Structural gymnastics of multifunctional metamorphic proteins

The classic structure–function paradigm holds that a protein exhibits a single well-defined native state that gives rise to its biological function. Nonetheless, over the past few decades, numerous examples of proteins exhibiting biological function arising from multiple structural states of varying disorder have been identified. Most recently, several examples of ‘metamorphic proteins’, able to interconvert between vastly different native-like topologies under physiological conditions, have been characterised with multiple functions. In this review, we look at the concept of protein metamorphosis in relation to the current understanding of the protein structure–function landscape. Although structural dynamism observed for metamorphic proteins provides a novel source of functional versatility, the dynamic nature of the metamorphic proteins generally makes them difficult to identify and probe using conventional protein structure determination methods. However, as the existence of metamorphic proteins has now been established and techniques enabling the analysis of multiple protein conformers are improving, it is likely that this class will continue to grow in number.     

Comparative proteomic profiling of culture filtrate proteins of less and highly virulent Francisella tularensis strains

The facultative intracellular bacterium Francisella tularensis is the causal agent of the serious infectious disease tularemia. Despite the dynamic progress, which has been made in last few years, important questions regarding Francisella pathogenicity still remain to be answered. Generally, secreted proteins play an important role in pathogenicity of intracellular microbes. In this study, we investigated the protein composition of the culture filtrate proteins of highly virulent F. tularensis subsp. tularensis, strain SCHU S4 and attenuated F. tularensis subsp. holarctica, live vaccine strain using a comparative proteomic analysis. The majority of proteins identified in this study have been implicated in virulence mechanisms of other pathogens, and several have been categorized as having moonlighting properties; those that have more than one unrelated function. This profiling study of secreted proteins resulted in the unique detection of acid phosphatase (precursor) A (AcpA), β-lactamase, and hypothetical protein FTT0484 in the highly virulent strain SCHU S4 secretome. The release of AcpA may be of importance for F. tularensis subsp. tularensis virulence due to the recently described AcpA role in the F. tularensis escape from phagosomes.     

Overexpression of CLIC1 in human gastric carcinoma and its clinicopathological significance

Gastric cancer is the second most common cancer worldwide and the fifth leading cause of cancer-related death in Taiwan. Identification of biomarkers is essential to improve patient survival. Fifty aberrantly expressed proteins were identified using 2-DE combined with MALDI TOF MS and were grouped based on their function. The overexpression of proteins was confirmed using real-time quantitative RT-PCR, Western blot, and immunohistochemical analysis. The clinicopathological correlations and prognostic significance of these aberrantly expressed proteins were evaluated to determine the novel gastric cancer biomarkers. In this study, expression of chloride intracellular channel 1 (CLIC1) is significantly up-regulated in 67.9% of gastric patients and was selected for further study. The CLIC1 expression in tumor tissues was increased by 1.95-fold (range, 0.01-6.19-fold) compared with that expressed by adjacent noncancerous mucosa. Elevated CLIC1 expression was strongly correlated with lymph node metastasis, lymphatic invasion, perineural invasion, and pathological staging. Additionally, the 5-year survival rate for the low CLIC1 expression group (n = 28; <1.72-fold) was higher than that for the high CLIC1 expression group (n = 28; >or=1.72-fold) (log rank, p = 0.0300). Experimental results indicate that overexpression of CLIC1 is a potential prognostic marker for gastric cancer.     

Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC

The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid.     

Keeping good friends close – The surface and secreted proteomes of a probiotic bacterium provide candidate proteins for intestinal attachment and communication with the host

Bacteria use cell surface proteins and secreted proteins to interact with host tissues. Several dozen previously published proteomics studies have identified cell surface proteins for pathogens. In this issue, Celebioglu and Svensson (Proteomics 2017, 17, 1700019) use 2D gel electrophoresis and mass spectrometry to identify secreted and cell surface proteins of a commensual gut bacterium, Lactobacillus acidophilus NCFM. Some of the proteins are known to have functions in the cytoplasm, and their presence on the cell surface suggests they might be moonlighting proteins. In addition, comparisons of proteins used by pathogenic and probiotic species to interact with their hosts could lead to improved treatments of infections and chronic diseases that are associated with an imbalance of pathogenic and probiotic gut bacteria.     

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito‐C. Mito‐C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito‐C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF‐1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER–mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito‐C counteracts dengue virus‐induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito‐C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti‐viral research.     

Mapping functional domains of chloride intracellular channel (CLIC) proteins in vivo

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A<70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the 'PTM' domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.     

Comparative studies on the changes of total soluble proteins and protease activity in Georgian commercial peanuts contaminated by Aspergillus flavus

The presence of Aspergillus species is an indicator of storage conditions, which also suggests the possibility of several biochemical changes in grains. A comparative change in total soluble proteins and protease activity was determined in commercial peanut seeds collected from Georgia State. Protein contents of healthy peanuts, naturally contaminated peanuts and then artificially inoculated peanut seeds with A. flavus were estimated by Bradford method, and protease activity was also determined by using the Protease Detection Kits. Protein contents and the protease activity of the peanuts varied from sample to sample. The soluble protein content of seeds was significantly higher in healthy peanuts than in artificially inoculated or naturally infected peanuts with A. flavus. Protease activity was found to be higher in artificially inoculated seeds than in either naturally infected or healthy peanuts. Level of soluble proteins in buffer extracts of contaminated seeds decreased with incubation time, and protease activity increased with incubation time. These changes may be attributed to host response due to infection, contribution by A. flavus or due to biochemical alterations that occur naturally during the transition from endosperm to seedling during incubation period.     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

