Novel Family Of Proteins Research Articles

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

A Degradation Motif in STAU1 Defines a Novel Family of Proteins Involved in Inflammation.

Cancer development is regulated by inflammation. Staufen1 (STAU1) is an RNA-binding protein whose expression level is critical in cancer cells as it is related to cell proliferation or cell death. STAU1 protein levels are downregulated during mitosis due to its degradation by the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). In this paper, we map the molecular determinant involved in STAU1 degradation to amino acids 38–50, and by alanine scanning, we shorten the motif to F39PxPxxLxxxxL50 (FPL-motif). Mutation of the FPL-motif prevents STAU1 degradation by APC/C. Interestingly, a search in databases reveals that the FPL-motif is shared by 15 additional proteins, most of them being involved in inflammation. We show that one of these proteins, MAP4K1, is indeed degraded via the FPL-motif; however, it is not a target of APC/C. Using proximity labeling with STAU1, we identify TRIM25, an E3 ubiquitin ligase involved in the innate immune response and interferon production, as responsible for STAU1 and MAP4K1 degradation, dependent on the FPL-motif. These results are consistent with previous studies that linked STAU1 to cancer-induced inflammation and identified a novel degradation motif that likely coordinates a novel family of proteins involved in inflammation. Data are available via ProteomeXchange with the identifier PXD036675.

Exercise training increases CISD family protein expression in murine skeletal muscle and white adipose tissue

Mitochondrial function in skeletal muscle and white adipose tissue (WAT) declines with aging and the progression of type 2 diabetes and insulin resistance. Although exercise increases mitochondrial biogenesis and function in both tissues, the molecular mechanisms are not fully understood. CDGSH iron sulfur domain-containing proteins (CISDs) are a novel family of proteins that regulate mitochondrial activity and biogenesis. However, the relationship between exercise and CISD expression is unclear. We addressed this in the present study by examining changes in the expression of CISDs and mitochondrial proteins in skeletal muscle and WAT of mice subjected to chronic exercise training. Mice were randomly assigned to either the sedentary or exercise group and were housed for 4 weeks in a standard cage without or with a running wheel, respectively. CISD and mitochondrial protein levels in the plantaris and soleus muscles and epididymal WAT were evaluated by western blotting. Chronic exercise increased CISD1 and CISD2 as well as mitochondrial protein expression in plantaris muscle and WAT but not soleus muscle. Moreover, this exercise-induced adaptation was strongly correlated with mitochondrial protein expression. Thus, mitochondrial biogenesis induced by chronic exercise coincides with the expression of CISDs in specific tissues, which may be critical for the maintenance of mitochondrial integrity.

Abstract 4537: Computational modeling and characterization of Diedel proteins that regulate highly-conserved immune signaling

Abstract The NF-kB and JAK/STAT pathways are well-established signal transduction pathways, conserved from insects to mammals. They regulate several key biological processes such as cellular proliferation, stem cell maintenance, hematopoiesis and innate immunity responses. Consequently, their dysregulation results in aberrant conditions, often with deleterious consequences. It is well documented that a variety of human diseases, especially cancer and immune-related conditions are associated with various components of these pathways. The focus of this study is the protein Diedel, which was identified as one of the novel genes involved in the regulation of the JAK/STAT pathway in a genome-wide RNA interference screen in Drosophila cells. Drosophila Diedel and its viral homolog also suppress the conserved Imd-mediated NF-kB pathway in flies. Diedel is a small cysteine-rich protein: It is comprised of only 115 amino acids out of which 10 are cysteines. A recent crystal structure has revealed that Diedel is composed of two sub-domains and incorporates two special structural features, i.e. a hydrophobic surface patch suggesting a putative interaction site, and a cis/trans conformation for a proline residue that may be relevant to its physiological mechanism. However, its precise molecular function and mechanism of action still remains unclear. Based on current sequence information in the databases, Diedel seems to be conserved in drosophilids, the aphid Acyrtosiphon pisum, and insect DNA viruses of the Baculoviridae and Ascoviridae families. While investigating the mechanism of infection of Drosophila melanogaster larvae by the parasitic wasp, Leptopilina heterotoma, we have uncovered two wasp venom sequences that are putative Diedel homologs. This study explores the detailed characterization of various Diedel homologs with an end to establishing structure-function correlations in this family of novel proteins, using a combined tool-kit of template based modeling, sequence and structure analyses algorithms. Our results confirm a conserved structural fold but there are significant sequence differences among the homologs. We present a comparative analysis of the theoretical models of Diedel homologs and their biophysical characterization. This study will help elucidate the molecular function of the parasite Diedel proteins in suppressing the fly’s immune response. Our findings will also point to new ways by which dysregulated immune signaling can be turned down and these findings will potentially have implications in treating inflammation and cancer in humans. Citation Format: Aulon Jerliu, Mary Ellen Heavner, Shubha Govind, Shaneen Singh. Computational modeling and characterization of Diedel proteins that regulate highly-conserved immune signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4537. doi:10.1158/1538-7445.AM2017-4537

