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Transcriptional activation by p53 provides powerful, organism-wide tumor suppression. We hypothesized that the local chromatin environment, including differential enhancer activities, contributes to various p53-dependent transcriptional activities in different cell types during stress-induced signaling. In this work, using ChIP-sequencing, immunoblotting, quantitative PCR, and computational analyses across various mammalian cell lines, we demonstrate that the p53-induced transcriptome varies by cell type, reflects cell type-specific activities, and is considerably broader than previously anticipated. We found that these molecular events are strongly influenced by p53's engagement with differentially active cell type-specific enhancers and promoters. We also observed that p53 activity depends on the p53 family member tumor protein p63 in epithelial cell types. Notably, we demonstrate that p63 is required for epithelial enhancer identity, including enhancers used by p53 during stress-dependent signaling. Loss of p63, but not p53, caused site-specific depletion of enhancer-associated chromatin modifications, suggesting that p63 functions as an enhancer maintenance factor in epithelial cells. Additionally, a subset of epithelial-specific enhancers depends on the activity of p63 providing a direct link between lineage determination and enhancer structure. These results suggest that a broad, cell-intrinsic mechanism controls p53-dependent cellular stress response through differential regulation of cis -regulatory elements., (© 2019 Karsli Uzunbas et al.)

We developed a molecular beacon targeting miR-141-3p, aberrantly increased in 5-fluorouracil-resistant colorectal cancer cells (R-CRCCs). It consists of a fluorophore-labeled oligonucleotide, antisense to miR-141-3p, and a quencher. It detected R-CRCCs and recovered the chemosensitivity of them to 5-fluorouracil by hybridization with miR-141-3p, which is applicable to cancer treatment.

The central regulator of the Wnt/β-catenin pathway is the Axin/APC/GSK3β destruction complex (DC), which, under unstimulated conditions, targets cytoplasmic β-catenin for degradation. How Wnt activation inhibits the DC to permit β-catenin-dependent signaling remains controversial, in part because the DC and its regulation have never been observed in vivo Using bimolecular fluorescence complementation (BiFC) methods, we have now analyzed the activity of the DC under near-physiological conditions in Drosophila By focusing on well-established patterns of Wnt/Wg signaling in the developing Drosophila wing, we have defined the sequence of events by which activated Wnt receptors induce a conformational change within the DC, resulting in modified Axin-GSK3β interactions that prevent β-catenin degradation. Surprisingly, the nucleus is surrounded by active DCs, which principally control the degradation of β-catenin and thereby nuclear access. These DCs are inactivated and removed upon Wnt signal transduction. These results suggest a novel mechanistic model for dynamic Wnt signal transduction in vivo ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Premature ovarian insufficiency involves amenorrhea and elevated follicle-stimulating hormone before age 40, and its genetic basis is poorly understood. Here, we study 13 premature ovarian insufficiency (POI) patients using whole-exome sequencing. We identify PREPL and TP63 causative variants, and variants in other potentially novel POI genes. PREPL deficiency is a known cause of syndromic POI, matching the patients' phenotype. A role for TP63 in ovarian biology has previously been proposed but variants have been described in multiorgan syndromes, and not isolated POI. One patient with isolated POI harbored a de novo nonsense TP63 variant in the terminal exon and an unrelated patient had a different nonsense variant in the same exon. These variants interfere with the repression domain while leaving the activation domain intact. We expand the phenotypic spectrum of TP63-related disorders, provide a new genotype:phenotype correlation for TP63 and identify a new genetic cause of isolated POI., (© 2019 Wiley Periodicals, Inc.)

The classical NF-κB transcription factor (RelA:p50) and the tumor suppressor Sef axis constitute a negative regulatory loop in which Sef, a target of NF-κB/RelA:p50, fine-tunes NF-κB/RelA:p50 transcriptional-activation in response to inflammatory stimuli trough binding to p50. Similar to the inhibitor IκBα, Sef sequesters NF-κB/RelA:p50 in the cytoplasm of unstimulated cells. Despite its key roles in regulating multiple cellular processes and its potential role as mediator between inflammation and cancer, Sef structural domains required to fulfill its tasks are poorly characterized, and how Sef specificity towards RelA:p50 is achieved is unknown. In-vitro binding assays using bacterially expressed Sef and Co-IP experiments, revealed that in addition to p50, Sef directly interacts with IκBα, and the IKKβ subunit of the IKK complex which mediates RelA:p50 induction by inflammatory stimuli. These interactions are ligand-independent and do not require Sef post-translational modifications. Deletion mutagenesis mapped binding site to IKKβ in a 74- residue segment juxtaposing Sef transmembrane domain, whereas several Sef regions seem to interact with IκBα. Moreover, we identified two new sites which together with the previously identified conserved tyrosine constitute three discontinuous Sef regions each indispensable for Sef binding to RelA:p50 and inhibiting its cytokine induced transcriptional activation. Contrary to IκBα, endogenous Sef is not degraded upon cytokine-stimulation, and its targeting in different cell types markedly enhances cytokine-induced NF-κB nuclear translocation. These results reveal Sef as the first scaffold that brings together the components of NF-κB/RelA:p50 signaling-module. Sef scaffolding function explains the basis for Sef specificity towards inhibiting inflammatory cytokine-induction of NF-κB/RelA:p50., (Copyright © 2019. Published by Elsevier Inc.)

The protein deacetylase SIRT1 is crucial to numerous physiological processes, such as aging, metabolism, and autoimmunity, and is repressed by various transcription factors, including HIC1. Conversely, we found that HIC2, which is highly homologous to HIC1, is a transcriptional activator of SIRT1 due to opposite activity of the intermediate domains of the two homologs. Importantly, this relationship between HIC2 and SIRT1 could be important for cardiac development, where both proteins are implicated. Here, we assessed whether ectopic expression of HIC2, and subsequent upregulation of SIRT1, might decrease apoptosis in H9c2 cardiomyocytes under simulated ischemia/reperfusion (I/R) injury conditions. Our results demonstrate that unlike its structural homolog HIC1, HIC2 is a pivotal transcriptional activator of SIRT1 and, consequently, may protect the heart from I/R injury., (© 2019 Federation of European Biochemical Societies.)

