 interact with the BIR2 and the linker region between BIR2 and BIR3 domains of XIAP. Modulation of expression and catalytic activity of USP7 in multiple type of cancer cell lines showed that USP7 stabilizes XIAP through its deubiquitinase activity. We have also observed that USP7 sensitizes cells against chemotherapeutic drugs through stabilization of XIAP. Thus, USP7 promotes tumorigenesis in multiple cancers, via stabilization of XIAP that results in apoptosis inhibition in caspase dependent pathway. Moreover, we observed that combinatorial inhibition of USP7 and XIAP can induce cellular apoptosis in a higher magnitude than their individual inhibition. Additionally, our results indicates that nanoformulated P5091 and P22077 showed higher potency for killing C6 cells in comparison to normal drugs. To the best of our knowledge, this is the first report on identification and validation of XIAP, a crucial E3 ubiquitin ligase, as a novel substrate of the deubiquitinase USP7 and they together involve in empowerment of the tumorigenic potential of cancer cells.

Non-Small Cell Lung Cancer (NSCLC) patients are diagnosed late when the disease has metastasized. Kras is a prevalent mutation in NSCLC besides EGFR and TP53 and targeted therapies against this have been challenging. We have explored deregulation of an endocytic adapter protein, Huntingtin Interacting Protein-1(HIP1) and its relevance in a Kras mutant lung adenocarcinoma cell line as a model system. HIP1 RNA expression is observed to be significantly reduced in high-grade and metastatic lung cancer patients as compared to low-grade tumours and this correlates with poor survival. HIP1 depletion followed by global proteome profiling in A549 cells identified metabolic pathways to be majorly upregulated, followed by RNA transport and surveillance, amongst others. HIP1 depletion also significantly increased anchorage independent growth and invasion of these cells. However, the EMT markers did not follow the canonical regulation. We observed E-Cadherin and Vimentin induction, which is suggestive of collective migration. Additionally, we observed a hypoxic microenvironment to induce HIP1 expression, mediated by Hypoxia Inducible Factor 2 (HIF2), suggesting that a HIF2-HIP1 axis can cause tumour suppression and needs further exploration.

Cancer cells display enhanced glycolytic activity and impaired oxidative phosphorylation even in the presence of adequate oxygen (Warburg effect). Mitochondrial physiology is a promising hit target for anti-cancer therapy because of its key role in Warburg effect and activating apoptosis in mammalian as well as yeast cells. Over-expression of human p53 in S.cerevisiae leads to cell cycle arrest and apotosis. In the present work we show that how S.cerevisiae escapes from p53 induced apoptosis in fermentable carbon source, whereas in case of non-fermentable carbon source this phenomenon is not observed. To shed the light on this aspect we performed a quantitative proteomic analysis of yeast mitochondria isolated from the cells grown on sucrose (fermentation) and glycerol (respiration) with and without p53 over-expression. Through this approach, we identified a total dataset of 1120 proteins with 1% FDR, of which 239(133+106) proteins are differentially experssed in both conditions. Interestingly, we observed that after over-expression of p53 in sucrose grown yeast cells, a complete set of pentose phosphate pathway (PPP) enzymes is up-regulated in the mitochondria that leads to enhanced mitochondrial NADPH production and ROS quenching. Increased association of a hexose transporter (HXT6) and a hexokinase (HXK2) with the mitochondria of fermenting yeast cells upon over-expression of p53, may direct glucose towards PPP inside the mitochondria. In conclusion, our results provide the evidence that up-regulated PPP inside the mitochondria is a key to evade apoptosis by S.cerevisiae upon p53 over-expression.

