Structural Mechanism for Cargo Recognition by the Retromer Complex.

Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.

Lung Cancer Gene Regulatory Network of Transcription Factors Related to the Hallmarks of Cancer.

The transcriptomic analysis of microarray and RNA-Seq datasets followed our own bioinformatic pipeline to identify a transcriptional regulatory network of lung cancer. Twenty-six transcription factors are dysregulated and co-expressed in most of the lung cancer and pulmonary arterial hypertension datasets, which makes them the most frequently dysregulated transcription factors. Co-expression, gene regulatory, coregulatory, and transcriptional regulatory networks, along with fibration symmetries, were constructed to identify common connection patterns, alignments, main regulators, and target genes in order to analyze transcription factor complex formation, as well as its synchronized co-expression patterns in every type of lung cancer. The regulatory function of the most frequently dysregulated transcription factors over lung cancer deregulated genes was validated with ChEA3 enrichment analysis. A Kaplan-Meier plotter analysis linked the dysregulation of the top transcription factors with lung cancer patients' survival. Our results indicate that lung cancer has unique and common deregulated genes and transcription factors with pulmonary arterial hypertension, co-expressed and regulated in a coordinated and cooperative manner by the transcriptional regulatory network that might be associated with critical biological processes and signaling pathways related to the acquisition of the hallmarks of cancer, making them potentially relevant tumor biomarkers for lung cancer early diagnosis and targets for the development of personalized therapies against lung cancer.

Selective Concurrence of the Long Non-Coding RNA MALAT1 and the Polycomb Repressive Complex 2 to Promoter Regions of Active Genes in MCF7 Breast Cancer Cells.

In cancer cells, the long non-coding RNA (lncRNA) MALAT1 has arisen as a key partner for the Polycomb Repressive Complex 2 (PRC2), an epigenetic modifier. However, it is unknown whether this partnership occurs genome-wide at the chromatin level, as most of the studies focus on single genes that are usually repressed. Due to the genomic binding properties of both macromolecules, we wondered whether there are binding sites shared by PRC2 and MALAT1. Using public genome-binding datasets for PRC2 and MALAT1 derived from independent ChIP- and CHART-seq experiments performed with the breast cancer cell line MCF7, we searched for regions containing PRC2 and MALAT1 overlapping peaks. Peak calls for each molecule were performed using MACS2 and then overlapping peaks were identified by bedtools intersect. Using this approach, we identified 1293 genomic sites where PRC2 and MALAT1 concur. Interestingly, 54.75% of those sites are within gene promoter regions (<3000 bases from the TSS). These analyses were also linked with the transcription profiles of MCF7 cells, obtained from public RNA-seq data. Hence, it is suggested that MALAT1 and PRC2 can concomitantly bind to promoters of actively-transcribed genes in MCF7 cells. Gene ontology analyses revealed an enrichment of genes related to categories including cancer malignancy and epigenetic regulation. Thus, by re-visiting occupancy and transcriptomic data, we identified a key gene subset controlled by the collaboration of MALAT1 and PRC2.

Long Noncoding RNA TALAM1 Is a Transcriptional Target of the RUNX2 Transcription Factor in Lung Adenocarcinoma.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide. It has been reported that genetic and epigenetic factors play a crucial role in the onset and evolution of lung cancer. Previous reports have shown that essential transcription factors in embryonic development contribute to this pathology. Runt-related transcription factor (RUNX) proteins belong to a family of master regulators of embryonic developmental programs. Specifically, RUNX2 is the master transcription factor (TF) of osteoblastic differentiation, and it can be involved in pathological conditions such as prostate, thyroid, and lung cancer by regulating apoptosis and mesenchymal-epithelial transition processes. In this paper, we identified TALAM1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) as a genetic target of the RUNX2 TF in lung cancer and then performed functional validation of the main findings. METHODS: We performed ChIP-seq analysis of tumor samples from a patient diagnosed with lung adenocarcinoma to evaluate the target genes of the RUNX2 TF. In addition, we performed shRNA-mediated knockdown of RUNX2 in this lung adenocarcinoma cell line to confirm the regulatory role of RUNX2 in TALAM1 expression. RESULTS: We observed RUNX2 overexpression in cell lines and primary cultured lung cancer cells. Interestingly, we found that lncRNA TALAM1 was a target of RUNX2 and that RUNX2 exerted a negative regulatory effect on TALAM1 transcription.