A Family of Negative Regulators Targets the Committed Step of de Novo Fatty Acid Biosynthesis

Acetyl-CoA carboxylase (ACCase) catalyzes the committed step of de novo fatty acid biosynthesis. In prokaryotes, green algae, and most plants, this enzyme is a heteromeric complex requiring four different subunits for activity. The plant complex is recalcitrant to conventional purification schemes and hence the structure and composition of the full assembly have been unclear. In vivo coimmunoprecipitation using subunit-specific antibodies identified a novel family of proteins in Arabidopsis thaliana annotated as biotin/lipoyl attachment domain containing (BADC) proteins. Results from yeast two-hybrid and coexpression in Escherichia coli confirmed that all three BADC isoforms interact with the two biotin carboxyl carrier protein (BCCP) isoforms of Arabidopsis ACCase. These proteins resemble BCCP subunits but are not biotinylated due to a mutated biotinylation motif. We demonstrate that BADC proteins significantly inhibit ACCase activity in both E. coli and Arabidopsis. Targeted gene silencing of BADC isoform 1 in Arabidopsis significantly increased seed oil content when normalized to either mass or individual seed. We conclude the BADC proteins are ancestral BCCPs that gained a new function as negative regulators of ACCase after initial loss of the biotinylation motif. A functional model is proposed.

Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires.

Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO (where RalFLLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.

Pyridoxamine is a substrate of the energy-coupling factor transporter HmpT

Energy-coupling factor (ECF) transporters belong to a novel family of proteins that forms a subset within the ATP-binding cassette (ABC) transporter family. These proteins are responsible for the uptake of micronutrients in bacteria. ECF transporters are composed of four proteins: the A- and A′-components, the T-component and the S-component. One of the ECF transporters, named HmpT, was crystallized in the apo form with all four components. It is currently unknown whether HmpT serves as a transporter for hydroxymethyl pyrimidine or the different forms of vitamin B6 (pyridoxine, pyridoxal or pyridoxamine). Using a combination of molecular dynamics (MD) simulations and mass spectrometry, we have identified pyridoxamine to be the preferred substrate of HmpT. Mass spectra show that the mass of the substrate from the HmpT–substrate complex matches that of pyridoxamine. MD simulations likewise indicate that pyridoxamine interacts most strongly with most of the conserved residues of the S-component (Glu 41, His 84 and Gln 43) compared with the other vitamin B6 forms. Furthermore, the simulations have implied that loops 1 and 5 of the S-component can participate in the gating action for HmpT.