Shank proteins represent a family of abundant scaffolds in the postsynaptic density. Their dysfunctions had been identified as possible causes behind autism spectrum disorders and various types of cancer. The remarkably promiscuous PDZ domain of the Shank family is highly conserved through isoforms, and contains a unique dynamic segment, the {beta}2-{beta}3 loop, which is likely to play an important role in ligand selectivity. We used the Shank1 PDZ as a model system to analyze the perturbing effects of five disease-associated missense mutations on the binding of different partner peptides. Using experimental methods and molecular dynamics simulations, we characterized the interactions in detail, focusing on their dynamic aspect. While the investigated mutations in general weaken most interactions, the R736Q mutant, unique in having increased thermal stability, also binds the GKAP peptide with higher affinity than the wild type. Overall, our results show that the perturbing effect of mutations is highly partner-specific and depends on the dynamic rearrangements of both uniformly occurring and ligand-specific residue-residue interactions. StatementThe Shank1 protein plays a possible role in autism spectrum disorders and also cancer, two conditions that affect many lives. Our paper provides a more-detailed-than-ever model of Shank1 PDZ - including disease-associated mutants - bound to its ligands, and we highlight the importance of flexibility and dynamics, which appear to be the main driving force behind its ligand selectivity, and reveal remarkable complexity hiding within this small protein domain.

IntroductionAutophagy is a dynamic, multi-step process that cells use to degrade damaged, abnormal, and potentially harmful cellular substances. While autophagy is maintained at a basal level in all cells, it is activated at a higher level in many cancer cells and promotes tumor growth, anti-tumor immune response, and resistance to cancer therapy. As a result, autophagy is increasingly being recognized to have an important role in cancer progression and emerging as a potential target for cancer therapy. We recently discovered that small GTPase Rab27B, a known regulator of vesicle trafficking and exosome secretion, is also involved in the autophagy process. MethodsRab27B was knocked out using CRISPR/Cas9 in CRC cell line HCT116. Western blotting, Immunofluorescence, MTT assay, spheroid formation assay, soft agar assay and xenograft studies were performed to analyze the effects of Rab27B deletion on CRC cells. ResultsCRISPR/Cas9 deletion or siRNA knockdown of Rab27B in colorectal cancer cells (CRC) showed an abnormal accumulation of autophagy vesicles. Additionally, we observed a significant increase in the autophagy markers LC3-II and p62 by immunocytochemistry and western blot analysis, suggesting a defect in the autophagy flux process. Lysotracker and mCherry-EGFP-LC3 fusion construct indicate an impairment in autophagosome and lysosome fusion when Rab27B is silenced. This defect was rescued by full-length and constitutively active GTP mutant of Rab27B. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that the observed defects in autophagy flux resulting from Rab27B loss would cause reduced stress response and tumor growth. Indeed, Rab27B knockout reduced cell viability in response to starvation and a 94% reduction in soft agar colony formation. Rab27B deletion also prevented spheroid formation in vitro. Finally, to analyze the effect of Rab27B deletion in tumor formation in vivo, we performed a xenograft study with wildtype and Rab27B knockout CRC cells, resulting in a dramatic loss of tumor growth (p<0.0001) in the KO cells. ConclusionsTogether, our results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Future studies will focus on investigating the mechanism of how Rab27B functions in the autophagy pathway and whether Rab27B can be targeted as a potential therapeutic strategy for CRC.

ARID1B, a key subunit of the SWI/SNF (also known as BAF) chromatin remodeling complex, is characterized as a canonical tumor suppressor across various cancer types. Although the downregulation of ARID1B transcript levels has been observed in many cancers, the regulation of ARID1B at the protein level is comparatively less studied. Here, we identify WWP2, an E3 ubiquitin ligase, as a novel interacting partner of ARID1B. We further show that using its WW domains, WWP2 interacts with the PPxY motif within the N-terminal intrinsically disordered region of ARID1B. The ability of wild-type (but not the catalytically inactive) WWP2 to modulate ARID1B protein stability was confirmed through cycloheximide chase assay. Interestingly, WWP2 appears to facilitate non-canonical K27- and K29-linked polyubiquitination of ARID1B, leading to the latters proteasomal degradation. Additionally, silencing WWP2 expression results in a decrease in ubiquitination and a subsequent increase in ARID1B protein levels, indicating that WWP2 plays a crucial role in regulating ARID1B stability. Finally, based on several tumorigenic assays, we show that WWP2 may modulate ARID1B-mediated tumor suppression. Our results therefore highlight a novel mechanism of post-translational regulation of ARID1B, which may have implications in ARID1B-mediated tumor suppression. HighlightsWWP2, an E3 ubiquitin ligase, is a novel interactor of ARID1B. WWP2 regulates ARID1B protein stability by K27- and K29-linked polyubiquitination mediated proteasomal degradation. WWP2 appears to modulate the tumor suppressor activity of ARID1B by controlling its abundance in tumor cells.

