ResearchPad - membrane-proteins https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[The <i>Caenorhabditis elegans</i> CUB-like-domain containing protein RBT-1 functions as a receptor for <i>Bacillus thuringiensis</i> Cry6Aa toxin]]> https://www.researchpad.co/article/elastic_article_14753 Bacillus thuringiensis (Bt) crystal proteins belong to pore-forming toxins (PFTs), which display virulence against target hosts by forming holes in the cell membrane. Cry6A is a nematicidal PFT, which exhibits unique protein structure and different mode of action than Cry5B, another nematicidal PFT. However, little is known about the mode of action of Cry6A. Although an intracellular nematicidal necrosis pathway of Cry6A was reported, its extracellular mode of action remains unknown. We here demonstrate that the CUB-like-domain containing protein RBT-1 acts as a functional receptor of Cry6A, which mediates the intestinal cell interaction and nematicidal activity of this toxin. RBT-1 represents a new class of crystal protein receptors. RBT-1 is dispensable for Cry5B toxicity against nematodes, consistent with that Cry6A and Cry5B have different nematicidal mechanisms. We also find that Cry6A kills nematodes by complex mechanism since rbt-1 mutation did not affect Cry6A-mediated necrosis signaling pathway. This work not only enhances the understanding of Bt crystal protein-nematode mechanism, but is also in favor for the application of Cry6A in nematode control.

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<![CDATA[Host interactors of effector proteins of the lettuce downy mildew <i>Bremia lactucae</i> obtained by yeast two-hybrid screening]]> https://www.researchpad.co/article/elastic_article_13834 Plant pathogenic bacteria, fungi and oomycetes secrete effector proteins to manipulate host cell processes to establish a successful infection. Over the last decade the genomes and transcriptomes of many agriculturally important plant pathogens have been sequenced and vast candidate effector repertoires were identified using bioinformatic analyses. Elucidating the contribution of individual effectors to pathogenicity is the next major hurdle. To advance our understanding of the molecular mechanisms underlying lettuce susceptibility to the downy mildew Bremia lactucae, we mapped physical interactions between B. lactucae effectors and lettuce candidate target proteins. Using a lettuce cDNA library-based yeast-two-hybrid system, 61 protein-protein interactions were identified, involving 21 B. lactucae effectors and 46 unique lettuce proteins. The top ten interactors based on the number of independent colonies identified in the Y2H and two interactors that belong to gene families involved in plant immunity, were further characterized. We determined the subcellular localization of the fluorescently tagged lettuce proteins and their interacting effectors. Importantly, relocalization of effectors or their interactors to the nucleus was observed for four protein-pairs upon their co-expression, supporting their interaction in planta.

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<![CDATA[The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases]]> https://www.researchpad.co/article/598bdfb5fa495b7488185485

Infection by enveloped coronaviruses (CoVs) initiates with viral spike (S) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound S proteins, which prompts S protein-mediated virus-cell membrane fusion. Infection therefore requires close proximity of receptors and proteases. We considered whether tetraspanins, scaffolding proteins known to facilitate CoV infections, hold receptors and proteases together on cell membranes. Using knockout cell lines, we found that the tetraspanin CD9, but not the tetraspanin CD81, formed cell-surface complexes of dipeptidyl peptidase 4 (DPP4), the MERS-CoV receptor, and the type II transmembrane serine protease (TTSP) member TMPRSS2, a CoV-activating protease. This CD9-facilitated condensation of receptors and proteases allowed MERS-CoV pseudoviruses to enter cells rapidly and efficiently. Without CD9, MERS-CoV viruses were not activated by TTSPs, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. Thus, we identified DPP4:CD9:TTSP as the protein complexes necessary for early, efficient MERS-CoV entry. To evaluate the importance of these complexes in an in vivo CoV infection model, we used recombinant Adenovirus 5 (rAd5) vectors to express human DPP4 in mouse lungs, thereby sensitizing the animals to MERS-CoV infection. When the rAd5-hDPP4 vectors co-expressed small RNAs silencing Cd9 or Tmprss2, the animals were significantly less susceptible, indicating that CD9 and TMPRSS2 facilitated robust in vivo MERS-CoV infection of mouse lungs. Furthermore, the S proteins of virulent mouse-adapted MERS-CoVs acquired a CD9-dependent cell entry character, suggesting that CD9 is a selective agent in the evolution of CoV virulence.

