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1.
《Journal of molecular biology》2022,434(2):167350
Maturation of adenoviruses is distinguished by proteolytic processing of several interior minor capsid proteins and core proteins by the adenoviral protease and subsequent reorganization of adenovirus core. We report the results derived from the icosahedrally averaged cryo-EM structure of a cell entry defective form of adenovirus, designated ts1, at a resolution of 3.7 Å as well as of the localized reconstructions of unique hexons and penton base. The virion structure revealed the structures and organization of precursors of minor capsid proteins, pIIIa, pVI and pVIII, which are closely associated with the hexons on the capsid interior. In addition to a well-ordered helical domain (a.a. 310–397) of pIIIa, highlights of the structure include the precursors of VIII display significantly different structures near the cleavage sites. Moreover, we traced residues 4–96 of the membrane lytic protein (pVI) that includes an amphipathic helix occluded deep in the hexon cavity suggesting the possibility of co-assembly of hexons with the precursors of VI. In addition, we observe a second copy of pVI ordered up to residue L40 in the peripentonal hexons and a few fragments of density corresponding to 2nd and 3rd copies of pVI in other hexons. However, we see no evidence of precursors of VII binding in the hexon cavity. These findings suggest the possibility that differently bound pVI molecules undergo processing at the N-terminal cleavage sites at varying efficiencies, subsequently creating competition between the cleaved and uncleaved forms of VI, followed by reorganization, processing, and release of VI molecules from the hexon cavities. 相似文献
2.
细胞自噬是进化上高度保守的细胞分解代谢途径. 在代谢应激下激活,产生双层膜结构的自噬小体,将胞浆内受损细胞器和蛋白质包裹、转运至溶酶体降解,维持细胞内环境平衡,是一种典型的细胞质量控制机制.目前,经典自噬通路中的主要蛋白质已经明确.但代谢应激信号的输入引起这些蛋白质怎样的活性和功能变化,这些变化对自噬产生怎样的影响,却是知之甚少.本文从翻译后修饰角度对代谢应激状态下自噬过程中相关蛋白质的调节进行综述,有助于深入了解自噬过程. 相似文献
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Comparative Genome Analysis Provides Insights into the Pathogenicity of Flavobacterium psychrophilum
Daniel Castillo Rói Hammershaimb Christiansen Inger Dalsgaard Lone Madsen Romilio Espejo Mathias Middelboe 《PloS one》2016,11(4)
Flavobacterium psychrophilum is a fish pathogen in salmonid aquaculture worldwide that causes cold water disease (CWD) and rainbow trout fry syndrome (RTFS). Comparative genome analyses of 11 F. psychrophilum isolates representing temporally and geographically distant populations were used to describe the F. psychrophilum pan-genome and to examine virulence factors, prophages, CRISPR arrays, and genomic islands present in the genomes. Analysis of the genomic DNA sequences were complemented with selected phenotypic characteristics of the strains. The pan genome analysis showed that F. psychrophilum could hold at least 3373 genes, while the core genome contained 1743 genes. On average, 67 new genes were detected for every new genome added to the analysis, indicating that F. psychrophilum possesses an open pan genome. The putative virulence factors were equally distributed among isolates, independent of geographic location, year of isolation and source of isolates. Only one prophage-related sequence was found which corresponded to the previously described prophage 6H, and appeared in 5 out of 11 isolates. CRISPR array analysis revealed two different loci with dissimilar spacer content, which only matched one sequence in the database, the temperate bacteriophage 6H. Genomic Islands (GIs) were identified in F. psychrophilum isolates 950106-1/1 and CSF 259–93, associated with toxins and antibiotic resistance. Finally, phenotypic characterization revealed a high degree of similarity among the strains with respect to biofilm formation and secretion of extracellular enzymes. Global scale dispersion of virulence factors in the genomes and the abilities for biofilm formation, hemolytic activity and secretion of extracellular enzymes among the strains suggested that F. psychrophilum isolates have a similar mode of action on adhesion, colonization and destruction of fish tissues across large spatial and temporal scales of occurrence. Overall, the genomic characterization and phenotypic properties may provide new insights to the mechanisms of pathogenicity in F. psychrophilum. 相似文献
5.
