Comparative analysis of PDZ-binding motifs in the diacylglycerol kinase family.

Diacylglycerol kinases (DGKs) control local and temporal amounts of diacylglycerol (DAG) and phosphatidic acid (PA) by converting DAG to PA through phosphorylation in cells. Certain DGK enzymes possess C-terminal sequences that encode potential PDZ-binding motifs (PBMs), which could be involved in their recruitment into supramolecular signaling complexes. In this study, we used two different interactomic approaches, quantitative native holdup (nHU) and qualitative affinity purification (AP), both coupled to mass spectrometry (MS) to investigate the PDZ partners associated with the potential PBMs of DGKs. Complementing these results with site-specific affinity interactomic data measured on isolated PDZ domain fragments and PBM motifs, as well as evolutionary conservation analysis of the PBMs of DGKs, we explored functional differences within different DGK groups. All our results indicate that putative PBM sequences of type II enzymes, namely DGKδ, DGKη, and DGKκ, are likely to be nonfunctional. In contrast, type IV enzymes, namely DGKζ and DGKι, possess highly promiscuous PBMs that interact with a set of PDZ proteins with very similar affinity interactomes. The combination of various interactomic assays and evolutionary analyses provides a useful strategy for identifying functional domains and motifs within diverse enzyme families.

IRE1 endoribonuclease signaling promotes myeloid cell infiltration in glioblastoma.

BACKGROUND: Intrinsic or environmental stresses trigger the accumulation of improperly folded proteins in the endoplasmic reticulum (ER), leading to ER stress. To cope with this, cells have evolved an adaptive mechanism named the unfolded protein response (UPR) which is hijacked by tumor cells to develop malignant features. Glioblastoma (GB), the most aggressive and lethal primary brain tumor, relies on UPR to sustain growth. We recently showed that IRE1 alpha (referred to IRE1 hereafter), 1 of the UPR transducers, promotes GB invasion, angiogenesis, and infiltration by macrophage. Hence, high tumor IRE1 activity in tumor cells predicts a worse outcome. Herein, we characterized the IRE1-dependent signaling that shapes the immune microenvironment toward monocytes/macrophages and neutrophils. METHODS: We used human and mouse cellular models in which IRE1 was genetically or pharmacologically invalidated and which were tested in vivo. Publicly available datasets from GB patients were also analyzed to confirm our findings. RESULTS: We showed that IRE1 signaling, through both the transcription factor XBP1s and the regulated IRE1-dependent decay controls the expression of the ubiquitin-conjugating E2 enzyme UBE2D3. In turn, UBE2D3 activates the NFκB pathway, resulting in chemokine production and myeloid infiltration in tumors. CONCLUSIONS: Our work identifies a novel IRE1/UBE2D3 proinflammatory axis that plays an instrumental role in GB immune regulation.

RNA polymerase II transcription with partially assembled TFIID complexes.

The recognition of core promoter sequences by the general transcription factor TFIID is the first step in the process of RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Inducible Taf7 knock out (KO) results in the formation of a Taf7-less TFIID complex, while Taf10 KO leads to serious defects within the TFIID assembly pathway. Either TAF7 or TAF10 depletions correlate with the detected TAF occupancy changes at promoters, and with the distinct phenotype severities observed in mouse embryonic stem cells or mouse embryos. Surprisingly however, under either Taf7 or Taf10 deletion conditions, TBP is still associated to the chromatin, and no major changes are observed in nascent Pol II transcription. Thus, partially assembled TFIID complexes can sustain Pol II transcription initiation, but cannot replace holo-TFIID over several cell divisions and/or development.

ATAC and SAGA co-activator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct.

To understand the function of multisubunit complexes, it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here, we demonstrate that the core modules of ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription co-activator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, a SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histone proteins. In contrast, ATAC complex subunits cannot be detected in the cytoplasm of mammalian cells. However, an endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related co-activators, ATAC and SAGA, assemble using co-translational pathways, but their subcellular localization, cytoplasmic abundance, and functions are distinct.

ATAC and SAGA coactivator complexes utilize co-translational assembly, but their cellular localization properties and functions are distinct.