UVRAG (UV radiation resistance associated) is an important regulator of mammalian macroautophagy/autophagy by interacting with BECN1, PIK3C3, and RUBCN. Phosphorylation of UVRAG by MTORC1 negatively regulates autophagosome maturation under nutrient-enriched conditions. However, how UVRAG ubiquitination is regulated is still unknown. Here we report that UVRAG is ubiquitinated by SMURF1 at lysine residues 517 and 559, which decreases the association of UVRAG with RUBCN and promotes autophagosome maturation. However, the deubiquitinase ZRANB1 specifically cleaves SMURF1-induced K29 and K33-linked polyubiquitin chains from UVRAG, thereby increasing the binding of UVRAG to RUBCN and inhibiting autophagy flux. We also demonstrate that CSNK1A1-mediated UVRAG phosphorylation at Ser522 disrupts the binding of SMURF1 to UVRAG through PPxY motif and blocks UVRAG ubiquitination-mediated autophagosome maturation. Interestingly, ZRANB1 is phosphorylated at Thr35, and Ser209 residues by CSNK1A1, and this phosphorylation activates its deubiquitinating activity. Importantly, we provide in vitro and in vivo evidence that UVRAG ubiquitination at lysine residues 517 and 559 or prevention of Ser522 phosphorylation by D4476, a CSNK1A1 inhibitor, enhances the lysosomal degradation of EGFR, which significantly inhibits hepatocellular carcinoma (HCC) growth. Furthermore, UVRAG S522 phosphorylation levels correlate with ZRANB1 T35/S209 phosphorylation levels and poor prognosis in HCC patients. These findings identify a novel molecular mechanism by which ubiquitination and phosphorylation of UVRAG regulate its function in autophagosome maturation and HCC growth, encouraging further study of their potential therapeutic implications. Abbreviations: ATG: autophagy related; BafA 1 : bafilomycin A 1 ; BECN1: beclin 1; CHX: cycloheximide; CSNK1A1/CK1α: casein kinase 1 alpha 1; CQ: chloroquine; DUB: deubiquitinase; EBSS: Earle's balanced salt solution; EGF: epidermal growth factor; GFP: green fluorescent protein; GST: glutathione S-transferase; HBSS: Hanks balanced salts solution; HCC: hepatocellular carcinoma; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MEFs: mouse embryo fibroblasts; mRFP: monomeric red fluorescent protein; PIK3C3 / VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PTMs: post-translational modifications; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; Ub-AMC: ubiquitin-7-amido-4-methylcoumarin: a fluorogenic substrate; UVRAG: UV radiation resistance associated; ZRANB1/TRABID: zinc finger RANBP2-type containing 1.

Mutations in the BRCA1 and BRCA2 genes predispose afflicted individuals to breast, ovarian, and other cancers. The BRCA-encoded products form complexes with other tumor suppressor proteins and with the recombinase enzyme RAD51 to mediate chromosome damage repair by homologous recombination and also to protect stressed DNA replication forks against spurious nucleolytic attrition. Understanding how the BRCA tumor suppressor network executes its biological functions would provide the foundation for developing targeted cancer therapeutics, but progress in this area has been greatly hampered by the challenge of obtaining purified BRCA complexes for mechanistic studies. In this article, we review how recent effort begins to overcome this technical challenge, leading to functional and structural insights into the biochemical attributes of these complexes and the multifaceted roles that they fulfill in genome maintenance. We also highlight the major mechanistic questions that remain.

Nucleoside diphosphate kinases (Nmes or NDPKs) have been implicated in a multitude of cellular processes, including an important role in metastasis suppression, and several enzymatic activities have been assigned to the Nme family. Nevertheless, for many of these processes, it has not been possible to establish a strong connection between Nme enzymatic activity and the relevant biological function. We hypothesized that, in addition to its known enzymatic functions, members of the Nme family might also regulate signaling cascades by acting on key signal transducers. Accordingly, here we show that Nme1 directly interacts with the calcium/calmodulin-dependent kinase II (CaMKII). Using purified proteins, we monitored the phosphorylation of a number of CaMKII substrates and determined that at nanomolar levels Nme1 enhances the phosphorylation of T-type substrates; this modulation shifts to inhibition at low micromolar concentrations. Specifically, the autophosphorylation of CaMKII at Thr286 is completely inhibited by 2 μM Nme1, a feature that distinguishes Nme1 from other known endogenous CaMKII inhibitors. Importantly, CaMKII inhibition does not require phosphotransfer activity by Nme1 because the kinase-dead Nme1 H118F mutant is as effective as the wild-type form of the enzyme. Our results provide a novel molecular mechanism whereby Nme1 could modulate diverse cellular processes in a manner that is independent of its known enzymatic activities.

We screened nearly 1000 synthetic peptides and found that JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI), which is derived from the BoxA domain in the Tob1 protein, activates both unfolded and folded proMMP-7. Interestingly, the smaller derivative of JAL-AK22, termed JAL-TA9 (YKGSGFRMI) possessed auto-proteolytic activity and cleaved three synthetic peptides fragment (MMP18-33, MMP18-40, and Aβ11-29) under physiological conditions. The k cat of JAL-TA9 was 4.58 × 10 -4  min -1 against MMP18-33 and 6.5 × 10 -4  min -1 against MMP18-40. These kinetic parameters are lower than those of general proteinases like trypsin, for which the k cat is 247.2 × 10 5  min -1 against benzoyl-l-arginine ethyl ester. In addition, a 5-mer peptide derived from JAL-TA9, GSGFR also cleaved Aβ11-29. These proteolytic activities were inhibited by AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a serine protease inhibitor. Our results demonstrate that some small synthetic peptides have protease activity. Thus, we propose calling small peptides possessing with protease activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications such as the development of strategic peptide drugs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

For many decades, kinases have predominantly been characterized as oncogenes and drivers of tumorigenesis, because activating mutations in kinases occur in cancer with high frequency. The oncogenic functions of kinases relate to their roles as growth factor receptors and as critical mediators of mitogen-activated pathways. Indeed, some of the most promising cancer therapeutic agents are kinase inhibitors. However, cancer genomics studies, especially screens that utilize high-throughput identification of loss-of-function somatic mutations, are beginning to shed light on a widespread role for kinases as tumor suppressors. The initial characterization of tumor-suppressing kinases- in particular members of the protein kinase C (PKC) family, MKK4 of the mitogen-activated protein kinase kinase family, and DAPK3 of the death-associated protein kinase family- laid the foundation for bioinformatic approaches that enable the identification of other tumor-suppressing kinases. In this review, we discuss the important role that kinases play as tumor suppressors, using several examples to illustrate the history of their discovery and highlight the modern approaches that presently aid in the identification of tumor-suppressing kinases. © 2018 IUBMB Life, 71(6):738-748, 2019., (© 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology.)