Wu Yin Stefano Romeo Shurong Chang Nick V. Grishin Helen H. Hobbs Jonathan C. Cohen 《The Journal of biological chemistry》2009,284(19):13213-13222
Angiopoietin-like protein 4 (ANGPTL4) is a secreted protein that modulates
the disposition of circulating triglycerides (TG) by inhibiting lipoprotein
lipase (LPL). Here we examine the steps involved in the synthesis and
post-translational processing of ANGPTL4, and the effects of a naturally
occurring sequence variant (E40K) that is associated with lower plasma TG
levels in humans. Expression of the wild-type and mutant proteins in HEK-293A
cells indicated that ANGPTL4 formed dimers and tetramers in cells prior to
secretion and cleavage of the protein. After cleavage at a canonical
proprotein convertase cleavage site (161RRKR164), the
oligomeric structure of the N-terminal domain was retained whereas the
C-terminal fibrinogen-like domain dissociated into monomers. Inhibition of
cleavage did not interfere with oligomerization of ANGPTL4 or with its ability
to inhibit LPL, whereas mutations that prevented oligomerization severely
compromised the capacity of the protein to inhibit LPL. ANGPTL4 containing the
E40K substitution was synthesized and processed normally, but no monomers or
oligomers of the N-terminal fragments accumulated in the medium; medium from
these cells failed to inhibit LPL activity. Parallel experiments performed in
mice recapitulated these results. Our findings indicate that oligomerization,
but not cleavage, of ANGPTL4 is required for LPL inhibition, and that the E40K
substitution destabilizes the protein after secretion, preventing the
extracellular accumulation of oligomers and abolishing the ability of the
protein to inhibit LPL activity.Angiopoietin-like protein 4
(ANGPTL4)4 is a 50-kDa
protein that is synthesized and secreted from several metabolically active
tissues and has been implicated in the trafficking of circulating TG
(1,
2). Triglycerides, either
acquired from the diet or synthesized endogenously, circulate in blood as
constituents of chylomicrons and very low density lipoproteins (VLDL). As
these lipoproteins circulate in tissues they encounter lipoprotein lipase
(LPL) at the vascular endothelial surfaces. LPL hydrolyzes the TG, producing
free fatty acids that are taken up by the surrounding tissues. ANGPTL4
inhibits the activity of LPL, thereby limiting the uptake of TG-derived fatty
acids by the underlying cells
(3,
4). Overexpression of ANGPTL4
in mice causes severe hypertriglyceridemia, whereas mice lacking ANGPTL4 have
increased LPL activity and low plasma levels of TG
(5,
6). In mice, ANGPTL4 is
predominantly expressed in adipose tissue and is strongly induced by fasting
(2). Accordingly it has been
proposed that ANGPTL4 inhibits LPL activity in adipose tissue to reroute fatty
acids away from fat to muscle and other tissues when food intake is low
(3,
4).ANGPTL4 belongs to a family of seven structurally similar secreted proteins
(ANGPTL1-ANGPTL7) that contain a signal sequence followed by an
α-helical region predicted to form a coiled-coil, and a globular
fibrinogen-like domain at the C terminus
(1). Gel filtration studies of
recombinant ANGPTL4 indicate that the protein assembles into oligomers that
are stabilized by disulfide bonds
(7). Substitution of two highly
conserved cysteine residues at positions 76 and 80 in the α-helical
domain prevents oligomerization of ANGPTL4 and impairs the ability of the
recombinant protein to increase plasma TG levels when overexpressed in the
livers of rats (7).Upon secretion into the circulation, ANGPTL4 is cleaved into an N-terminal
domain and a C-terminal fibrinogen-like domain
(8). The N-terminal peptide
circulates as an oligomer, and the fibrinogen-like domain circulates as a
monomer (8). The N-terminal
helical region of ANGPTL4 is necessary and sufficient for inhibition of LPL
(9). A peptide corresponding to
amino acids 1-187 of the protein binds LPL with high affinity and converts the
enzyme from catalytically active dimers to inactive monomers, thereby
inhibiting LPL activity (10).
After disrupting the LPL dimer, ANGPTL4 is released. The LPL monomers remain
folded and stable but fail to re-form active dimers. These data suggest that
the N-terminal domain of ANGPTL4 interacts directly but transiently with LPL,
triggering a stable conformational switch in LPL that irreversibly inactivates
the enzyme.Recently, we used a population-based resequencing strategy to examine the
metabolic role of ANGPTL4 in humans
(11). Resequencing the coding
region of ANGPTL4 in a large (n = 3,501), multiethnic sample
revealed multiple rare sequence variations that alter an amino acid in the
protein and are associated with low plasma TG levels. In addition, we
identified a more common variant (E40K), that was present in ∼3% of
European-Americans and was associated with significantly lower plasma levels
of TG and low density lipoprotein-cholesterol (LDL-C), and higher levels of
high density lipoprotein (HDL)-C in two large epidemiological studies
(11). These association
studies confirmed that ANGPTL4 is involved in TG metabolism in humans, and
also revealed additional roles in humans in the metabolism of HDL and LDL,
which were not apparent from studies in genetically modified mice.Here we examined the synthesis, secretion, and processing of ANGPTL4 and
determine the mechanism by which substitution of a basic (lysine) for an
acidic (glutamate) residue at residue 40 affects the function of the
protein. 相似文献
6.
Friederike Schmidt Nora Treiber Georg Zocher Sasa Bjelic Michel O. Steinmetz Hubert Kalbacher Thilo Stehle Gabriele Dodt 《The Journal of biological chemistry》2010,285(33):25410-25417
The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance. 相似文献
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Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill E. coli cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death. 相似文献
11.