To understand the function of multisubunit complexes it is of key importance to uncover the precise mechanisms that guide their assembly. Nascent proteins can find and bind their interaction partners during their translation, leading to co-translational assembly. Here we demonstrate that the core modules of ATAC (ADA-Two-A-Containing) and SAGA (Spt-Ada-Gcn5-acetyltransferase), two lysine acetyl transferase-containing transcription coactivator complexes, assemble co-translationally in the cytoplasm of mammalian cells. In addition, SAGA complex containing all of its modules forms in the cytoplasm and acetylates non-histones proteins. In contrast, fully assembled ATAC complex cannot be detected in the cytoplasm of mammalian cells. However, endogenous ATAC complex containing two functional modules forms and functions in the nucleus. Thus, the two related coactivators, ATAC and SAGA, assemble by using co-translational pathways, but their subcellular localization, cytoplasmic abundance and functions are distinct.

Changes in social behavior with MAPK2 and KCTD13/CUL3 pathways alterations in two new outbred rat models for the 16p11.2 syndromes with autism spectrum disorders.

Copy number variations (CNVs) of the human 16p11.2 locus are associated with several developmental/neurocognitive syndromes. Particularly, deletion and duplication of this genetic interval are found in patients with autism spectrum disorders, intellectual disability and other psychiatric traits. The high gene density associated with the region and the strong phenotypic variability of incomplete penetrance, make the study of the 16p11.2 syndromes extremely complex. To systematically study the effect of 16p11.2 CNVs and identify candidate genes and molecular mechanisms involved in the pathophysiology, mouse models were generated previously and showed learning and memory, and to some extent social deficits. To go further in understanding the social deficits caused by 16p11.2 syndromes, we engineered deletion and duplication of the homologous region to the human 16p11.2 genetic interval in two rat outbred strains, Sprague Dawley (SD) and Long Evans (LE). The 16p11.2 rat models displayed convergent defects in social behavior and in the novel object test in male carriers from both genetic backgrounds. Interestingly major pathways affecting MAPK1 and CUL3 were found altered in the rat 16p11.2 models with additional changes in males compared to females. Altogether, the consequences of the 16p11.2 genetic region dosage on social behavior are now found in three different species: humans, mice and rats. In addition, the rat models pointed to sexual dimorphism with lower severity of phenotypes in rat females compared to male mutants. This phenomenon is also observed in humans. We are convinced that the two rat models will be key to further investigating social behavior and understanding the brain mechanisms and specific brain regions that are key to controlling social behavior.

A novel IRE1 kinase inhibitor for adjuvant glioblastoma treatment.

Inositol-requiring enzyme 1 (IRE1) is a major mediator of the unfolded protein response (UPR), which is activated upon endoplasmic reticulum (ER) stress. Tumor cells experience ER stress due to adverse microenvironmental cues, a stress overcome by relying on IRE1 signaling as an adaptive mechanism. Herein, we report the discovery of structurally new IRE1 inhibitors identified through the structural exploration of its kinase domain. Characterization in in vitro and in cellular models showed that they inhibit IRE1 signaling and sensitize glioblastoma (GB) cells to the standard chemotherapeutic, temozolomide (TMZ). Finally, we demonstrate that one of these inhibitors, Z4P, permeates the blood-brain barrier (BBB), inhibits GB growth, and prevents relapse in vivo when administered together with TMZ. The hit compound disclosed herein satisfies an unmet need for targeted, non-toxic IRE1 inhibitors and our results support the attractiveness of IRE1 as an adjuvant therapeutic target in GB.

The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells.

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

Metalloprotease-mediated cleavage of CD95 ligand.

CD95 is a member of the TNF receptor superfamily that is ubiquitously expressed in healthy and pathological tissues. Stimulation of CD95 by its physiological ligand CD95L induces its oligomerization leading in turn to the transduction of either apoptotic or nonapoptotic signals. CD95L can exist as both membrane-anchored and soluble forms (sCD95L), the latter resulting from the proteolytic cleavage of the former. Candidate proteases able to achieve CD95L cleavage were identified as matrix metalloproteases (MMP) due to their demonstrated ability to cleave other TNF superfamily ligands. The main goal of this study was to systematically identify the MMP family members capable of cleaving CD95L and subsequently determine the corresponding cleavage sites. By using different orthogonal biochemical approaches and combining them with molecular modelling, we confirmed data from the literature regarding CD95L cleavage by MMP-3 and MMP-7. Moreover, we found that MMP-2 and MMP-12 can cleave CD95L and characterized their resulting cleavage sites. This study provides a systematic approach to analyse the cleavage of CD95L, which until now had only been poorly described.