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<![CDATA[Analysis of Epichloë festucae small secreted proteins in the interaction with Lolium perenne]]> https://www.researchpad.co/article/5c6dc9b2d5eed0c48452a041

Epichloë festucae is an endophyte of the agriculturally important perennial ryegrass. This species systemically colonises the aerial tissues of this host where its growth is tightly regulated thereby maintaining a mutualistic symbiotic interaction. Recent studies have suggested that small secreted proteins, termed effectors, play a vital role in the suppression of host defence responses. To date only a few effectors with important roles in mutualistic interactions have been described. Here we make use of the fully assembled E. festucae genome and EffectorP to generate a suite of 141 effector candidates. These were analysed with respect to their genome location and expression profiles in planta and in several symbiosis-defective mutants. We found an association between effector candidates and a class of transposable elements known as MITEs, but no correlation with other dynamic features of the E. festucae genome, such as transposable element-rich regions. Three effector candidates and a small GPI-anchored protein were chosen for functional analysis based on their high expression in planta compared to in culture and their differential regulation in symbiosis defective E. festucae mutants. All three candidate effector proteins were shown to possess a functional signal peptide and two could be detected in the extracellular medium by western blotting. Localization of the effector candidates in planta suggests that they are not translocated into the plant cell, but rather, are localized in the apoplastic space or are attached to the cell wall. Deletion and overexpression of the effector candidates, as well as the putative GPI-anchored protein, did not affect the plant growth phenotype or restrict growth of E. festucae mutants in planta. These results indicate that these proteins are either not required for the interaction at the observed life stages or that there is redundancy between effectors expressed by E. festucae.

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<![CDATA[RPGRIP1L is required for stabilizing epidermal keratinocyte adhesion through regulating desmoglein endocytosis]]> https://www.researchpad.co/article/5c58d63dd5eed0c48403195b

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.

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<![CDATA[New interfaces on MiD51 for Drp1 recruitment and regulation]]> https://www.researchpad.co/article/5c5ca2b5d5eed0c48441e93f

Mitochondrial fission is facilitated by dynamin-related protein Drp1 and a variety of its receptors. However, the molecular mechanism of how Drp1 is recruited to the mitochondrial surface by receptors MiD49 and MiD51 remains elusive. Here, we showed that the interaction between Drp1 and MiD51 is regulated by GTP binding and depends on the polymerization of Drp1. We identified two regions on MiD51 that directly bind to Drp1, and found that dimerization of MiD51, relevant to residue C452, is required for mitochondrial dynamics regulation. Our Results have suggested a multi-faceted regulatory mechanism for the interaction between Drp1 and MiD51 that illustrates the potentially complicated and tight regulation of mitochondrial fission.

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<![CDATA[Collective radioresistance of T47D breast carcinoma cells is mediated by a Syncytin-1 homologous protein]]> https://www.researchpad.co/article/5c5b524ad5eed0c4842bc5fc

It is generally accepted that radiotherapy must target clonogenic cells, i.e., those cells in a tumour that have self-renewing potential. Focussing on isolated clonogenic cells, however, may lead to an underestimate or even to an outright neglect of the importance of biological mechanisms that regulate tumour cell sensitivity to radiation. We develop a new statistical and experimental approach to quantify the effects of radiation on cell populations as a whole. In our experiments, we change the proximity relationships of the cells by culturing them in wells with different shapes, and we find that the radiosensitivity of T47D human breast carcinoma cells in tight clusters is different from that of isolated cells. Molecular analyses show that T47D cells express a Syncytin-1 homologous protein (SyHP). We observe that SyHP translocates to the external surface of the plasma membrane of cells killed by radiation treatment. The data support the fundamental role of SyHP in the formation of intercellular cytoplasmic bridges and in the enhanced radioresistance of surviving cells. We conclude that complex and unexpected biological mechanisms of tumour radioresistance take place at the cell population level. These mechanisms may significantly bias our estimates of the radiosensitivity of breast carcinomas in vivo and thereby affect treatment plans, and they call for further investigations.