A better Amazon road network for people and the environment.

The rapidly expanding network of roads into the Amazon is permanently altering the world's largest tropical forest. Most proposed road projects lack rigorous impact assessments or even basic economic justification. This study analyzes the expected environmental, social and economic impacts of 75 road projects, totaling 12 thousand kilometers of planned roads, in the region. We find that all projects, although in different magnitudes, will negatively impact the environment. Forty-five percent will also generate economic losses, even without accounting for social and environmental externalities. Canceling economically unjustified projects would avoid 1.1 million hectares of deforestation and US$ 7.6 billion in wasted funding for development projects. For projects that exceed a basic economic viability threshold, we identify the ones that are comparatively better not only in terms of economic return but also have lower social and environmental impacts. We find that a smaller set of carefully chosen projects could deliver 77% of the economic benefit at 10% of the environmental and social damage, showing that it is possible to have efficient tradeoff decisions informed by legitimately determined national priorities.

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD.

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection.

Structural Analysis of the Black-Legged Tick Saliva Protein Salp15.

Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4+ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1-21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed α + ß secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine.

The L-Rhamnose Biosynthetic Pathway in Trichomonas vaginalis: Identification and Characterization of UDP-D-Glucose 4,6-dehydratase.

Trichomonas vaginalis is the causative agent of one of the most widespread sexually transmitted diseases in the world. The adhesion of the parasite to the vaginal epithelial cells is mediated by specific proteins and by a complex glycan structure, the lipoglycan (TvLG), which covers the pathogen surface. L-rhamnose is an important component of TvLG, comprising up to 40% of the monosaccharides. Thus, the inhibition of its production could lead to a severe alteration in the TvLG structure, making the L-rhamnose biosynthetic pathway an attractive pharmacologic target. We report the identification and characterization of the first committed and limiting step of the L-rhamnose biosynthetic pathway, UDP-D-glucose 4,6-dehydratase (UGD, EC 4.2.1.76). The enzyme shows a strong preference for UDP-D-glucose compared to dTDP-D-glucose; we propose that the mechanism underlying the higher affinity for the UDP-bound substrate is mediated by the differential recognition of ribose versus the deoxyribose of the nucleotide moiety. The identification of the enzymes responsible for the following steps of the L-rhamnose pathway (epimerization and reduction) was more elusive. However, sequence analyses suggest that in T. vaginalis L-rhamnose synthesis proceeds through a mechanism different from the typical eukaryotic pathways, displaying intermediate features between the eukaryotic and prokaryotic pathways and involving separate enzymes for the epimerase and reductase activities, as observed in bacteria. Altogether, these results form the basis for a better understanding of the formation of the complex glycan structures on TvLG and the possible use of L-rhamnose biosynthetic enzymes for the development of selective inhibitors.

The p12 subunit of human polymerase δ uses an atypical PIP box for molecular recognition of proliferating cell nuclear antigen (PCNA).

Human DNA polymerase δ is essential for DNA replication and acts in conjunction with the processivity factor proliferating cell nuclear antigen (PCNA). In addition to its catalytic subunit (p125), pol δ comprises three regulatory subunits (p50, p68, and p12). PCNA interacts with all of these subunits, but only the interaction with p68 has been structurally characterized. Here, we report solution NMR-, isothermal calorimetry-, and X-ray crystallography-based analyses of the p12-PCNA interaction, which takes part in the modulation of the rate and fidelity of DNA synthesis by pol δ. We show that p12 binds with micromolar affinity to the classical PIP-binding pocket of PCNA via a highly atypical PIP box located at the p12 N terminus. Unlike the canonical PIP box of p68, the PIP box of p12 lacks the conserved glutamine; binds through a 2-fork plug made of an isoleucine and a tyrosine residue at +3 and +8 positions, respectively; and is stabilized by an aspartate at +6 position, which creates a network of intramolecular hydrogen bonds. These findings add to growing evidence that PCNA can bind a diverse range of protein sequences that may be broadly grouped as PIP-like motifs as has been previously suggested.