Giovan F. Gómez Sara A. Bickersmith Ranulfo González Jan E. Conn Margarita M. Correa 《PloS one》2015,10(3)
Phylogenetic analysis of partial mitochondrial cytochrome oxidase c subunit I (COI) and nuclear internal transcribed spacer 2 (ITS2) sequences were used to evaluate initial identification and to investigate phylogenetic relationships of seven Anopheles morphospecies of the Arribalzagia Series from Colombia. Phylogenetic trees recovered highly supported clades for An. punctimaculas.s., An. calderoni, An. malefactor s.l., An. neomaculipalpus, An. apicimacula s.l., An. mattogrossensis and An. peryassui. This study provides the first molecular confirmation of An. malefactorfrom Colombia and discovered conflicting patterns of divergence for the molecular markers among specimens from northeast and northern Colombia suggesting the presence of two previously unrecognized Molecular Operational Taxonomic Units (MOTUs). Furthermore, two highly differentiated An. apicimacula MOTUs previously found in Panama were detected. Overall, the combined molecular dataset facilitated the detection of known and new Colombian evolutionary lineages, and constitutes the baseline for future research on their bionomics, ecology and potential role as malaria vectors. 相似文献
12.
Eduard Hofsetz Fatih Demir Karolina Szczepanowska Alexandra Kukat Jayachandran N. Kizhakkedathu Aleksandra Trifunovic Pitter F. Huesgen 《Molecular & cellular proteomics : MCP》2020,19(8):1330-1345
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- •Mitochondrial heart N terminome shows aminopeptidase processing after MTS cleavage.
- •CLPP-deficiency alters protein processing patterns in mouse heart mitochondria.
- •Candidate substrates identified by N termini accumulation and interaction with inactive ClpXP.
- •UQCRC1, HSPA9 and OAT validated biochemically as high confidence ClpXP substrates.
13.
《Journal of molecular biology》2022,434(24):167874
Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca2+) sensing protein that regulates store-operated calcium entry (SOCE). In SOCE, STIM1 activates Orai1-composed Ca2+ channels in the plasma membrane (PM) after ER stored Ca2+ depletion. S-Glutathionylation of STIM1 at Cys56 evokes constitutive SOCE in DT40 cells; however, the structural and biophysical mechanisms underlying the regulation of STIM1 by this modification are poorly defined. By establishing a protocol for site-specific STIM1 S-glutathionylation using reduced glutathione and diamide, we have revealed that modification of STIM1 at either Cys49 or Cys56 induces thermodynamic destabilization and conformational changes that result in increased solvent-exposed hydrophobicity. Further, S-glutathionylation or point-mutation of Cys56 reduces Ca2+ binding affinity, as measured by intrinsic fluorescence and far-UV circular dichroism spectroscopies. Solution NMR showed S-glutathionylated-induced perturbations in STIM1 are localized to the α1 helix of the canonical EF-hand, the α3 and α4 helices of the non-canonical EF-hand and α6 and α8 helices of the SAM domain. Finally, we designed an S-glutathiomimetic mutation that strongly recapitulates the structural, biophysical and functional effects within the STIM1 luminal domain and we envision to be another tool for understanding the effects of protein S-glutathionylation in vitro, in cellulo and in vivo. 相似文献
14.
Microtubules are nano-machines that grow and shrink stochastically, making use of the coupling between chemical kinetics and mechanics of its constituent protofilaments (PFs). We investigate the stability and shrinkage of microtubules taking into account inter-protofilament interactions and bending interactions of intrinsically curved PFs. Computing the free energy as a function of PF tip position, we show that the competition between curvature energy, inter-PF interaction energy and entropy leads to a rich landscape with a series of minima that repeat over a length-scale determined by the intrinsic curvature. Computing Langevin dynamics of the tip through the landscape and accounting for depolymerization, we calculate the average unzippering and shrinkage velocities of GDP protofilaments and compare them with the experimentally known results. Our analysis predicts that the strength of the inter-PF interaction () has to be comparable to the strength of the curvature energy () such that , and questions the prevalent notion that unzippering results from the domination of bending energy of curved GDP PFs. Our work demonstrates how the shape of the free energy landscape is crucial in explaining the mechanism of MT shrinkage where the unzippered PFs will fluctuate in a set of partially peeled off states and subunit dissociation will reduce the length. 相似文献
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Mikael Altun Thomas S. Walter Holger B. Kramer Patrick Herr Alexander Iph?fer Johan Bostr?m Yael David Alia Komsany Nicola Ternette Ami Navon David I. Stuart Jingshan Ren Benedikt M. Kessler 《PloS one》2015,10(1)
Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways. 相似文献
18.
Salmonella Paratyphi A (S. Paratyphi A) is a highly adapted, human-specific pathogen that causes paratyphoid fever. Cases of paratyphoid fever have recently been increasing, and the disease is becoming a major public health concern, especially in Eastern and Southern Asia. To investigate the genomic variation and evolution of S. Paratyphi A, a pan-genomic analysis was performed on five newly sequenced S. Paratyphi A strains and two other reference strains. A whole genome comparison revealed that the seven genomes are collinear and that their organization is highly conserved. The high rate of substitutions in part of the core genome indicates that there are frequent homologous recombination events. Based on the changes in the pan-genome size and cluster number (both in the core functional genes and core pseudogenes), it can be inferred that the sharply increasing number of pseudogene clusters may have strong correlation with the inactivation of functional genes, and indicates that the S. Paratyphi A genome is being degraded. 相似文献
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