Basal cell carcinomas (BCC) have been recently included into the spectrum of BAP1-tumor predisposition syndrome (TPDS). Uveal melanoma (UM) is also a tumor often observed in patients with this hereditary tumor syndrome, in particular bilateral UM is highly suspicious for BAP1-TPDS although no patient has been reported yet. Based on our index patient with BAP1-TPDS with bilateral UM (choroid OD, oculus dexter; iris OS, oculus sinister), several BCCs and thyroid cancer as well as a family history for cancer, this paper analyzes hints and pitfalls to diagnose this syndrome clinically and histologically. A previously undescribed germline variant, namely a heterozygous deletion of a single nucleotide on position 2001 (c.2001delG;p.[Thr668Profs*24] in exon 16 of the BAP1 gene), was identified. Structural changes in the C-terminal of the BAP1 protein were observed by in silico analysis. While the excised iris melanoma showed loss of BAP1 nuclear staining by immunohistochemical staining, the BCCs of our patient (and in the control group, n = 13) were BAP1 positive. Genetic analysis of the BCC of the ocular adnexae confirmed a remaining intact BAP1 copy. The constellation of (bilateral) UM in combination with BCC should raise suspicion for a BAP1-TPDS. As our BCCs probably developed independently from the BAP1-TPDS and UMs frequently show loss of nuclear BAP1 staining, genetic analysis is mandatory to diagnose this syndrome., (© 2018 Wiley Periodicals, Inc.)

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

SAM and SH3 domain-containing 1 (SASH1), a scaffold protein, is regarded as a tumor suppressor. Recent studies have verified the decreased expression of SASH1 in many tumors. Our previous clinical investigation found that SASH1 was widely expressed in normal brain tissues but reduced or absent in glioma tissues. However, the functions of SASH1 in normal astrocytes and the reasons for the reductions in SASH1 levels in glioma tissues are unclear. In this study, we found that in astrocytes, SASH1 functions in cell adhesion. We observed that knockdown of SASH1 expression in cultured astrocytes significantly decreased cell adhesion and increased invasion. Conversely, overexpression of SASH1 in C6 cells markedly promoted cell adhesion and decreased cell invasion. In addition, we found that the expression level of one member of the integrin family, integrin β8, was significantly reduced in SASH1-downregulated astrocytes and elevated in SASH1-upregulated C6 cells. Furthermore, the results of methylation and ChIP assays showed that the methylation level of the SASH1 gene was markedly higher in C6 cells than in astrocytes and that HMGB1 could bind to the CpG islands of the SASH1 gene. HMGB1 overexpression in astrocytes significantly increased the methylation level of the SASH1 gene. This study reveals, for the first time, that HMGB1 contributes to the methylation of the SASH1 gene, and our findings suggest that methylation downregulates the expression of the SASH1 gene and later reduces integrin β8 expression, thereby reducing cell adhesion and promoting cell migration.

p53, the guardian of the genome, requires chaperoning by Hsp70 and Hsp90. However, how the two chaperone machineries affect p53 conformation and regulate its function remains elusive. We found that Hsp70, together with Hsp40, unfolds p53 in an ATP-dependent reaction. This unfolded state of p53 is susceptible to aggregation after release induced by the nucleotide exchange factor Bag-1. However, when Hsp90 and the adaptor protein Hop are present, p53 is transferred from Hsp70 to Hsp90, allowing restoration of the native state upon ATP hydrolysis. Our results suggest that the p53 conformation is constantly remodeled by the two major chaperone machineries. This connects p53 activity to stress, and the levels of free molecular chaperones are important factors regulating p53 activity. Together, our findings reveal an intricate interplay and cooperation of Hsp70 and Hsp90 in regulating the conformation of a client., (Copyright © 2019 Elsevier Inc. All rights reserved.)

PDCD4 is a novel tumor suppressor to show multi-functions inhibiting cell growth, tumor invasion, metastasis, and inducing apoptosis. PDCD4 protein binds to the translation initiation factor eIF4A, some transcription factors, and many other factors and modulates the function of the binding partners. PDCD4 downregulation stimulates and PDCD4 upregulation inhibits the TPA-induced transformation of cells. However, PDCD4 gene mutations have not been found in tumor cells but gene expression was post transcriptionally downregulated by micro environmental factors such as growth factors and interleukins. In this review, we focus on the suppression mechanisms of PDCD4 protein that is induced by the tumor promotors EGF and TPA, and in the inflammatory conditions. PDCD4-protein is phosphorylated at 2 serines in the SCF βTRCP ubiquitin ligase binding sequences via EGF and/or TPA induced signaling pathway, ubiquitinated, by the ubiquitin ligase and degraded in the proteasome system. The PDCD4 protein synthesis is inhibited by microRNAs including miR21.

STAT family proteins are important mediators of cell signaling and represent therapeutic targets for the treatment of human diseases. Most STAT inhibitors target the protein-protein interaction domain, the SH2 domain, but specificity for a single STAT protein is often limited. Recently, we developed catechol bisphosphates as the first inhibitors of STAT5b demonstrated to exhibit a high degree of selectivity over the close homologue STAT5a. Here, we show that the amino acid in position 566 of the linker domain, not the SH2 domain, is the main determinant of specificity. Arg566 in wild-type STAT5b favors tight binding of catechol bisphosphates, while Trp566 in wild-type STAT5a does not. Amino acid 566 also determines the affinity for a tyrosine-phosphorylated peptide derived from the EPO receptor for STAT5a and STAT5b, demonstrating the functional relevance of the STAT5 linker domain for the adjacent SH2 domain. These results provide the first demonstration that a residue in the linker domain can determine the affinity of nonpeptidic small-molecule inhibitors for the SH2 domain of STAT proteins. We propose targeting the interface between the SH2 domain and linker domain as a novel design approach for the development of potent and selective STAT inhibitors. In addition, our data suggest that the linker domain could contribute to the enigmatically divergent biological functions of the two STAT5 proteins.