Editorial: RNA in bacteria: biogenesis, regulatory mechanisms and functions

The advent of high-throughput computational and RNA sequencing approaches in the past decade has highlighted an increasing number of RNA molecules in bacteria with critical functions in metabolic, physiological and virulence processes. Accordingly, proteins that interact with RNA have also benefited from a regained interest. A multitude of mechanisms involved in the regulation of gene expression by RNA molecules and in the interaction of RNAs with molecular partners have been described. There is a growing curiosity in understanding how RNA interferes or interacts with target DNA, RNA and proteins, and in the identification and characterization of nucleases, RNA helicases or RNA chaperones responsible for the activation, stability, decay or modes of action of RNA molecules. Remarkably, the recent analysis of CRISPR-Cas systems as RNA-mediated adaptive immune pathways in bacteria and archaea has resulted in the identification of novel families of proteins that function alone or in complexes to interact with RNA and/or DNA, and novel mechanisms of nucleic acid-based interference herewith. Information is now available on a large and still growing number of bacterial RNA molecules, the targets and functions of which are largely unknown. Given the enormous heterogeneity among different bacteria and the constant pressure for adaptation to multiple stresses and environments, we expect to discover a wide variety of novel RNAs with regulatory functions and associated proteins. We predict exciting findings and innovation in the field of RNA research in bacteria for a number of years to come. In this issue, FEMS Microbiology Reviews has selected a series of articles that highlight some of the recent findings on RNA biology in bacteria focusing on RNA biogenesis, modes of action, regulatory mechanisms and functions. The continuous effort to adapt innovative technologies and biological approaches to the bacterial RNA field has advanced our understanding of how regulatory RNAs and their … [↵][1]* Corresponding editors. E-mail: emmanuelle.charpentier{at}helmholtz-hzi.de and wolfgang.hess{at}biologie.uni-freiburg.de [1]: #xref-corresp-1-1

FAM129B, a Novel Protein, Suppresses the TNFα Apoptotic pathway and Promotes the NF‐kB Survival Pathway in Cancer Cells by Interacting with KEAP1

FAM129B, is a member of a family of novel proteins that include FAM129A and FAM129C.Little is known about the structure and functions of these proteins. Previously; we have shown that FAM129B suppresses the TNFα induced apoptotic pathway in HeLa cells (Song, Evans, & Evans (2010), JBC 286, 10201‐09). To elucidate the mechanism by which FAM129B suppresses this apoptotic pathway, we investigated the effect of FAM129B on several components of the TNFα apoptotic pathway: IkB kinase (IKKβ), the transcription factor NF‐kB, and the anti‐apoptotic proteins c‐FLIP, and cIAP. Silencing FAM129B expression using siRNA in HeLa cells promoted IKKβ degradation and decreased NF‐kB translocation to the nucleus and the expression of c‐FLIP and cIAP following induction of apoptosis. Also, over‐expression of recombinant FAM129B in HEK293 cells decreased IKKβ degradation, thus promoting NF‐kb activation. We also show that KEAP1, a protein that promotes IKKβ degradation, forms a complex with FAM129B in vivo and in vitro. The ETGE motif in FAM129B and the KELCH repeats in KEAP1 are the sites of interaction between the two proteins. Using site directed mutagenesis, we changed the ETGE motif into ETAA. This mutation disrupted the interaction with KEAP1 in vivo and in vitro .Over expression of recombinant FAM129B with the mutated ETGE motif in HEK293 cells, in contrast to the wild type protein, did not suppress apoptosis, inhibit IKKβ degradation or activate NF‐κB.We propose that FAM‐129B sequesters KEAP‐1, and blocks apoptosis by inhibiting the NF‐κB signaling pathway and the expression of anti‐apoptotic proteins.

Distribution in microbial genomes of genes similar to lodA and goxA which encode a novel family of quinoproteins with amino acid oxidase activity.