Our work presented here showed that MelB can be crystallized in the conditions as similar as that of other membrane transporter protein of known structure. To identify a rigid protein by modifying the protein structure is the critical factor for facilitating MelB crystallization. It is necessary to perform extensive crystallization screens to obtain crystals. MelB-MelB interaction in the DDM containing solution will be affect by protein preparation, which may lead to reduce in reproducibility of crystallization experiment. Using a detergent mixture is essential for improve protein contact in the crystals, then improve crystallizability. R149C MelB crystal can be obtained in DDM, but these crystals were only diffracted to about 8Å resolution limit. MelB wide type crystal also can be obtained from the condition as that of R149C mutant, but the resolution is weaker than that of mutant. Although MelB and other transporters of known structure share common feature of the crystallization, the emphasis was as much on the protein itself, as it was on detergent type or efficient screening and refinement of the crystallization conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ShcD was previously found to promote cell motility in melanoma cells. Screening of a yeast two hybrid mouse embryo cDNA library identified Nischarin, a negative regulator to cell motility, as an interacting partner to the ShcD-CH2 domain. Therefore, we aimed to investigate the interaction between Nischarin and ShcD in mammalian cells and to determine their functional impact on cell migration. The Nischarin/ShcD interaction was confirmed by transfection and co-immunoprecipitation assays using full-length constructs in HEK293, MCF7 and MM253 cell lines. Deletion of the first 93 amino acids of ShcD abrogated the interaction indicating the importance of these residues for Nischarin binding. Co-expression of Nischarin and ShcD demonstrated an inhibitory effect on the levels of phospho-ERK and phospho-LIMK. In support of this, Nischarin was found to block the migratory activities of ShcD. A brief in silico analysis of publicly available breast cancer patient data was performed to elucidate the effect of Nischarin/ShcD co-expression on the patients overall survival. Patients with high expression of both proteins had better survival than those with only ShcD overexpression. Our results reveal that the novel protein Nischarin is an interacting partner to ShcD. In addition, we report that the tumour suppressive abilities of Nischarin can overcome ShcD-mediated cell migration when both proteins are concomitantly expressed. *This abstract was presented in the National Cancer Research Institute (NCRI)-2019

Transforming Growth Factor {beta} 1 Stimulated Clone 22 D4 (TSC22D4) is an intrinsically disordered protein that regulates cellular and physiological processes such as cell proliferation, cellular senescence as well as hepatic glucose and lipid metabolism. The molecular mechanism of TSC22D4 action in these cellular and metabolic functions, however, remains largely elusive. Here, we identified TSC22D4 as a novel protein kinase B/Akt1 interacting protein, a critical mediator of insulin/PI3K signaling pathway implicated in diverse set of diseases including type 2 diabetes, obesity and cancer. TSC22D4 interacts with Akt1 not constitutively but rather in a regulatory manner. While glucose and insulin stimulation of cells or refeeding of mice impair the hepatic TSC22D4-Akt1 interaction, inhibition of mitochondria and oxidative stress, promote it; indicating that extra- and intra-cellular cues play a key role in controlling TSC22D4-Akt1 interaction. Our results also demonstrate that together with its dimerization domain, i.e. the TSC box, TSC22D4 requires its intrinsically disordered region (D2 domain) to interact with Akt1. To understand regulation of TSC22D4 function further, we employed tandem mass spectrometry and identified 15 novel phosphorylation sites on TSC22D4. Similar to TSC22D4-Akt1 interaction, TSC22D4 phosphorylation also responds to environmental signals such as starvation, mitochondrial inhibition and oxidative stress. Interestingly, 6 out of the 15 novel phosphorylation sites lie within the TSC22D4 D2 domain, which is required for TSC22D4-Akt1 interaction. Characterization of the regulation and function of these novel phosphorylation sites, in the future, will shed light on our understanding of the role of TSC22D4-Akt1 interaction in both cell biological and physiological functions. Overall, our findings postulate a model whereby TSC22D4 acts as an environmental sensor and interacts with Akt1 to regulate cell proliferation, cellular senescence as well as maintain metabolic homeostasis.