Polybromo-1 (PBRM1) is an important tumor suppressor in kidney cancer. It contains six tandem bromodomains (BDs), which are specialized structures that recognize acetyl-lysine residues. While BD2 has been found to bind acetylated histone H3 lysine 14 (H3K14ac), it is not known whether other BDs collaborate with BD2 to generate strong binding to H3K14ac, and the importance of H3K14ac recognition for the molecular and tumor suppressor function of PBRM1 is also unknown. We discovered that full-length PBRM1, but not its individual BDs, strongly binds H3K14ac. BDs 2, 4, and 5 were found to collaborate to facilitate strong binding to H3K14ac. Quantitative measurement of the interactions between purified BD proteins and H3K14ac or nonacetylated peptides confirmed the tight and specific association of the former. Interestingly, while the structural integrity of BD4 was found to be required for H3K14ac recognition, the conserved acetyl-lysine binding site of BD4 was not. Furthermore, simultaneous point mutations in BDs 2, 4, and 5 prevented recognition of H3K14ac, altered promoter binding and gene expression, and caused PBRM1 to relocalize to the cytoplasm. In contrast, tumor-derived point mutations in BD2 alone lowered PBRM1's affinity to H3K14ac and also disrupted promoter binding and gene expression without altering cellular localization. Finally, overexpression of PBRM1 variants containing point mutations in BDs 2, 4, and 5 or BD2 alone failed to suppress tumor growth in a xenograft model. Taken together, our study demonstrates that BDs 2, 4, and 5 of PBRM1 collaborate to generate high affinity to H3K14ac and tether PBRM1 to chromatin. Mutations in BD2 alone weaken these interactions, and this is sufficient to abolish its molecular and tumor suppressor functions., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Multidomain proteins account for 70% of the eukaryotic proteome. In genetic disease, multidomain proteins are often affected by numerous mutations, but the effects of these mutations on protein stability and their roles in genetic disease are not well understood. Here, we analyzed protein globular domains to understand how genetic mutations affect the stability of multidomain proteins in inherited disease. In total, 291 domain atomic structures from nine multidomain proteins were modeled by homology, equilibrated using molecular dynamics in water, and subjected to global computational mutagenesis. The domains were separated into 7 groups based on protein fold homology. Mutation propensities within each group of domains were then averaged to select residues critical for domain fold stability. The consensus derived from the sequence alignment shows that the critical residues determined by global mutagenesis are conserved within each group. From this analysis, we concluded that 80% of known disease-related genetic variants are associated with critical residues and are expected to have significant destabilizing effects on domain structure. Our work provides an in silico quantification of protein stability and could help to analyze the complex relationship among missense mutations, multidomain protein stability, and disease phenotypes in inherited eye disease.

Glioma is the most common brain malignancy. Standard first-line therapy for glioma includes surgery, radiotherapy and systemic administration of temozolomide. However, temozolomide does not reach the brain in sufficient doses when administered orally and has poor efficiency in more than half of the patients. Strategies to improve the treatment of glial malignancies are therefore needed. We have recently developed a system (Temodex) for local administration of temozolomide by encapsulating the drug in a biologically inert matrix. Here, we assessed the effect of Temodex in combination with standard therapy in a small-scale clinical study. Since the efficacy of temozolomide therapy is known to depend on the methylation status of the O6-methylguanine-DNA methyltransferase gene (MGMT) promoter, we also analyzed whether the effect of Temodex was influenced by the methylation status of MGMT. Our data show that the combination of standard therapy and Temodex was more efficient than standard therapy alone, promoting the overall patient survival by up to 33 weeks. Moreover, the efficacy of Temodex was not dependent on the methylation status of MGMT. Local Temodex administration in combination with standard therapy thereby emerges as a novel therapeutic option, with applicability that is independent on the methylation status of the MGMT promoter.

Scribble (SCRIB) is an important adaptor protein that controls the establishment and maintenance of apico-basal cell polarity. To better understand how SCRIB controls cell polarity signalling via its PDZ domains, we investigated human SCRIB interactions with adenomatous polyposis coli (APC). We show that SCRIB PDZ1, PDZ2 and PDZ3 are the major interactors with the APC PDZ-binding motif (PBM), whereas SCRIB PDZ4 does not show detectable binding to APC. We then determined the crystal structure of SCRIB PDZ1 domain bound to the APC PBM. Our findings reveal a previously unreported pattern of interactions between the SCRIB PDZ domain region with the C-terminal PDZ binding motif of APC, where SCRIB PDZ1 domain is the highest affinity site., (© 2019 Federation of European Biochemical Societies.)

mTOR complex 1 (mTORC1) is a major regulator of cell growth and proliferation that coordinates nutrient inputs with anabolic and catabolic processes. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which directly recruit mTORC1 onto the lysosomal surface, its site of activation. The Rag GTPase heterodimer has a unique architecture that consists of two GTPase subunits, RagA or RagB bound to RagC or RagD. Their nucleotide-loading states are strictly controlled by several lysosomal or cytosolic protein complexes that directly detect and transmit the amino acid signals. GATOR1 (GTPase-activating protein (GAP) activity toward Rags-1), a negative regulator of the cytosolic branch of the nutrient-sensing pathway, comprises three subunits, Depdc5 (DEP domain-containing protein 5), Nprl2 (NPR2-like GATOR1 complex subunit), and Nprl3 (NPR3-like GATOR1 complex subunit), and is a GAP for RagA. GATOR1 binds the Rag GTPases via two modes: an inhibitory mode that holds the Rag GTPase heterodimer and has previously been captured by structural determination, and a GAP mode that stimulates GTP hydrolysis by RagA but remains structurally elusive. Here, using site-directed mutagenesis, GTP hydrolysis assays, coimmunoprecipitation experiments, and structural analysis, we probed the GAP mode and found that a critical residue on Nprl2, Arg-78, is the arginine finger that carries out GATOR1's GAP function. Substitutions of this arginine residue rendered mTORC1 signaling insensitive to amino acid starvation and are found frequently in cancers such as glioblastoma. Our results reveal the biochemical bases of mTORC1 inactivation through the GATOR1 complex., (© 2019 Shen et al.)

Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 μM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cell-culture assays revealed the mechanism of RRM3 ARE recognition and dimerization. While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuR mRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Protein-templated fragment ligations have been established as a powerful method for the assembly and detection of optimized protein ligands. Initially developed for reversible ligations, the method has been expanded to irreversible reactions enabling the formation of super-additive fragment combinations. Here, protein-induced Mannich ligations are discovered as a biocatalytic reaction furnishing inhibitors of the transcription factor STAT5. STAT5 protein catalyzes multicomponent reactions of a phosphate mimetic, formaldehyde, and 1H-tetrazoles yielding protein ligands with greatly increased binding affinity and ligand efficiency. Reactions are induced under physiological conditions selectively by native STAT5 but not by other proteins. Formation of ligation products and (auto-)inhibition of the reaction are quantified and the mechanism is investigated. Inhibitors assembled by STAT5 block specifically the phosphorylation of this protein in a cellular model of acute myeloid leukemia (AML), DNA-binding of STAT5 dimers, expression of downstream targets of the transcription factor, and the proliferation of cancer cells in mice.

Apical-basal polarity is a common trait that underlies epithelial function. Although the asymmetric distribution of cortical polarity proteins works in a functioning equilibrium, it also retains plasticity to accommodate cell division, during which the basolateral determinant Lgl is released from the cortex. Here, we investigated how Lgl restores its cortical localization to maintain the integrity of dividing epithelia. We show that cytoplasmic Lgl is reloaded to the cortex at mitotic exit in Drosophila epithelia. Lgl cortical localization depends on protein phosphatase 1, which dephosphorylates Lgl on the serines phosphorylated by aPKC and Aurora A kinases through a mechanism that relies on the regulatory subunit Sds22 and a PP1-interacting RVxF motif of Lgl. This mechanism maintains epithelial polarity and is of particular importance at mitotic exit to couple Lgl cortical reloading with the polarization of the apical domain. Hence, PP1-mediated dephosphorylation of Lgl preserves the apical-basal organization of proliferative epithelia., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the current study, we have combined molecular simulations and energetic analysis with dynamics-based network modeling and perturbation response scanning to determine molecular signatures of mutational hotspot residues in the p53, PTEN, and SMAD4 tumor suppressor proteins. By examining structure, energetics and dynamics of these proteins, we have shown that inactivating mutations preferentially target a group of structurally stable residues that play a fundamental role in global propagation of dynamic fluctuations and mediating allosteric interaction networks. Through integration of long-range perturbation dynamics and network-based approaches, we have quantified allosteric potential of residues in the studied proteins. The results have revealed that mutational hotspot sites often correspond to high centrality mediating centers of the residue interaction networks that are responsible for coordination of global dynamic changes and allosteric signaling. Our findings have also suggested that structurally stable mutational hotpots can act as major effectors of allosteric interactions and mutations in these positions are typically associated with severe phenotype. Modeling of shortest inter-residue pathways has shown that mutational hotspot sites can also serve as key mediating bridges of allosteric communication in the p53 and PTEN protein structures. Multiple regression models have indicated that functional significance of mutational hotspots can be strongly associated with the network signatures serving as robust predictors of critical regulatory positions responsible for loss-of-function phenotype. The results of this computational investigation are compared with the experimental studies and reveal molecular signatures of mutational hotspots, providing a plausible rationale for explaining and localizing disease-causing mutations in tumor suppressor genes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Transcription factor p63 is a key regulator of epidermal keratinocyte proliferation and differentiation. Mutations in the p63 DNA-binding domain are associated with ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome. However, the underlying molecular mechanism of these mutations remains unclear. Here, we characterized the transcriptome and epigenome of p63 mutant keratinocytes derived from EEC patients. The transcriptome of p63 mutant keratinocytes deviated from the normal epidermal cell identity. Epigenomic analyses showed an altered enhancer landscape in p63 mutant keratinocytes contributed by loss of p63-bound active enhancers and unexpected gain of enhancers. The gained enhancers were frequently bound by deregulated transcription factors such as RUNX1. Reversing RUNX1 overexpression partially rescued deregulated gene expression and the altered enhancer landscape. Our findings identify a disease mechanism whereby mutant p63 rewires the enhancer landscape and affects epidermal cell identity, consolidating the pivotal role of p63 in controlling the enhancer landscape of epidermal keratinocytes., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early-onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N-terminal ubiquitin-like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho-ubiquitin-loaded C-terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re-modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

The major facilitator superfamily domain-containing protein 2A (MFSD2A) is a constituent of the blood-brain barrier and functions to transport lysophosphatidylcholines (LPCs) into the central nervous system. LPCs such as that derived from docosahexanoic acid (DHA) are indispensable to neurogenesis and maintenance of neurons, yet cannot be synthesized within the brain and are dependent on MFSD2A for brain uptake. Recent studies have implicated MFSD2A mutations in lethal and non-lethal microcephaly syndromes, with the severity correlating to the residual activity of the transporter. We describe two siblings with shared parental ancestry, in whom we identified a homozygous missense mutation (c.1205C > A; p.Pro402His) in MFSD2A. Both affected individuals had microcephaly, hypotonia, appendicular spasticity, dystonia, strabismus, and global developmental delay. Neuroimaging revealed paucity of white matter with enlarged lateral ventricles. Plasma lysophosphatidylcholine (LPC) levels were elevated, reflecting reduced brain transport. Cell-based studies of the p.Pro402His mutant protein indicated complete loss of activity of the transporter despite the non-lethal, attenuated phenotype. The aggregate data of MFSD2A-associated genotypes and phenotypes suggest that additional factors, such as nutritional supplementation or modifying genetic factors, may modulate the severity of disease and call for consideration of treatment options for affected individuals.

Human antigen R (HuR) functions as a major post-transcriptional regulator of gene expression through its RNA-binding activity. HuR is composed by three RNA recognition motifs, namely RRM1, RRM2, and RRM3. The two N-terminal RRM domains are disposed in tandem and contribute mostly to HuR interaction with adenine and uracil-rich elements (ARE) in mRNA. Here, we used a combination of NMR and electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) to characterize the structure, dynamics, RNA recognition, and dimerization of HuR RRM1. Our solution structure reveals a canonical RRM fold containing a 19-residue, intrinsically disordered N-terminal extension, which is not involved in RNA binding. NMR titration results confirm the primary RNA-binding site to the two central β-strands, β1 and β3, for a cyclooxygenase 2 (Cox2) ARE I-derived, 7-nucleotide RNA ligand. We show by 15 N relaxation that, in addition to the N- and C-termini, the β2-β3 loop undergoes fast backbone dynamics (ps-ns) both in the free and RNA-bound state, indicating that no structural ordering happens upon RNA interaction. ESI-IMS-MS reveals that HuR RRM1 dimerizes, however dimer population represents a minority. Dimerization occurs via the α-helical surface, which is oppositely orientated to the RNA-binding β-sheet. By using a DNA analog of the Cox2 ARE I, we show that DNA binding stabilizes HuR RRM1 monomer and shifts the monomer-dimer equilibrium toward the monomeric species. Altogether, our results deepen the current understanding of the mechanism of RNA recognition employed by HuR.