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<![CDATA[Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes]]> https://www.researchpad.co/article/5c61e8c6d5eed0c48496f14c

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.

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<![CDATA[Interaction between the transmembrane domains of Sho1 and Opy2 enhances the signaling efficiency of the Hog1 MAP kinase cascade in Saccharomyces cerevisiae]]> https://www.researchpad.co/article/5c57e68cd5eed0c484ef36b5

To cope with increased extracellular osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK), which controls a variety of adaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of a core MAPK cascade and two independent upstream branches (SHO1 and SLN1 branches) containing distinct osmosensing machineries. In the SHO1 branch, a homo-oligomer of Sho1, the four-transmembrane (TM) osmosensor, interacts with the transmembrane co-osmosensors, Hkr1 and Msb2, and the membrane anchor protein Opy2, through their TM domains, and activates the Ste20-Ste11-Pbs2-Hog1 kinase cascade. In this study, we isolated and analyzed hyperactive mutants of Sho1 and Opy2 that harbor mutations within their TM domains. Several hyperactive mutations enhanced the interaction between Sho1 and Opy2, indicating the importance of the TM-mediated interaction between Sho1 and Opy2 for facilitating effective signaling. The interaction between the TM domains of Sho1 and Opy2 will place their respective cytoplasmic binding partners Pbs2 and Ste11 in close proximity. Indeed, genetic analyses of the mutants showed that the Sho1-Opy2 interaction enhances the activation of Pbs2 by Ste11, but not Hog1 by Pbs2. Some of the hyperactive mutants had mutations at the extracellular ends of either Sho1 TM4 or Opy2 TM, and defined the Sho1-Opy2 binding site 1 (BS1). Chemical crosslinking and mutational analyses revealed that the cytoplasmic ends of Sho1 TM1 and Opy2 TM also interact with each other, defining the Sho1-Opy2 binding site 2 (BS2). A geometric consideration constrains that one Opy2 molecule must interact with two adjacent Sho1 molecules in Sho1 oligomer. These results raise a possibility that an alteration of the conformation of the Sho1-Opy2 complex might contributes to the osmotic activation of the Hog1 MAPK cascade.

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<![CDATA[Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells]]> https://www.researchpad.co/article/5c4b7f2dd5eed0c484840b72

Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult F. hepatica, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the F. hepatica EVs were resistant to exo- and endo-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by F. hepatica and sheds light on the molecular interaction between parasite EVs and host cells.

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<![CDATA[Structural basis of human ORP1-Rab7 interaction for the late-endosome and lysosome targeting]]> https://www.researchpad.co/article/5c63397ed5eed0c484ae68d3

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of lipid transfer proteins conserved in eukaryotes. ORP1 transports cholesterol at the interface between the late endosomes/lysosomes (LELs) and the endoplasmic reticulum (ER). ORP1 is targeted to the endosomal membranes by forming a tripartite complex with the LE GTPase Rab7 and its effector RILP (Rab7-interacting lysosomal protein). Here, we determined the crystal structure of human ORP1 ANK domain in complex with the GTP-bound form of Rab7. ORP1 ANK binds to the helix α3 of Rab7 located away from the switching regions, which makes the interaction independent of the nucleotide-binding state of Rab7. Thus, the effector-interacting switch regions of Rab7 are accessible for RILP binding, allowing formation of the ORP1-Rab7-RILP complex. ORP1 ANK binds to Rab7 and the Rab7-RILP complex with similar micro-molar affinities, which is consistent with the independence binding of ORP1 and RILP to Rab7. The structural model of the ORP1-Rab7-RILP complex correlates with the recruitment of ORP1 at the LEL-ER interface and the role in lipid transport and regulation.