Metabolic reprogramming is one of the hallmarks of cancer. Glutamine is one of the most important nutrients that fuels the TCA cycle and therefore takes part in the production of energy. Glutamine is used as starting metabolite for the synthesis of nucleotides, fatty acids and non-essential amino acids. Since nutrients are uptaken from the blood stream, and considering the 3-dimensional state of solid tumors, access of nutrients is highly dependent on the location of individual cells within a tumor, which results in affecting their metabolic activity. This gives rise to two disctincts cell population: the ones that have access to nutrient and the ones that are nutrient-deprived. We studied the effect of the lack of glutamine by creating glutamine-resistent hepatocellular carcinoma cell lines chosen based on their epithelial (Hep3B) or mesenchymal phenotype (SNU-499 and HLF). We found that glutamine deprivation decreased the proliferation rate, clonogenicity and stemness frequency of the three cell lines but in a greater extent of the mesenchymal cells. Transcriptomic analysis performed in HLF cells showed that glutamine deprivation decreased the activation of signaling pathways involved in cell-cell junction, cell-extracellular matrix interactions and decreased the expression of the hallmarks of epithelial-to-mesenchymal transition. We therefore investigated the role of TGF{beta}, a master regulator of these three processes, by transcriptomic and functional analyses in epithelial (Hep3B) and mesenchymal cells (HLF). We found that the lack of glutamine strongly impared the activation of TGF{beta} signaling which correlated with an altered regulation of TGF{beta} target genes: the expression of mesenchymal genes was no longer induced by TGF{beta} while the epithelial genes were more strongly induced. Functional analyses showed that glutamine deprivation abolished the invasive capacities of HCCs and decreased cell adhesion. Altogehter, our results show that glutamine metabolism is necessary to maintain a mesenchymal phenotype and to maintain an efficient TGF{beta} signaling in hepatocellularcarcinoma.

The ability to progress and invade through the extracellular matrix is a characteristic shared by both normal and cancer cells through the formation of structures called invadosomes gathering invadopodia and podosomes. These invadosomes are plastic and dynamic structures that can adopt different organizations depending on the cell types and the environment such as rosettes, dots or linear invadosomes. In this study, we used the specific invadosome marker Tks5 (SH3PXD2A), to identify common features in these different organizations. Tks5 immunoprecipitation coupled with mass spectrometry analysis allowed us to identify common proteins in these different models. We identified elements of the translation machinery, in particular the EIF4B protein, but also endoplasmic reticulum (ER) proteins as part of the invadosome structure. Providing new data on invadosome molecular composition through Tks5 interactome, we identified that ER-associated translation machinery is recruited to invadosome and involved in their formation, persistence and function in all types of invadosomes. SummaryInvadosomes are invasive F-actin structures exhibiting different organizations that degrade the extracellular matrix. The study uses their universal marker, Tks5, to provide new data about invadosome molecular composition and reveal the role of ER-associated translation machinery in invadosome formation and function.

mTOR regulates cell growth by forming the mTORC1 and mTORC2 complexes. DEPTOR partially inhibits mTORC1, which in turn phosphorylates and inactivates it. Despite the mTORC1-DEPTOR structures, the exact mechanism remains unclear largely because functionally flexible key elements, DEPTORs linker in particular, are unresolved. By taking DEPTORs dimerisation into consideration, our modelling of these missing loops suggests that monomeric DEPTOR bound to mTORC1 in a non-inhibitory mode, while the domain-swapped dimeric DEPTOR could interact with mTORC1s FRB domain and block the kinases catalytic site with its linker. These two states indicate that linker phosphorylation inactivates DEPTOR possibly by disrupting its dimerisation, which could tether the linker to the kinase domain to enhance mTORC1 inhibition. In addition to DEPTOR, mTORs k9b-k10 loop, which harbours the S2481 autophosphorylation site, and mSIN1s flexible domains in mTORC2 might act as inhibitory elements too.