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases.

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.

Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL.

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.

Mll-COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts.

Expression of Runx2/p57 is a hallmark of the osteoblast-lineage identity. Although several regulators that control the expression of Runx2/p57 during osteoblast-lineage commitment have been identified, the epigenetic mechanisms that sustain this expression in differentiated osteoblasts remain to be completely determined. Here, we assess epigenetic mechanisms associated with Runx2/p57 gene transcription in differentiating MC3T3 mouse osteoblasts. Our results show that an enrichment of activating histone marks at the Runx2/p57 P1 promoter is accompanied by the simultaneous interaction of Wdr5 and Utx proteins, both are components of COMPASS complexes. Knockdown of Wdr5 and Utx expression confirms the activating role of both proteins at the Runx2-P1 promoter. Other chromatin modifiers that were previously described to regulate Runx2/p57 transcription in mesenchymal precursor cells (Ezh2, Prmt5, and Jarid1b proteins) were not found to contribute to Runx2/p57 transcription in full-committed osteoblasts. We also determined the presence of additional components of COMPASS complexes at the Runx2/p57 promoter, evidencing that the Mll2/COMPASS- and Mll3/COMPASS-like complexes bind to the P1 promoter in osteoblastic cells expressing Runx2/p57 to modulate the H3K4me1 to H3K4me3 transition.

Role of RUNX2 transcription factor in epithelial mesenchymal transition in non-small cell lung cancer lung cancer: Epigenetic control of the RUNX2 P1 promoter.

Lung cancer has a high mortality rate in men and women worldwide. Approximately 15% of diagnosed patients with this type of cancer do not exceed the 5-year survival rate. Unfortunately, diagnosis is established in advanced stages, where other tissues or organs can be affected. In recent years, lineage-specific transcription factors have been associated with a variety of cancers. One such transcription factor possibly regulating cancer is RUNX2, the master gene of early and late osteogenesis. In thyroid and prostate cancer, it has been reported that RUNX2 regulates expression of genes important in tumor cell migration and invasion. In this study, we report on RUNX2/ p57 overexpression in 16 patients with primary non-small cell lung cancer and/or metastatic lung cancer associated with H3K27Ac at P1 gene promoter region. In some patients, H3K4Me3 enrichment was also detected, in addition to WDR5, MLL2, MLL4, and UTX enzyme recruitment, members of the COMPASS-LIKE complex. Moreover, transforming growth factor-ß induced RUNX2/ p57 overexpression and specific RUNX2 knockdown supported a role for RUNX2 in epithelial mesenchymal transition, which was demonstrated through loss of function assays in adenocarcinoma A549 lung cancer cell line. Furthermore, RUNX2 increased expression of epithelial mesenchymal transition genes VIMENTIN, TWIST1, and SNAIL1, which reflected increased migratory capacity in lung adenocarcinoma cells.

Gene dosage of DAX-1, determining in sexual differentiation: duplication of DAX-1 in two sisters with gonadal dysgenesis.

Two sisters phenotypically normal females, presenting with tumor abdominal mass with histopathological findings of teratoma and gonadoblastoma associated to 46,XY male-to-female sex reversal syndrome, secondary to a duplication in DAX-1, possibly inherited of maternal gonadal mosaicism. Copy number variation and functional effects of the duplication were done by MLPA multiplex ligation-dependent probe amplification and real time PCR. DAX-1, also known as dosage sensitive sex reversal gene (DSS), is considered the most likely candidate gene involved in XY gonadal dysgenesis when overexpressed. The excess of DAX-1 gene disturbs testicular development by down regulation of SF-1, WT1, and SOX9. This is the first report of 46,XY sex reversal in two siblings who have a maternally inherited duplication of DAX-1 associated with reduced levels of expression of downstream genes as SOX9-SF1.