Deficits in DNA damage-repair pathways are the root cause of several human cancers. In mammalian cells, DNA double-strand break repair is carried out by multiple mechanisms, including homologous recombination (HR). The partner and localizer of BRCA2 (PALB2), which is an essential factor for HR, binds to the breast cancer susceptibility 1 (BRCA1) protein at DNA double-strand breaks. At the break site, PALB2 also associates with the breast cancer susceptibility 2 (BRCA2) protein to form a multiprotein complex that facilitates HR. The BRCA1-PALB2 interaction is mediated by association of predicted helical coiled-coil regions in both proteins. PALB2 can also homodimerize through the formation of a coiled coil by the self-association of helical elements at the N-terminus of the PALB2 protein, and this homodimerization has been proposed to regulate the efficiency of HR. We have produced a segment of PALB2, designated PALB2cc (PALB2 coiled coil segment) that forms α-helical structures, which assemble into stable homodimers. PALB2cc also forms heterodimers with a helical segment of BRCA1, called BRCA1cc (BRCA1 coiled coil segment). The three-dimensional structure of the homodimer formed by PALB2cc was determined by solution NMR spectroscopy. This PALB2cc homodimer is a classical antiparallel coiled-coil leucine zipper. NMR chemical-shift perturbation studies were used to study dimer formation for both the PALB2cc homodimer and the PALB2cc/BRCA1cc heterodimer. The mutation of residue Leu24 of PALB2cc  significantly reduces its homodimer stability, but has a more modest effect on the stability of the heterodimer formed between PALB2cc and BRCA1cc. We show that mutation of Leu24 leads to genomic instability and reduced cell viability after treatment with agents that induce DNA double-strand breaks. These studies may allow the identification of distinct mutations of PALB2cc that selectively disrupt homodimeric versus heterodimeric interactions, and reveal the specific role of PALB2cc homodimerization in HR.

The Hippo pathway controls cell proliferation, differentiation, and survival by regulating the Yes-associated protein (YAP) transcriptional coactivator in response to various stimuli, including the mechanical environment. The major YAP regulators are the LATS1/2 kinases, which phosphorylate and inhibit YAP. LATS1/2 are activated by phosphorylation on a hydrophobic motif (HM) outside of the kinase domain by MST1/2 and other kinases. Phosphorylation of the HM motif then triggers autophosphorylation of the kinase in the activation loop to fully activate the kinase, a process facilitated by MOB1. The angiomotin family of proteins (AMOT, AMOTL1, and AMOTL2) bind LATS1/2 and promote its kinase activity and YAP phosphorylation through an unknown mechanism. Here we show that angiomotins increase Hippo signaling through multiple mechanisms. We found that, by binding LATS1/2, SAV1, and YAP, angiomotins function as a scaffold that connects LATS1/2 to both its activator SAV1-MST1 and its target YAP. Deletion of all three angiomotins reduced the association of LATS1 with SAV1-MST1 and decreased MST1/2-mediated LATS1/2-HM phosphorylation. Angiomotin deletion also reduced LATS1/2's ability to associate with and phosphorylate YAP. In addition, we found that angiomotins have an unexpected function along with MOB1 to promote autophosphorylation of LATS1/2 on the activation loop motif independent of HM phosphorylation. These results indicate that angiomotins enhance Hippo signaling by stimulating LATS1/2 autophosphorylation and by connecting LATS1/2 with both its activator SAV1-MST1/2 and its substrate YAP., (© 2018 Mana-Capelli and McCollum.)

Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.

Tumor initiation, progression, and therapeutic resistance have been proposed to originate from a subset of tumor cells, cancer stem cells (CSCs). However, the current understanding of the mechanisms involved in their self-renewal and tumor initiation capacity remains limited. Here, we report that expression of LANO/LRRC1, the vertebrate paralog of SCRIB tumor suppressor, is associated with a stem cell signature in normal and tumoral mammary epithelia. Through in vitro and in vivo experiments including a Lano/Lrrc1 knockout mouse model, we demonstrate its involvement in the regulation of breast CSC (bCSC) fate. Mechanistically, we demonstrate that Lano/LRRC1-depleted cells secrete increased levels of WNT ligands, which act in a paracrine manner to positively deregulate the WNT/β-catenin pathway in bCSCs. In addition to describing the first function of LANO/LRRC1, our results suggest that its expression level could be used as a biomarker to stratify breast cancer patients who could benefit from WNT/β-catenin signaling inhibitors., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein., (© 2018 Aubol et al.)

The MuvB transcriptional regulatory complex, which controls cell-cycle-dependent gene expression, cooperates with B-Myb to activate genes required for the G2 and M phases of the cell cycle. We have identified the domain in B-Myb that is essential for the assembly of the Myb-MuvB (MMB) complex. We determined a crystal structure that reveals how this B-Myb domain binds MuvB through the adaptor protein LIN52 and the scaffold protein LIN9. The structure and biochemical analysis provide an understanding of how oncogenic B-Myb is recruited to regulate genes required for cell-cycle progression, and the MMB interface presents a potential therapeutic target to inhibit cancer cell proliferation., Competing Interests: The authors declare no conflict of interest.