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<![CDATA[Tobramycin reduces key virulence determinants in the proteome of Pseudomonas aeruginosa outer membrane vesicles]]> https://www.researchpad.co/article/5c57e695d5eed0c484ef385a

Tobramycin is commonly used to treat Pseudomonas aeruginosa lung infections in patients with Cystic Fibrosis (CF). Tobramycin treatment leads to increased lung function and fewer clinical exacerbations in CF patients, and modestly reduces the density of P. aeruginosa in the lungs. P. aeruginosa resides primarily in the mucus overlying lung epithelial cells and secretes outer membrane vesicles (OMVs) that diffuse through the mucus and fuse with airway epithelial cells, thus delivering virulence factors into the cytoplasm that modify the innate immune response. The goal of this study was to test the hypothesis that Tobramycin reduces the abundance of virulence factors in OMVs secreted by P. aeruginosa. Characterization of the proteome of OMVs isolated from control or Tobramycin-exposed P. aeruginosa strain PAO1 revealed that Tobramycin reduced several OMV-associated virulence determinants, including AprA, an alkaline protease that enhances P. aeruginosa survival in the lung, and is predicted to contribute to the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion by primary human bronchial epithelial cells. Deletion of the gene encoding AprA reduced the inhibitory effect of P. aeruginosa on Phe508del-CFTR Cl- secretion. Moreover, as predicted by our proteomic analysis, OMVs isolated from Tobramycin treated P. aeruginosa had a diminished inhibitory effect on Phe508del-CFTR Cl- secretion compared to OMVs isolated from control P. aeruginosa. Taken together, our proteomic analysis of OMVs and biological validation suggest that Tobramycin may improve lung function in CF patients infected with P. aeruginosa by reducing several key virulence factors in OMVs that reduce CFTR Cl- secretion, which is essential for bacterial clearance from the lungs.

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<![CDATA[Sugar and iron: Toward understanding the antibacterial effect of ciclopirox in Escherichia coli]]> https://www.researchpad.co/article/5c42438cd5eed0c4845e0573

New antibiotics are needed against antibiotic-resistant gram-negative bacteria. The repurposed antifungal drug, ciclopirox, equally blocks antibiotic-susceptible or multidrug-resistant Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates, indicating that it is not affected by existing resistance mechanisms. Toward understanding how ciclopirox blocks growth, we screened E. coli mutant strains and found that disruption of genes encoding products involved in galactose salvage, enterobacterial common antigen synthesis, and transport of the iron binding siderophore, enterobactin, lowered the minimum inhibitory concentration of ciclopirox needed to block growth of the mutant compared to the isogenic parent strain. We found that ciclopirox induced enterobactin production and that this effect is strongly affected by the deletion of the galactose salvage genes encoding UDP-galactose 4-epimerase, galE, or galactose-1-phosphate uridylyltransferase, galT. As disruption of ECA synthesis activates the regulation of capsular synthesis (Rcs) phosphorelay, which inhibits bacterial swarming and promotes biofilm development, we test whether ciclopirox prevents activation of the Rcs pathway. Sub-inhibitory concentrations of ciclopirox increased swarming of the E. coli laboratory K12 strain BW25113 but had widely varying effects on swarming or surface motility of clinical isolate E. coli, A. baumannii, and K. pneumoniae. There was no effect of ciclopirox on biofilm production, suggesting it does not target Rcs. Altogether, our data suggest ciclopirox-mediated alteration of lipopolysaccharides stimulates enterobactin production and affects bacterial swarming.