Crystallography captures catalytic steps in human methionine adenosyltransferase enzymes.

The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a "structural movie" of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE.

Novel homozygous mutation in a colombian patient with persistent müllerian duct syndrome: expanded phenotype.

The anti-Müllerian hormone triggers the regression of uterus and fallopian tubes in male embryos; if there are problems in the synthesis or action of this protein, Müllerian structures persist in an otherwise phenotypic male. The most frequent clinical presentation of Persistent Mullerian Duct syndrome is cryptorchidism and inguinal hernia. The few cases reported in adults are incidental findings or inguinal hernias. However, we present an adult male with history of bilateral cryptorchidism with unsuccessful orchidopexy, who presents with a large abdominal mass with the finding of a seminomatous tumor and persistence of Müllerian structures, in whom the variant c.916delC (p.Leu306Cysfs*29) in the AMHR2 gene not previously reported was documented.

Structural basis of pyrrole polymerization in human porphobilinogen deaminase.

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.

Polycomb PRC2 complex mediates epigenetic silencing of a critical osteogenic master regulator in the hippocampus.

During hippocampal neuron differentiation, the expression of critical inducers of non-neuronal cell lineages must be efficiently silenced. Runx2 transcription factor is the master regulator of mesenchymal cells responsible for intramembranous osteoblast differentiation and formation of the craniofacial bone tissue that surrounds and protects the central nervous system (CNS) in mammalian embryos. The molecular mechanisms that mediate silencing of the Runx2 gene and its downstream target osteogenic-related genes in neuronal cells have not been explored. Here, we assess the epigenetic mechanisms that mediate silencing of osteoblast-specific genes in CNS neurons. In particular, we address the contribution of histone epigenetic marks and histone modifiers on the silencing of the Runx2/p57 bone-related isoform in rat hippocampal tissues at embryonic to adult stages. Our results indicate enrichment of repressive chromatin histone marks and of the Polycomb PRC2 complex at the Runx2/p57 promoter region. Knockdown of PRC2 H3K27-methyltransferases Ezh2 and Ezh1, or forced expression of the Trithorax/COMPASS subunit Wdr5 activates Runx2/p57 mRNA expression in both immature and mature hippocampal cells. Together these results indicate that complementary epigenetic mechanisms progressively and efficiently silence critical osteoblastic genes during hippocampal neuron differentiation.

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5.

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD-Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.

Epigenetic Control of the Bone-master Runx2 Gene during Osteoblast-lineage Commitment by the Histone Demethylase JARID1B/KDM5B.

Transcription factor Runx2 controls bone development and osteoblast differentiation by regulating expression of a significant number of bone-related target genes. Here, we report that transcriptional activation and repression of the Runx2 gene via its osteoblast-specific P1 promoter (encoding mRNA for the Runx2/p57 isoform) is accompanied by selective deposition and elimination of histone marks during differentiation of mesenchymal cells to the osteogenic and myoblastic lineages. These epigenetic profiles are mediated by key components of the Trithorax/COMPASS-like and Polycomb group complexes together with histone arginine methylases like PRMT5 and lysine demethylases like JARID1B/KDM5B. Importantly, knockdown of the H3K4me2/3 demethylase JARID1B, but not of the demethylases UTX and NO66, prevents repression of the Runx2 P1 promoter during myogenic differentiation of mesenchymal cells. The epigenetically forced expression of Runx2/p57 and osteocalcin, a classical bone-related target gene, under myoblastic-differentiation is accompanied by enrichment of the H3K4me3 and H3K27ac marks at the Runx2 P1 promoter region. Our results identify JARID1B as a key component of a potent epigenetic switch that controls mesenchymal cell fate into myogenic and osteogenic lineages.