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Host Proteins (host + protein)
Terms modified by Host Proteins Selected AbstractsLow free energy cost of very long loop insertions in proteinsPROTEIN SCIENCE, Issue 2 2003Michelle Scalley-Kim Abstract Long insertions into a loop of a folded host protein are expected to have destabilizing effects because of the entropic cost associated with loop closure unless the inserted sequence adopts a folded structure with amino- and carboxy-termini in close proximity. A loop entropy reduction screen based on this concept was used in an attempt to retrieve folded sequences from random sequence libraries. A library of long random sequences was inserted into a loop of the SH2 domain, displayed on the surface of M13 phage, and the inserted sequences that did not disrupt SH2 function were retrieved by panning using beads coated with a phosphotyrosine containing SH2 peptide ligand. Two sequences of a library of 2 × 108 sequences were isolated after multiple rounds of panning, and were found to have recovery levels similar to the wild-type SH2 domain and to be relatively intolerant to further mutation in PCR mutagenesis experiments. Surprisingly, although these inserted sequences exhibited little nonrandom structure, they do not significantly destabilize the host SH2 domain. Additional insertion variants recovered at lower levels in the panning experiments were also found to have a minimal effect on the stability and peptide-binding function of the SH2 domain. The additional level of selection present in the panning experiments is likely to involve in vivo folding and assembly, as there was a rough correlation between recovery levels in the phage-panning experiments and protein solubility. The finding that loop insertions of 60,80 amino acids have minimal effects on SH2 domain stability suggests that the free energy cost of inserting long loops may be considerably less than polymer theory estimates based on the entropic cost of loop closure, and, hence, that loop insertion may have provided an evolutionary route to multidomain protein structures. [source] The N-terminal region of Pseudomonas type III effector AvrPtoB elicits Pto-dependent immunity and has two distinct virulence determinantsTHE PLANT JOURNAL, Issue 4 2007Fangming Xiao Summary Resistance to bacterial speck disease in tomato is activated by the physical interaction of the host Pto kinase with either of the sequence-dissimilar type III effector proteins AvrPto or AvrPtoB (HopAB2) from Pseudomonas syringae pv. tomato. Pto-mediated immunity requires Prf, a protein with a nucleotide-binding site and leucine-rich repeats. The N-terminal 307 amino acids of AvrPtoB were previously reported to interact with the Pto kinase, and we show here that this region (AvrPtoB1-307) is sufficient for eliciting Pto/Prf-dependent immunity against P. s. pv. tomato. AvrPtoB1-307 was also found to be sufficient for a virulence activity that enhances ethylene production and increases growth of P. s. pv. tomato and severity of speck disease on susceptible tomato lines lacking either Pto or Prf. Moreover, we found that residues 308,387 of AvrPtoB are required for the previously reported ability of AvrPtoB to suppress pathogen-associated molecular patterns-induced basal defenses in Arabidopsis. Thus, the N-terminal region of AvrPtoB has two structurally distinct domains involved in different virulence-promoting mechanisms. Random and targeted mutagenesis identified five tightly clustered residues in AvrPtoB1-307 that are required for interaction with Pto and for elicitation of immunity to P. s. pv. tomato. Mutation of one of the five clustered residues abolished the ethylene-associated virulence activity of AvrPtoB1-307. However, individual mutations of the other four residues, despite abolishing interaction with Pto and avirulence activity, had no effect on AvrPtoB1-307 virulence activity. None of these mutations affected the basal defense-suppressing activity of AvrPtoB1-387. Based on sequence alignments, estimates of helical propensity, and the previously reported structure of AvrPto, we hypothesize that the Pto-interacting domains of AvrPto and AvrPtoB1-307 have structural similarity. Together, these data support a model in which AvrPtoB1-307 promotes ethylene-associated virulence by interaction not with Pto but with another unknown host protein. [source] A Robust Protein Host for Anchoring Chelating Ligands and OrganocatalystsCHEMBIOCHEM, Issue 4 2008Manfred T. Reetz Prof. Dr. Abstract In order to put the previously proposed concept of directed evolution of hybrid catalysts (proteins that harbor synthetic transition-metal catalysts or organocatalysts) into practice, several prerequisites must be met. The availability of a robust host protein that can be expressed in sufficiently large amounts, and that can be purified in a simple manner is crucial. The thermostable enzyme tHisF from Thermotoga maritima, which constitutes the synthase subunit of a bi-enzyme complex that is instrumental in the biosynthesis of histidine, fulfills these requirements. In the present study, fermentation has been miniaturized and parallelized, as has purification of the protein by simple heat treatment. Several mutants with strategically placed cysteines for