BackgroundL-Amino acid oxidases (LAOs) have been generally described as flavoproteins that oxidize amino acids releasing the corresponding ketoacid, ammonium and hydrogen peroxide. The generation of hydrogen peroxide gives to these enzymes antimicrobial characteristics. They are involved in processes such as biofilm development and microbial competition. LAOs are of great biotechnological interest in different applications such as the design of biosensors, biotransformations and biomedicine.The marine bacterium Marinomonas mediterranea synthesizes LodA, the first known LAO that contains a quinone cofactor. LodA is encoded in an operon that contains a second gene coding for LodB, a protein required for the post-translational modification generating the cofactor. Recently, GoxA, a quinoprotein with sequence similarity to LodA but with a different enzymatic activity (glycine oxidase instead of lysine-ε-oxidase) has been described. The aim of this work has been to study the distribution of genes similar to lodA and/or goxA in sequenced microbial genomes and to get insight into the evolution of this novel family of proteins through phylogenetic analysis.ResultsGenes encoding LodA-like proteins have been detected in several bacterial classes. However, they are absent in Archaea and detected only in a small group of fungi of the class Agaromycetes. The vast majority of the genes detected are in a genome region with a nearby lodB-like gene suggesting a specific interaction between both partner proteins.Sequence alignment of the LodA-like proteins allowed the detection of several conserved residues. All of them showed a Cys and a Trp that aligned with the residues that are forming part of the cysteine tryptophilquinone (CTQ) cofactor in LodA. Phylogenetic analysis revealed that LodA-like proteins can be clustered in different groups. Interestingly, LodA and GoxA are in different groups, indicating that those groups are related to the enzymatic activity of the proteins detected.ConclusionsGenome mining has revealed for the first time the broad distribution of LodA-like proteins containing a CTQ cofactor in many different microbial groups. This study provides a platform to explore the potentially novel enzymatic activities of the proteins detected, the mechanisms of post-translational modifications involved in their synthesis, as well as their biological relevance.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1455-y) contains supplementary material, which is available to authorized users.

GK4, a G‐protein‐coupled receptor with a phosphatidylinositol phosphate kinase domain in Phytophthora infestans, is involved in sporangia development and virulence

For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G-protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven-transmembrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.

Orai channels – New insights, new ideas

The basic mechanism of agonist-activated Ca2+ entry in non-excitable cells was first defined by Jim Putney some 25 years ago (Putney, 1986). This mechanism of ‘capacitative’, or store-operated, Ca2+ entry described how such entry was strictly a consequence of the depletion of intracellular Ca2+ stores, typically those formed by the endoplasmic reticulum (ER). During agonist action such depletion would normally result from the generation of InsP3, and the resulting release of ER Ca2+, but the essential dependence on store depletion per se was demonstrated by the fact that the same entry could be induced by any means that resulted in a corresponding decline in the concentration of free Ca2+ in the ER, including inhibition of SERCA pumps, or increasing Ca2+ buffer levels in the ER. Subsequently, a highly Ca2+-selective conductance representing such an entry pathway was identified, initially in mast cells and T lymphocytes, and its essential biophysical properties described and defined (Hoth & Penner, 1992; Zweifach & Lewis, 1993). This was the CRAC channel (Ca2+ release-activated Ca2+ channel). However, despite extensive analysis of the properties and behaviour of these channels, their molecular identity and the actual processes that link the depletion of intracellular Ca2+ stores with activation of the channels remained unknown for almost 15 years. All of this changed dramatically with a series of papers that appeared in 2005 and 2006. First, the protein STIM1 was identified as the molecule linking the detection of Ca2+ store depletion with the subsequent activation of the CRAC channels (Liou et al. 2005; Roos et al. 2005). Then, a year later, a novel family of proteins named Orai proteins were shown to form the channels themselves (Feske et al. 2006; Vig et al. 2006; Zhang et al. 2006). The resulting surge of new findings that resulted from this identification of the molecular basis of store-operated Ca2+ entry was nicely summarized in a symposium organized by Anant Parekh and sponsored by The Journal of Physiology, which was held at the 52nd Annual Biophysical Society meeting at Long Beach, California in 2008.

Different expression patterns of Phactr family members in normal and injured mouse brain

Phosphatase and actin regulators (Phactrs) are a novel family of proteins expressed in the brain, and they exhibit both strong modulatory activity of protein phosphatase 1 and actin-binding activity. Phactrs are comprised of four family members (Phactr1–4), but their detailed expression patterns during embryonic and postnatal development are not well understood. We found that these family members exhibit different spatiotemporal mRNA expression patterns. Phactr4 mRNA was found in neural stem cells in the developing and adult brains, whereas Phactr1 and 3 appeared to be expressed in post-mitotic neurons. Following traumatic brain injury which promotes neurogenesis in the neurogenic region and gliogenesis in the injury penumbra, the mRNA expression of phactr2 and 4 was progressively increased in the injury penumbra, and phactr4 mRNA and protein induction was observed in reactive astrocytes. These differential expression patterns of phactrs imply specific functions for each protein during development, and the importance of Phactr4 in the reactive gliosis following brain injury.