Native holdup (nHU) to measure binding affinities from cell extracts.

Characterizing macromolecular interactions is essential for understanding cellular processes, yet most methods currently used to detect protein interactions from cells are qualitative. Here, we introduce the native holdup (nHU) approach to estimate equilibrium binding constants of protein interactions directly from cell extracts. Compared to other pull-down-based assays, nHU requires less sample preparation and can be coupled to any analytical methods as readouts, such as Western blotting or mass spectrometry. We use nHU to explore interactions of SNX27, a cargo adaptor of the retromer complex and find good agreement between in vitro affinities and those measured directly from cell extracts using nHU. We discuss the strengths and limitations of nHU and provide simple protocols that can be implemented in most laboratories.

Quantitative fragmentomics allow affinity mapping of interactomes.

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

Oxidative Transformation of Dihydroflavonols and Flavan-3-ols by Anthocyanidin Synthase from Vitis vinifera.

Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate-cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C3-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed.

Androgen receptor-mediated transcriptional repression targets cell plasticity in prostate cancer.

Androgen receptor (AR) signaling remains the key therapeutic target in the management of hormone-naïve-advanced prostate cancer (PCa) and castration-resistant PCa (CRPC). Recently, landmark molecular features have been reported for CRPC, including the expression of constitutively active AR variants that lack the ligand-binding domain. Besides their role in CRPC, AR variants lead to the expression of genes involved in tumor progression. However, little is known about the specificity of their mode of action compared with that of wild-type AR (AR-WT). We performed AR transcriptome analyses in an androgen-dependent PCa cell line as well as cross-analyses with publicly available RNA-seq datasets and established that transcriptional repression capacity that was marked for AR-WT was pathologically lost by AR variants. Functional enrichment analyses allowed us to associate AR-WT repressive function to a panel of genes involved in cell adhesion and epithelial-to-mesenchymal transition. So, we postulate that a less documented AR-WT normal function in prostate epithelial cells could be the repression of a panel of genes linked to cell plasticity and that this repressive function could be pathologically abrogated by AR variants in PCa.

Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders: The polyG diseases.

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disease characterized by the presence of intranuclear inclusions of unknown origin. NIID is caused by an expansion of GGC repeats in the 5' UTR of the NOTCH2NLC (N2C) gene. We found that these repeats are embedded in a small upstream open reading frame (uORF) (uN2C), resulting in their translation into a polyglycine-containing protein, uN2CpolyG. This protein accumulates in intranuclear inclusions in cell and mouse models and in tissue samples of individuals with NIID. Furthermore, expression of uN2CpolyG in mice leads to locomotor alterations, neuronal cell loss, and premature death of the animals. These results suggest that translation of expanded GGC repeats into a novel and pathogenic polyglycine-containing protein underlies the presence of intranuclear inclusions and neurodegeneration in NIID.

Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription.

Co-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3-/- embryos show developmental delay already at embryonic day (E) 7.5, while Usp22-/- embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3-/- mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3-/- mESCs, or Atxn7l3-/- MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3-/- cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

Reflux of Endoplasmic Reticulum proteins to the cytosol inactivates tumor suppressors.

In the past decades, many studies reported the presence of endoplasmic reticulum (ER)-resident proteins in the cytosol. However, the mechanisms by which these proteins relocate and whether they exert cytosolic functions remain unknown. We find that a subset of ER luminal proteins accumulates in the cytosol of glioblastoma cells isolated from mouse and human tumors. In cultured cells, ER protein reflux to the cytosol occurs upon ER proteostasis perturbation. Using the ER luminal protein anterior gradient 2 (AGR2) as a proof of concept, we tested whether the refluxed proteins gain new functions in the cytosol. We find that refluxed, cytosolic AGR2 binds and inhibits the tumor suppressor p53. These data suggest that ER reflux constitutes an ER surveillance mechanism to relieve the ER from its contents upon stress, providing a selective advantage to tumor cells through gain-of-cytosolic functions-a phenomenon we name ER to Cytosol Signaling (ERCYS).

Citrullination of pyruvate kinase M2 by PADI1 and PADI3 regulates glycolysis and cancer cell proliferation.

Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.

TBPL2/TFIIA complex establishes the maternal transcriptome through oocyte-specific promoter usage.

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.