subsequent bioconjugation have been produced. One of the tHisF mutants, Cys9Ala/Asp11Cys, was subjected to bioconjugation by the introduction of a variety of ligands for potential metal ligation, of a ligand/metal moiety, and of several organocatalytic entities that comprise a flavin or thiazolium salts. Characterization by mass spectrometry and tryptic digestion was achieved. As a result of this study, a platform for performing future directed evolution of these hybrid catalysts is now available. [source] A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situFEBS JOURNAL, Issue 12 2008Fanjie Meng To measure mechanical stress in real time, we designed a fluorescence resonance energy transfer (FRET) cassette, denoted stFRET, which could be inserted into structural protein hosts. The probe was composed of a green fluorescence protein pair, Cerulean and Venus, linked with a stable ,-helix. We measured the FRET efficiency of the free cassette protein as a function of the length of the linker, the angles of the fluorophores, temperature and urea denaturation, and protease treatment. The linking helix was stable to 80 °C, unfolded in 8 m urea, and rapidly digested by proteases, but in all cases the fluorophores were unaffected. We modified the ,-helix linker by adding and subtracting residues to vary the angles and distance between the donor and acceptor, and assuming that the cassette was a rigid body, we calculated its geometry. We tested the strain sensitivity of stFRET by linking both ends to a rubber sheet subjected to equibiaxial stretch. FRET decreased proportionally to the substrate strain. The naked cassette expressed well in human embryonic kidney-293 cells and, surprisingly, was concentrated in the nucleus. However, when the cassette was located into host proteins such ,-actinin, nonerythrocyte spectrin and filamin A, the labeled hosts expressed well and distributed normally in cell lines such as 3T3, where they were stressed at the leading edge of migrating cells and relaxed at the trailing edge. When collagen-19 was labeled near its middle with stFRET, it expressed well in Caenorhabditis elegans, distributing similarly to hosts labeled with a terminal green fluorescent protein, and the worms behaved normally. [source] Immunization of mice with Lactobacillus casei expressing intimin fragments produces antibodies able to inhibit the adhesion of enteropathogenic Escherichia coli to cultivated epithelial cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2008Patrícia C.D. Ferreira Abstract Enteropathogenic Escherichia coli (EPEC) are frequently isolated as a cause of infantile diarrhea in developing countries. Its pathogenicity is distinguished by histopathological alterations at the site of infection, known as attaching and effacing (A/E) lesions, in which bacterial virulence factors and host proteins participate. Intimin, a bacterial adhesin expressed by all EPEC described to date, is responsible for the intimate adherence of the bacteria to host cells and is essential for the formation of A/E lesions. Mucosal vaccination may represent an efficacious intervention to prevent EPEC infection and lower morbidity and mortality rates. Strategies for mucosal vaccinations that use lactic acid bacteria for the delivery of heterologous antigens rely on their safety profile and ability to stimulate the immune system. In the present work, we have constructed Lactobacillus casei strains expressing different fragments of intimin ,, a subtype that is frequently expressed by EPEC strains. Mucosal immunization of mice with L. casei expressing intimin fragments induced specific systemic and mucosal antibodies. These antibodies were able to recognize native intimin on the surface of EPEC and to inhibit in vitro EPEC binding to epithelial cells. [source] A teratocyte gene from a parasitic wasp that is associated with inhibition of insect growth and development inhibits host protein synthesisINSECT MOLECULAR BIOLOGY, Issue 5 2003D. L. Dahlman Abstract After parasitization, some wasps induce hosts prematurely to initiate metamorphic development that is then suspended in a postwandering, prepupal state. Following egression of the parasite larva, the host remains in this developmentally arrested state until death. Teratocytes, cells released at egg hatch from extra-embryonic serosal membranes of some wasp parasites, inhibit growth and development when injected into host larvae independent of other parasite factors (e.g. venom, polydnavirus). Synthesis of some developmentally regulated, abundantly expressed Heliothis virescens host proteins is inhibited in hosts parasitized by Microplitis croceipes and by teratocyte injection. A cDNA encoding a 13.9 kDa protein (TSP14) that inhibited protein synthesis, growth and development was isolated from a protein fraction secreted by teratocytes. TSP14 appears to be responsible, in part, for the teratocyte-mediated inhibition of host growth and development. Interestingly, this cDNA encoded a cysteine-rich amino acid motif similar to that described from Campoletis sonorensis polydnavirus, a mutualistic virus that enables wasp parasitization of lepidopteran larvae. Moreover, TSP14 inhibited protein synthesis in a dose-dependent manner in rabbit reticulocyte lysate and wheat germ extract translation