Background & Aims: The tumor-suppressor sterile α motif- and Src-homology 3-domain containing 1 (SASH1) has clinical relevance in colorectal carcinoma and is associated specifically with metachronous metastasis. We sought to identify the molecular mechanisms linking decreased SASH1 expression with distant metastasis formation., Methods: SASH1-deficient, SASH1-depleted, or SASH1-overexpressing HCT116 colon cancer cells were generated by the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9-method, RNA interference, and transient plasmid transfection, respectively. Epithelial-mesenchymal transition (EMT) was analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblotting, immunofluorescence microscopy, migration/invasion assays, and 3-dimensional cell culture. Yeast 2-hybrid assays and co-immunoprecipitation/mass-spectrometry showed V-Crk avian sarcoma virus CT10 oncogene homolog-like (CRKL) as a novel interaction partner of SASH1, further confirmed by domain mapping, site-directed mutagenesis, co-immunoprecipitation, and dynamic mass redistribution assays. CRKL-deficient cells were generated in parental or SASH1-deficient cells. Metastatic capacity was analyzed with an orthotopic mouse model. Expression and significance of SASH1 and CRKL for survival and response to chemotherapy was assessed in patient samples from our department and The Cancer Genome Atlas data set., Results: SASH1 expression is down-regulated during cytokine-induced EMT in cell lines from colorectal, pancreatic, or hepatocellular cancer, mediated by the putative SASH1 promoter. Deficiency or knock-down of SASH1 induces EMT, leading to an aggressive, invasive phenotype with increased chemoresistance. SASH1 counteracts EMT through interaction with the oncoprotein CRKL, inhibiting CRKL-mediated activation of SRC kinase, which is crucially required for EMT. SASH1-deficient cells form significantly more metastases in vivo, depending entirely on CRKL. Patient tumor samples show significantly decreased SASH1 and increased CRKL expression, associated with significantly decreased overall survival. Patients with increased CRKL expression show significantly worse response to adjuvant chemotherapy., Conclusions: We propose SASH1 as an inhibitor of CRKL-mediated SRC signaling, introducing a potentially druggable mechanism counteracting chemoresistance and metastasis formation.

Serine/threonine kinase 11 (STK11) is a protein kinase that is encoded by Stk11 gene located on chromosome 19 and 10 in humans and mouse respectively. It acts as a master kinase of adenine monophosphate-activated protein kinase (AMPK) pathway that coordinates the regulation of cellular energy metabolism and cell division. STK11 exerts effect by activating more than 14 kinases including AMPK and AMPK-related kinases. It is also known to regulate cell polarity and acts as tumor suppressor. Alternative splicing of pre-mRNA is a mechanism which results in multiple transcript variants of a single gene. In human, two STK11 isoforms have been reported, an alternatively spliced isoform which has variation at its C-terminal and mostly expressed in testis (LKB1 S ). Another isoform exhibiting oncogenic properties lacks few residues at its N-terminal (ΔN-LKB1). In the present study, we report the identification of a new transcript variant Stk11N which is generated through alternative splicing. The new variant was found to have differential and tissue specific expression at Postnatal-7 and adult stages of mouse. As compared to the known variant Stk11C, the conceptually translated amino acid sequences of the new variant differ from exon-E2 onwards. In silico post translational studies of the new and published variant show similarity in some of the properties while differ in properties like nuclear export signals, phosphorylation, glycosylation, etc. Thus, alternative splicing of Stk11 gene generating new variant with heterogeneous properties suggests for complex regulation of these variants in controlling the AMPK pathway and other functions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Despite high sequence homology among the p53 family members, the regulation of their transactivation potential is based on strikingly different mechanisms. Previous studies revealed that the activity of TAp63α is regulated via an autoinhibitory mechanism that keeps inactive TAp63α in a dimeric conformation. While all p73 isoforms are constitutive tetramers, their basal activity is much lower compared with tetrameric TAp63. We show that the dimeric state of TAp63α not only reduces DNA binding affinity, but also suppresses interaction with the acetyltransferase p300. Exchange of the transactivation domains is sufficient to transfer the regulatory characteristics between p63 and p73. Structure determination of the transactivation domains of p63 and p73 in complex with the p300 Taz2 domain further revealed that, in contrast to p53 and p73, p63 has a single transactivation domain. Sequences essential for stabilizing the closed dimer of TAp63α have evolved into a second transactivation domain in p73 and p53., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The human WWOX (WW domain-containing oxidoreductase) gene, originally known as a tumor suppressor gene, has been shown to be important for brain function and development. In recent years, mutations in WWOX have been associated with a wide phenotypic spectrum of autosomal recessively inherited neurodevelopmental disorders. Whole exome sequencing was completed followed by Sanger sequencing to verify segregation of the identified variants. Functional WWOX analysis was performed in fibroblasts of one patient. Transcription and translation were assessed by quantitative real-time PCR and Western blotting. We report two related patients who presented with early epilepsy refractory to treatment, progressive microcephaly, profound developmental delay, and brain MRI abnormalities. Additionally, one of the patients showed bilateral optic atrophy. Whole exome sequencing revealed homozygosity for a novel missense variant affecting the evolutionary conserved amino acid Gln230 in the catalytic short-chain dehydrogenase/reductase (SDR) domain of WWOX in both girls. Functional studies showed normal levels of WWOX transcripts but absence of WWOX protein. To our knowledge, our patients are the first individuals presenting the more severe end of the phenotypic spectrum of WWOX deficiency, although they were only affected by a single missense variant of WWOX. This could be explained by the functional data indicating an impaired translation or premature degradation of the WWOX protein.

Accumulating evidence suggests a key role of BAP1 in oncogenesis, but mechanisms regulating BAP1 gene expression remain unexplored. In this report, we revealed that the BAP1 promoter contains multiple G-tracts in its negative strand with high potential of forming G-quadruplex (G4) structures. In circular dichroism studies, synthesized oligonucleotides within these G-rich regions upstream the BAP1 transcription start site showed molar ellipticity at specific wavelengths characteristic of G4 structures. Analyses of these oligonucleotides by native polyacrylamide gel electrophoresis revealed formation of multiple types of G4 structures. In reporter assays, mutations or deletion of predicted G4 structures reduced BAP1 promoter activity. Additionally, DNA helicases CHD2 and CHD7 could reduce BAP1 promoter activity, likely through unwinding its G4 structures., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TG n oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a "mechanical staple" that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics.

Trafficking regulator of GLUT4 1 (TRARG1) was recently identified to localize to glucose transporter type 4 (GLUT4) storage vesicles (GSVs) and to positively regulate GLUT4 trafficking. Our knowledge of TRARG1 structure and membrane topology is limited to predictive models, hampering efforts to further our mechanistic understanding of how it carries out its functions. Here, we use a combination of bioinformatics prediction tools and biochemical assays to define the membrane topology of the 173-amino acid mouse TRARG1. These analyses revealed that, contrary to the consensus prediction, the N-terminus is cytosolic and that a short segment at the C-terminus resides in the luminal/extracellular space. Based on our biochemical analyses including membrane association and antibody accessibility assays, we conclude that TRARG1 has one transmembrane domain (TMD) (145-172) and a re-entrant loop between residues 101 and 127.