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<![CDATA[Plasma membrane architecture protects Candida albicans from killing by copper]]> https://www.researchpad.co/article/5c424382d5eed0c4845e0414

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

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<![CDATA[Computational characterization of the peptidome in transporter associated with antigen processing (TAP)-deficient cells]]> https://www.researchpad.co/article/5c478ca9d5eed0c484bd3c18

The transporter associated with antigen processing (TAP) is a key element of the major histocompatibility complex (MHC) class I antigen processing and presentation pathway. Nonfunctional TAP complexes impair the translocation of cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen. This drastic reduction in the available peptide repertoire leads to a significant decrease in MHC class I cell surface expression. Using mass spectrometry, different studies have analyzed the cellular MHC class I ligandome from TAP-deficient cells, but the analysis of the parental proteins, the source of these ligands, still deserves an in-depth analysis. In the present report, several bioinformatics protocols were applied to investigate the nature of parental proteins for the previously identified TAP-independent MHC class I ligands. Antigen processing in TAP-deficient cells mainly focused on small, abundant or highly integral transmembrane proteins of the cellular proteome. This process involved abundant proteins of the central RNA metabolism. In addition, TAP-independent ligands were preferentially cleaved from the N- and C-terminal ends with respect to the central regions of the parental proteins. The abundance of glycine, proline and aromatic residues in the C-terminal sequences from TAP-independently processed proteins allows the accessibility and specificity required for the proteolytic activities that generates the TAP-independent ligandome. This limited proteolytic activity towards a set of preferred proteins in a TAP-negative environment would therefore suffice to promote the survival of TAP-deficient individuals.

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<![CDATA[The apical protein Apnoia interacts with Crumbs to regulate tracheal growth and inflation]]> https://www.researchpad.co/article/5c478c97d5eed0c484bd34d1

Most organs of multicellular organisms are built from epithelial tubes. To exert their functions, tubes rely on apico-basal polarity, on junctions, which form a barrier to separate the inside from the outside, and on a proper lumen, required for gas or liquid transport. Here we identify apnoia (apn), a novel Drosophila gene required for tracheal tube elongation and lumen stability at larval stages. Larvae lacking Apn show abnormal tracheal inflation and twisted airway tubes, but no obvious defects in early steps of tracheal maturation. apn encodes a transmembrane protein, primarily expressed in the tracheae, which exerts its function by controlling the localization of Crumbs (Crb), an evolutionarily conserved apical determinant. Apn physically interacts with Crb to control its localization and maintenance at the apical membrane of developing airways. In apn mutant tracheal cells, Crb fails to localize apically and is trapped in retromer-positive vesicles. Consistent with the role of Crb in apical membrane growth, RNAi-mediated knockdown of Crb results in decreased apical surface growth of tracheal cells and impaired axial elongation of the dorsal trunk. We conclude that Apn is a novel regulator of tracheal tube expansion in larval tracheae, the function of which is mediated by Crb.

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<![CDATA[BRASSINOSTEROID-SIGNALING KINASE 3, a plasma membrane-associated scaffold protein involved in early brassinosteroid signaling]]> https://www.researchpad.co/article/5c3d00fcd5eed0c4840374aa

Brassinosteroids (BRs) are steroid hormones essential for plant growth and development. The BR signaling pathway has been studied in some detail, however, the functions of the BRASSINOSTEROID-SIGNALING KINASE (BSK) family proteins in the pathway have remained elusive. Through forward genetics, we identified five semi-dominant mutations in the BSK3 gene causing BSK3 loss-of-function and decreased BR responses. We therefore investigated the function of BSK3, a receptor-like cytoplasmic kinase, in BR signaling and plant growth and development. We find that BSK3 is anchored to the plasma membrane via N-myristoylation, which is required for its function in BR signaling. The N-terminal kinase domain is crucial for BSK3 function, and the C-terminal three tandem TPR motifs contribute to BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation. Interestingly, the effects of BSK3 on BR responses are dose-dependent, depending on its protein levels. Our genetic studies indicate that kinase dead BSK3K86R protein partially rescues the bsk3-1 mutant phenotypes. BSK3 directly interacts with the BSK family proteins (BSK3 and BSK1), BRI1 receptor kinase, BSU1 phosphatase, and BIN2 kinase. BIN2 phosphorylation of BSK3 enhances BSK3/BSK3 homodimer and BSK3/BSK1 heterodimer formation, BSK3/BRI1 interaction, and BSK3/BSU1 interaction. Furthermore, we find that BSK3 upregulates BSU1 transcript and protein levels to activate BR signaling. BSK3 is broadly expressed and plays an important role in BR-mediated root growth, shoot growth, and organ separation. Together, our findings suggest that BSK3 may function as a scaffold protein to regulate BR signaling. The results of our studies provide new insights into early BR signaling mechanisms.