systems. We hypothesize that some wasp parasites inhibit translation as a general means to regulate and redirect lepidopteran host physiology to support endoparasite development. [source] Autonomous plasmid-like replication of a conjugative transposonMOLECULAR MICROBIOLOGY, Issue 2 2010Catherine A. Lee Summary Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra-chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid-like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1 -encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid-like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations. [source] Escherichia coli Hsp31 functions as a holding chaperone that cooperates with the DnaK-DnaJ-GrpE system in the management of protein misfolding under severe stress conditionsMOLECULAR MICROBIOLOGY, Issue 3 2004Mirna Mujacic Summary Escherichia coli Hsp31 is a homodimeric protein that exhibits chaperone activity in vitro and is a representative member of a recently recognized family of heat shock proteins (Hsps). To gain insights on Hsp31 cellular function, we deleted the hchA gene from the MC4100 chromosome and combined the resulting null allele with lesions in other cytoplasmic chaperones. Although the hchA mutant only exhibited growth defects when cultivated at 48°C, loss of Hsp31 had a strong deleterious effect on the ability of cells to survive and recover from transient exposure to 50°C, and led to the enhanced aggregation of a subset of host proteins at this temperature. The absence of Hsp31 did not significantly affect the ability of the ClpB-DnaK-DnaJ-GrpE system to clear thermally aggregated proteins at 30°C suggesting that Hsp31 does not possess disaggregase activity. Although it had no effect on the growth of groES30, ,clpB or ,ibpAB cells at high temperatures, the hchA deletion aggravated the temperature sensitive phenotype of dnaK756 and grpE280 mutants and led to increased aggregation in stressed dnaK756 cells. On the basis of biochemical, structural and genetic data, we propose that Hsp31 acts as a modified holding chaperone that captures early unfolding intermediates under prolonged conditions of severe stress and releases them when cells return to physiological conditions. This additional line of defence would complement the roles of DnaK-DnaJ-GrpE, ClpB and IbpB in the management of thermally induced cellular protein misfolding. [source] Post-translational modification of host proteins in pathogen-triggered defence signalling in plantsMOLECULAR PLANT PATHOLOGY, Issue 4 2008IRIS J. E. STULEMEIJER SUMMARY Microbial plant pathogens impose a continuous threat to global food production. Similar to animals, an innate immune system allows plants to recognize pathogens and swiftly activate defence. To activate a rapid response, receptor-mediated pathogen perception and subsequent downstream signalling depends on post-translational modification (PTM) of components essential for defence signalling. We discuss different types of PTMs that play a role in mounting plant immunity, which include phosphorylation, glycosylation, ubiquitination, sumoylation, nitrosylation, myristoylation, palmitoylation and glycosylphosphatidylinositol (GPI)-anchoring. PTMs are rapid, reversible, controlled and highly specific, and provide a tool to regulate protein stability, activity and localization. Here, we give an overview of PTMs that modify components essential for defence signalling at the site of signal perception, during secondary messenger production and during signalling in the cytoplasm. In addition, we discuss effectors from pathogens that suppress plant defence responses by interfering with host PTMs. [source] Host,pathogen protein interactions predicted by comparative modelingPROTEIN SCIENCE, Issue 12 2007Fred P. Davis Abstract Pathogens have evolved numerous strategies to infect their hosts, while hosts have evolved immune responses and other defenses to these foreign challenges. The vast majority of host,pathogen interactions involve protein,protein recognition, yet our current understanding of these interactions is limited. Here, we present and apply a computational whole-genome protocol that generates testable predictions of host,pathogen protein interactions. The protocol first scans the host and pathogen genomes for proteins with similarity to known protein complexes, then assesses these putative interactions, using structure if available, and, finally, filters the remaining interactions using biological context, such as the stage-specific expression of pathogen proteins and tissue expression of host proteins. The technique was applied to 10 pathogens, including species of Mycobacterium, apicomplexa, and kinetoplastida, responsible for "neglected" human diseases. The method was assessed by (1) comparison to a set of known host,pathogen interactions, (2) comparison to gene expression and essentiality data describing host and pathogen genes involved in infection, and (3) analysis of the functional properties of the human proteins predicted to interact with pathogen proteins, demonstrating an enrichment for functionally relevant host,pathogen interactions. We present several specific predictions that warrant experimental follow-up, including interactions from previously characterized mechanisms, such