Helicobacter pylori Has an Unprecedented Nitric Oxide Detoxifying System

The ability of pathogens to cope with the damaging effects of nitric oxide (NO), present in certain host niches and produced by phagocytes that support innate immunity, relies on multiple strategies that include the action of detoxifying enzymes. As for many other pathogens, these systems remained unknown for Helicobacter pylori. This work aimed at identifying and functionally characterizing an H. pylori system involved in NO protection. In the present work, the hp0013 gene of H. pylori is shown to be related to NO resistance, as its inactivation increases the susceptibility of H. pylori to nitrosative stress, and significantly decreases the NADPH-dependent NO reduction activity of H. pylori cells. The recombinant HP0013 protein is able to complement an NO reductase-deficient Escherichia coli strain and exhibits significant NO reductase activity. Mutation of hp0013 renders H. pylori more vulnerable to nitric oxide synthase-dependent macrophage killing, and decreases the ability of the pathogen to colonize mice stomachs. Phylogenetic studies reveal that HP0013, which shares no significant amino acid sequence similarity to the other so far known microbial NO detoxifiers, belongs to a novel family of proteins with a widespread distribution in the microbial world. H. pylori HP0013 represents an unprecedented enzymatic NO detoxifying system for the in vivo microbial protection against nitrosative stress.

The highly conserved spermatophyte cell wall DUF642 protein family: Phylogeny and first evidence of interaction with cell wall polysaccharides in vitro

The evolution of spermatophyte plants involved fundamental changes in cell wall structure and function which resulted from diversification of carbohydrates and proteins. Cell wall proteomic analyses identified a novel family of proteins of yet unknown function, the DUF642 (Domain of Unknown Function 642) proteins. To investigate the evolution of the DUF642 gene family, 154 gene sequences from 24 plant species were analyzed, and phylogenetic inferences were conducted using the Maximum Likelihood and Bayesian Inference methods. Orthologous genes were detected in spermatophyte species and absent in non-seed known plant genomes. Protein sequences shared conserved motifs that defined the signature of the family. Distribution of conserved motifs indicated an ancestral intragenic duplication event. Gene phylogeny documented paleoduplication events originating three or four clades, depending on root position. When based on mid-point rooting, it retrieved four monophyletic clades: A, B, C, and D. A glycosylphosphatidylinositol (GPI)-anchor site and one or two galactose-binding domains-like (GBDLs) could be predicted for some DUF642 proteins. The B, C, and D clades grouped the predicted GPI-anchored proteins. First evidence of in vitro interaction of a DUF642 protein with a cell wall polysaccharide fraction is provided. A competition assay with cellulose prevented this interaction. The degree of diversification and the conservation of the family suggested that DUF642 proteins are key components in seed plant evolution.

Abstract IA21: Role of Tet1-catalyzed 5mC hydroxylation in transcriptional regulation and ES cell maintenance

Abstract Epigenetic modifications play important roles in diverse biological processes that range from regulation of gene expression, embryonic development, stem cell reprogramming, and human diseases such as cancers. One of the epigenetic modifications is DNA methylation. Although enzymes responsible for DNA methylation have been well characterized, enzymes responsible for active DNA demethylation in mammalian cells have remained elusive. Recent studies have demonstrated that a novel family of proteins— Tet1–3—have the capacity to convert 5mC to 5hmC, raising the possibility that Tet proteins may catalyze the initial step leading to DNA demethylation. Interestingly, Tet1 plays an important role in stem cell self-renewal and maintenance. To understand how Tet1 contributes to these processes, we profiled the genome-wide location of Tet1 and 5hmC in ES cells. This study revealed that Tet1 has a dual function in activating pluripotency stem cell genes and silencing lineage-specific differentiation genes. Further studies revealed that Tet1 contributes to gene silencing by facilitating recruitment of PRC2. Thus, our studies establish a link between Tet1 and PRC2 and revealed a dual function for Tet1 and 5hmC in transcriptional regulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA21.