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<![CDATA[The ESCRT and autophagy machineries cooperate to repair ESX-1-dependent damage at the Mycobacterium-containing vacuole but have opposite impact on containing the infection]]> https://www.researchpad.co/article/5c6059d0d5eed0c4847cbf91

Phagocytic cells capture and kill most invader microbes within the bactericidal phagosome, but some pathogens subvert killing by damaging the compartment and escaping to the cytosol. To prevent the leakage of pathogen virulence and host defence factors, as well as bacteria escape, host cells have to contain and repair the membrane damage, or finally eliminate the cytosolic bacteria. All eukaryotic cells engage various repair mechanisms to ensure plasma membrane integrity and proper compartmentalization of organelles, including the Endosomal Sorting Complex Required for Transport (ESCRT) and autophagy machineries. We show that during infection of Dictyostelium discoideum with Mycobacterium marinum, the ESCRT-I component Tsg101, the ESCRT-III protein Snf7/Chmp4/Vps32 and the AAA-ATPase Vps4 are recruited to sites of damage at the Mycobacterium-containing vacuole. Interestingly, damage separately recruits the ESCRT and the autophagy machineries. In addition, the recruitment of Vps32 and Vps4 to repair sterile membrane damage depends on Tsg101 but appears independent of Ca2+. Finally, in absence of Tsg101, M. marinum accesses prematurely the cytosol, where the autophagy machinery restricts its growth. We propose that ESCRT has an evolutionary conserved function to repair small membrane damage and to contain intracellular pathogens in intact compartments.

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<![CDATA[Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis]]> https://www.researchpad.co/article/5c390bb5d5eed0c48491df0f

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.

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<![CDATA[The Vaccinia virion: Filling the gap between atomic and ultrastructure]]> https://www.researchpad.co/article/5c3d00e1d5eed0c484036549

We have investigated the molecular-level structure of the Vaccinia virion in situ by protein-protein chemical crosslinking, identifying 4609 unique-mass crosslink ions at an effective FDR of 0.33%, covering 2534 unique pairs of crosslinked protein positions, 625 of which were inter-protein. The data were statistically non-random and rational in the context of known structures, and showed biological rationality. Crosslink density strongly tracked the individual proteolytic maturation products of p4a and p4b, the two major virion structural proteins, and supported the prediction of transmembrane domains within membrane proteins. A clear sub-network of four virion structural proteins provided structural insights into the virion core wall, and proteins VP8 and A12 formed a strongly-detected crosslinked pair with an apparent structural role. A strongly-detected sub-network of membrane proteins A17, H3, A27 and A26 represented an apparent interface of the early-forming virion envelope with structures added later during virion morphogenesis. Protein H3 seemed to be the central hub not only for this sub-network but also for an ‘attachment protein’ sub-network comprising membrane proteins H3, ATI, CAHH(D8), A26, A27 and G9. Crosslinking data lent support to a number of known interactions and interactions within known complexes. Evidence is provided for the membrane targeting of genome telomeres. In covering several orders of magnitude in protein abundance, this study may have come close to the bottom of the protein-protein crosslinkome of an intact organism, namely a complex animal virus.

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