as cytoadhesion and protease inhibition, as well as suspected interactions in hypothesized networks, such as apoptotic pathways. Our computational method provides a means to mine whole-genome data and is complementary to experimental efforts in elucidating networks of host,pathogen protein interactions. [source] Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone SkpPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23-24 2008Svenja Jarchow Abstract The "seventeen kilodalton protein" (Skp) is a predominant periplasmic chaperone of Escherichia coli, which is involved in the biogenesis of abundant outer membrane proteins (OMPs) such as OmpA, PhoE, and LamB. In this study the substrate profile of Skp was investigated in a proteomics approach. Skp was overexpressed in a deficient E. coli strain as a fusion protein with the Strep,tag and captured, together with any host proteins associated with it, from the periplasmic cell extract under mild conditions via one-step Strep,Tactin affinity chromatography. Copurified substrate proteins were then identified by high resolution 2-DE with immobilized pH-gradients, followed by MALDI-TOF MS. Apart from the known Skp substrates, including OmpA and LamB, more than 30 other interacting proteins were detected, especially from the outer membrane, among these FadL and BtuB, and from the periplasm such as MalE and OppA. Thus, Skp does not only serve as a specialized chaperone for a small set of OMPs, but it seems to exhibit a broader substrate spectrum, including soluble periplasmic proteins. These findings should prompt further investigation into the physiological role of Skp and may promote its use for the bacterial production of biochemically active heterologous proteins whose folding requires secretion into the oxidizing milieu of the periplasm. [source] Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and ,-enolase: Implications for autoimmunity in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 9 2010Natalia Wegner Objective To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA). Methods The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis,knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and ,-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry. Results Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or ,-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry. Conclusion Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis,mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA. [source] Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCR, fragmentsACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2010Walter M. Kim HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling , subunit of the T-cell receptor (TCR,). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCR, fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCR, polypeptide (Leu51,Asp93) was determined to 3.7,Ĺ resolution (Rwork = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCR, polypeptide (Ala63,Arg80) was determined to 2.05,Ĺ resolution (Rwork = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCR, polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, ,l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning. [source] Enemy at the gates: traffic at the plant cell pathogen interfaceCELLULAR MICROBIOLOGY, Issue 12 2008Caroline Hoefle Summary The plant apoplast constitutes a space for early recognition of potentially harmful non-self. Basal pathogen recognition operates via dynamic sensing of conserved microbial patterns by pattern recognition receptors or of elicitor-active molecules released from plant cell walls during infection. Recognition elicits defence reactions depending on cellular export via SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex-mediated vesicle fusion or plasma membrane transporter activity. Lipid rafts appear also involved in focusing immunity-associated proteins to the site of pathogen contact. Simultaneously, pathogen effectors target recognition, apoplastic host proteins and transport for cell wall-associated defence. This microreview highlights most recent reports on the arms race for plant disease and immunity at the cell surface. [source] Proteases in pathogenesis and plant defenceCELLULAR MICROBIOLOGY, Issue 10 2004Yiji Xia Summary Plant pathogens deliver a variety of virulence factors to host cells to suppress basal defence responses and create suitable environments for their propagation. Plants have in turn evolved disease resistance genes whose products detect the virulence factors as a signal of invasion and activate effective defence responses. Understanding how a virulence effector contributes to virulence on susceptible hosts but becomes an avirulence factor that triggers defence responses on resistance hosts has been a major focus in plant research. Recent studies have shown that a growing list of pathogen-encoded effectors functions as proteases that are secreted into plant cells to modify host proteins. In addition, several plant proteases have been found to function in activation of the defence mechanism. These findings reveal that post-translational modification of host proteins through proteolytic processing is a widely used mechanism in regulating the plant defence response. [source] | ||||||||||||||||