Phox homology band 4.1/ezrin/radixin/moesin-like proteins function as molecular scaffolds that interact with cargo receptors and Ras GTPases

Following endocytosis, the fates of receptors, channels, and other transmembrane proteins are decided via specific endosomal sorting pathways, including recycling to the cell surface for continued activity. Two distinct phox-homology (PX)-domain-containing proteins, sorting nexin (SNX) 17 and SNX27, are critical regulators of recycling from endosomes to the cell surface. In this study we demonstrate that SNX17, SNX27, and SNX31 all possess a novel 4.1/ezrin/radixin/moesin (FERM)-like domain. SNX17 has been shown to bind to Asn-Pro-Xaa-Tyr (NPxY) sequences in the cytoplasmic tails of cargo such as LDL receptors and the amyloid precursor protein, and we find that both SNX17 and SNX27 display similar affinities for NPxY sorting motifs, suggesting conserved functions in endosomal recycling. Furthermore, we show for the first time that all three proteins are able to bind the Ras GTPase through their FERM-like domains. These interactions place the PX-FERM-like proteins at a hub of endosomal sorting and signaling processes. Studies of the SNX17 PX domain coupled with cellular localization experiments reveal the mechanistic basis for endosomal localization of the PX-FERM-like proteins, and structures of SNX17 and SNX27 determined by small angle X-ray scattering show that they adopt non-self-assembling, modular structures in solution. In summary, this work defines a novel family of proteins that participate in a network of interactions that will impact on both endosomal protein trafficking and compartment specific Ras signaling cascades.

Molecular mechanisms of mechanotransduction in mammalian sensory neurons

The somatosensory system mediates fundamental physiological functions, including the senses of touch, pain and proprioception. This variety of functions is matched by a diverse array of mechanosensory neurons that respond to force in a specific fashion. Mechanotransduction begins at the sensory nerve endings, which rapidly transform mechanical forces into electrical signals. Progress has been made in establishing the functional properties of mechanoreceptors, but it has been remarkably difficult to characterize mechanotranducer channels at the molecular level. However, in the past few years, new functional assays have provided insights into the basic properties and molecular identity of mechanotransducer channels in mammalian sensory neurons. The recent identification of novel families of proteins as mechanosensing molecules will undoubtedly accelerate our understanding of mechanotransduction mechanisms in mammalian somatosensation.

Cell type specific expression of the apoptosis stimulating protein (ASPP-2) in human tissues

Apoptosis stimulating proteins of p53 (ASPP-l and ASPP-2) are a novel family of proteins that have been found to co-stimulate p53 activation of Bax (Bcl-2 associated protein X) inducing caspase-mediated apoptosis. Therefore, these proteins may play an important role in regulating apoptosis in normal and neoplastic cells. However, their cellular and tissue distribution has not been documented. The aim of this study was to determine the localization pattern of ASPP-2 in a variety of normal and malignant human tissues, including liver, lung, prostate, small intestine, kidney, ovary, bladder, cervix, breast, stomach, bowel, gallbladder, endometrium, pancreas, spleen and thyroid.The distribution and expression of ASPP-2 was assessed by immunohistochemistry in a range of formalin-fixed, paraffin embedded, benign and malignant human tissues, using a mouse monoclonal antibody against ASPP-2.The results showed a variable pattern of positivity of ASPP-2 within the tissues studied. ASPP-2 expression was localized in the cytoplasmic paranuclear granules in the epithelial cells of most of the organs we studied. The pattern of staining intensity of ASPP-2 correlated to the maturation state in benign tissue and to the differentiation state in the context of bladder cancer.This study indicates that ASPP-2 has a specific distribution pattern within tissues and cells in a way that appears to be related to differentiation. However, the patterns are neither simplistic nor straightforward and will require further investigation in order to appreciate fully their physiological/pathological significance.

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

BackgroundThymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages.Methodology/Principal FindingsHere we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo.Conclusions/SignificanceWe hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.