Nuclear pores control nucleo-cytoplasmic trafficking and directly or indirectly regulate vital cellular processes. Nup88, important for Crm1 mediated nuclear export process, is overexpressed in many cancers. A positive correlation exists between progressive stages of cancer and Nup88 expression. However, links between Nup88 overexpression and head and neck cancer are insignificant, and mechanistic details are non-existent. Here, we report that Nup88 exhibits positive correlation in head and neck cancer in addition to elevated Nup62 levels. We demonstrate that Nup88 interacts with Nup62 in a cell-cycle and glycosylation independent manner. The overexpression of Nup88 or Nup62 imparts proliferation and migration advantages to cells. We further report that the interaction with Nup62 stabilizes Nup88 by inhibiting proteasome-mediated degradation of overexpressed Nup88. Overexpressed Nup88 is stabile and partly inside the nucleus and can interact with NF{kappa}B (p65). Nup88 overexpression induces proliferative and inflammatory responses downstream of p65. Altogether, we suggest that simultaneous overexpression of Nup62 and Nup88 in head and neck cancer stabilizes overexpressed Nup88. Stable Nup88 interacts with p65 and induces inflammatory, proliferative, and migratory advantages to cells, which perhaps is the underlying mechanism driving tumorigenic transformations.

Mitochondrial translation is a critical regulatory step in mitochondrial genome expression. In Saccharomyces cerevisiae, translational activators are believed to bind to the 5 UTRs of their target mRNAs to position the mitochondrial ribosome at the start codon. Pet309 and Mss51 are translational activators of COX1 mRNA, which encodes subunit one of cytochrome c oxidase. Pet309 physically interacts with COX1 mRNA, but no direct interaction of Mss51 with its target mRNA has been detected. Currently, the mechanisms underlying translational activation of COX1, or any other mitochondrial gene, remain poorly understood. To explore in depth the mechanism of COX1 mRNA translational activation, we studied the association of Pet309 and Mss51 with the mitochondrial ribosome. Both Pet309 and Mss51 interact with the mitoribosome regardless of the presence of COX1 mRNA or of each other. Pet309s association with the ribosome and with COX1 mRNA depends on its N-terminal domain. These findings indicate that Pet309 and Mss51 stably interact with the mitoribosome independently of an active translation. By integrating our data with previously published research, we propose a new mechanism of COX1 mRNA translation activation. SUMMARY STATEMENTYeast mitochondrial mRNAs require translational activators by an almost unknown mechanism. Based on our findings on Mss51 and Pet309 function, we present a new model for translation of the COX1 mRNA

Defective intracellular trafficking and export of miRNAs has been observed in senescent mammalian cells having impaired mitochondrial potential. Similar to what happens in senescent cells, Uncoupling Protein 2 mediated depolarization of mitochondrial membrane potential results in progressive sequestration of miRNAs with polysomes and lowered release of miRNAs through extracellular vesicles. Supporting importance of mitochondrial membrane potential on miRNAs fate determination, impaired miRNA-trafficking process in growth retarded human cells has been found to be reversed in presence of Genipin an inhibitor of Uncoupling Protein 2. Mitochondrial detethering of endoplasmic reticulum in mitochondria depolarized cells, found to be responsible for defective compartmentalization of translation initiation factor eIF4E to ER attached polysomes. It causes retarded translation process of target mRNAs with rER attached polysomes to ensure reduced intracellular trafficking and extracellular export of miRNAs. We have identified a reduced activity of mTORC1 complex in mitochondria defective cells to cause reduced phosphorylation of eIF4E-BP1 to cause retarded eIF-4E targeting to ER attached polysome. Cumulatively, these data suggest intricate involvement of mitochondrial membrane potential and dynamics to determine stability of miRNAs in mammalian cells by affecting sub-cellular locations and export of miRNPs by affecting mTORC1 complex, the regulator of the protein translational machinery. Significance statementHow the reduced mitochondrial activity in growth retarded cells causes defective miRNA export is an open question. Mitochondrial defects induces a retarded subcellular miRNP trafficking in human cells to cause an upregulation in cellular miRNA content by reducing extracellular vesicle-mediated export of miRNA. We have identified a defective compartmentalization of translation initiation factor eIF4E in mitochondria-ER detethered mammalian cells to cause the retarded intracellular miRNA movement and export Activity of mTORC1 complex, a key regulator of protein translation in mammalian cells, is found to be responsible for ER-compartmentalization of eIF4E. mTORC1 activity reduction in growth retarded and mitochondria detethered cells influences the cell fate by acting on miRNA-mRNA axis. This is a unique way how mitochondrial activity is linked with protein translation and gene repression control in mammalian cells.