Intrinsically disordered regions are present in one-third of eukaryotic proteins and are overrepresented in cellular processes such as signaling, suggesting that intrinsically disordered proteins (IDPs) may have a functional advantage over folded proteins. Upon interacting with a partner macromolecule, a subset of IDPs can fold and bind to form a well-defined three-dimensional conformation. For example, disordered BH3-only proteins bind promiscuously to a large number of homologous BCL-2 family proteins, where they fold to a helical structure in a groove on the BCL-2-like protein surface. As two protein chains are involved in the folding reaction, and the structure is only formed in the presence of the partner macromolecule, this raises the question of where the folding information is encoded. Here, we examine these coupled folding and binding reactions to determine which component determines the folding and binding pathway. Using Φ value analysis to compare transition state interactions between the disordered BH3-only proteins PUMA and BID and the folded BCL-2-like proteins A1 and MCL-1, we found that, even though the BH3-only protein is disordered in isolation and requires a stabilizing partner to fold, its folding and binding pathway is encoded in the IDP itself; the reaction is not templated by the folded partner. We suggest that, by encoding both its transition state and level of residual structure, an IDP can evolve a specific kinetic profile, which could be a crucial functional advantage of disorder., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Crabtree et al.)

In the present study whole-exome sequencing using the Complete Genomics platform was employed to scan a proband from a split‑hand/split‑foot malformation (SHFM) 4 family. The missense mutation c.728G>A (p.Arg243Gln) in the TP63 gene was revealed to be associated with SHFM. Sanger sequencing confirmed the sequences of the proband and his father. The father was diagnosed with SHFM and harbored a CGG‑to‑CAG mutation in exon 5, which produced a R243Q substitution in the zinc binding site and dimerization site of TP63. The R243Q mutation was predicted to be pathogenic by PolyPhen‑2. The proband, who was diagnosed with four digit SHFM, exhibited a more severe phenotype. X‑ray analysis returned the following results: Absence of third phalange bilaterally and third metacarpus of the left hand; absence of the second toes bilaterally and partial third toes; and partial fusion of the second, third and metatarsal bones of the right side with deformity of the second metatarsal of the right side. Osteochondroma was present in the fourth proximal radial metacarpal of the left hand and the basal and proximal parts of the second metatarsal of the right side. The proband's father had five digits in both feet. These results indicate that the R243Q mutation produces a novel phenotype named SHFM4. The present study revealed that the R243Q mutation in the TP63 gene produced a novel phenotype named SHFM4, thereby demonstrating the mutational overlap between ectrodactyly‑ectodermal dysplasia‑cleft syndrome and SHFM4.

Background: The expression of desmosomal genes in lung adenocarcinoma and lung squamous carcinoma is different. However, the regulatory mechanism of desmosomal gene expression in lung adenocarcinoma and lung squamous carcinoma remains unknown., Methods: The correlation between expression of desmosomal gene expression and SOX30 expression were analyzed by bioinformatics. The expression of SOX30, DSP, JUP and DSC3 were detected in lung cancer cell lines, lung tissues of mice and patients' tissues by qPCR, WB, Immunofluorescence and Immunohistochemistry. A chromatin Immunoprecipitation assay was used to investigate the mechanisms of the SOX30 regulation on desmosomal gene expression. In vitro proliferation, migration and invasion assays, and an in vivo nude mice model were utilized to assess the important role of desmosomal genes on SOX30-induced tumor suppression. A WB assay and TOP/FOP flash reporter assay was used to investigate the downstream pathway regulated by the SOX30-desmosomal gene axis. A chemical carcinogenic model of SOX30-knockout mice was generated to confirm the role of the SOX30-desmosomal gene axis in tumorigenesis., Results: The expression of desmosomal genes were upregulated by SOX30 in lung adenocarcinoma but not in lung squamous carcinoma. Further mechanism studies showed that SOX30 acts as a key transcriptional regulator of desmosomal genes by directly binding to the ACAAT motif of desmosomal genes promoter region and activating their transcription in lung adenocarcinoma. Knockdown of the expression of related desmosomal genes by miRNA significantly attenuated the inhibitory effect of SOX30 on cell proliferation, migration and invasion in vitro and on tumor growth and metastasis in vivo. In addition, knockout of SOX30 promotes lung tumor development and loss the inhibition of desmosomal genes on downstream Wnt and ERK signal in urethane-induced lung carcinogenesis in SOX30-knockout mice., Conclusions: Overall, these findings demonstrate for the first time that SOX30 acts as a master switch of desmosomal genes, inhibits lung adenocarcinoma cell proliferation, migration and invasion by activating the transcription of desmosomal genes. This study provides novel insights on the regulatory mechanism of desmosomal genes in lung adenocarcinoma.

MOAP-1 is a pro-apoptotic tumor suppressor molecule with a growing set of known interacting partners. We have demonstrated that during death receptor-dependent apoptosis, MOAP-1 is recruited to TNF-R1 or TRAIL-R1, followed by RASSF1A and Bax association. MOAP-1/Bax association promotes Bax conformational change resulting in the translocation of Bax into the mitochondrial membrane, mitochondrial membrane insertion and dysregulation resulting in several hallmark events that execute apoptosis. Although a role in apoptosis is established, it is currently unknown how MOAP-1 is regulated and how it links to Bax to promote apoptosis. In this study, we demonstrate robust association with RACK1, a versatile scaffolding protein that responds to activation of protein kinase C. Furthermore, we can demonstrate that RACK1 functions to bring the E3 ligase, TRAF2, to MOAP-1 in order to undergo a K63-dependent ubiquitination. Furthermore, RACK1 associates with MOAP-1 via electrostatic associations similar to those observed between MOAP-1/RASSF1A and MOAP-1/TNF-R1. These events illustrate the complex nature of MOAP-1 regulation and characterizes the important role of the scaffolding protein, RACK1, in influencing MOAP-1 biology., (Copyright © 2018 Elsevier B.V. All rights reserved.)