An in vitro enzymatic assay coupled to proteomics analysis reveals a new DNA processing activity for Ewing sarcoma and TAF(II)68 proteins

Based on structural and functional similarities, translocated in liposarcoma/fusion (TLS/FUS) protein, Ewing sarcoma (EWS) protein and human TATA binding protein-associated factor (hTAF(II)68) have been grouped in the TLS-EWS-TAF(II)68 (TET) protein family. Translocations involving their genes lead to sarcomas. Polypyrimidine tract-binding protein-associated splicing factor (PSF), although not grouped in this family, presents structural and functional similarities with TET proteins and is involved in translocation leading to carcinoma. Beside their role in RNA metabolism, the precise cellular functions of these multifunctional proteins are not yet fully elucidated. We previously showed that both TLS/FUS and PSF display activities able to pair homologous DNA on membrane in an in vitro assay. In the present study, we address the question whether EWS and hTAF(II)68 also display pairing on membrane activities, and to a larger extent whether other proteins also exhibit such activity. We applied the pairing on membrane assay to 2-DE coupled to MS analysis for a global screening of DNA pairing on membrane activities. In addition to TLS/FUS and PSF, this test allowed us to identify EWS and hTAF(II)68, but no other proteins, indicating a feature specific to a protein family whose members share extensive structural similarities. This common activity suggests a role for TET proteins and PSF in genome plasticity control.     

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.     

Structural biochemistry of nuclear actin-related proteins 4 and 8 reveals their interaction with actin

Nuclear actin and actin-related proteins (Arps) are integral components of various chromatin-remodelling complexes. Actin in such nuclear assemblies does not form filaments but associates in defined complexes, for instance with Arp4 and Arp8 in the INO80 remodeller. To understand the relationship between nuclear actin and its associated Arps and to test the possibility that Arp4 and Arp8 help maintain actin in defined states, we structurally analysed Arp4 and Arp8 from Saccharomyces cerevisiae and tested their biochemical effects on actin assembly and disassembly. The solution structures of isolated Arp4 and Arp8 indicate them to be monomeric and the crystal structure of ATP-Arp4 reveals several differences to actin that explain why Arp4 does not form filaments itself. Remarkably, Arp4, assisted by Arp8, influences actin polymerization in vitro and is able to depolymerize actin filaments. Arp4 likely forms a complex with monomeric actin via the barbed end. Our data thus help explaining how nuclear actin is held in a discrete complex within the INO80 chromatin remodeller.     

Spatial features of proteins related to their phosphorylation and associated structural changes

Protein phosphorylation is widely used in biological regulatory processes. The study of spatial features related to phosphorylation sites is necessary to increase the efficacy of recognition of phosphorylation patterns in protein sequences. Using the data on phosphosites found in amino acid sequences, we mapped these sites onto 3D structures and studied the structural variability of the same sites in different PDB entries related to the same proteins. Solvent accessibility was calculated for the residues known to be phosphorylated. A significant change in accessibility was shown for many sites, but several ones were determined as buried in all the structures considered. Most phosphosites were found in coil regions. However, a significant portion was located in the structurally stable ordered regions. Comparison of structures with the same sites in modified and unmodified states showed that the region surrounding a site could be significantly shifted due to phosphorylation. Comparison between non‐modified structures (as well as between the modified ones) suggested that phosphorylation stabilizes one of the possible conformations. The local structure around the site could be changed due to phosphorylation, but often the initial conformation of the site surrounding is not altered within bounds of a rather large substructure. In this case, we can observe an extensive displacement within a protein domain. Phosphorylation without structural alteration seems to provide the interface for domain‐domain or protein‐protein interactions. Accounting for structural features is important for revealing more specific patterns of phosphorylation. It is also necessary for explaining structural changes as a basis for regulatory processes.     

杆状病毒的结构蛋白及其功能

综述了昆虫杆状病毒的主要结构蛋白及其功能。髓核是由大约120 kb的双链DNA分子和与其密切相关的碱性蛋白所组成,碱性蛋白同DNA紧密结合以维持其复杂有序的超螺旋结构,并且能够增强杆状病毒DNA的感染性;衣壳蛋白是杆状病毒粒子的主要结构蛋白,主要有VP 39、VP 78/83、VP 87、VP 80、VP 24;囊膜蛋白有PDV-E 25、PDV-6 e、PDV-E 66、PDV-E 56、PDV-E 18、PDV-EC 27、PDV-E 35、PDV-EC 27、BV/PDV-E 26、PDV-43、gp 64-67、VP 40/41;包涵体的基质蛋白;多角体（或颗粒体）膜主要是由糖类构成,VP 32-36是与之相关的蛋白。     

The secretory proteins in the plerocercoids of the cestode Digramma interrupta and their enzymatic activity]

A composition of secretory proteins of plerocercoids of the cestode Digramma interrupta and their enzyme activity have been studied. It has been shown that plerocercoids produce 26 secretory proteins with molecular weight range of 3.9 to 112.2 kDa. These secretory polypeptides possess protease, DNAase and RNAase activities.     

Nanopore sequencing reveals full-length Tropomyosin 1 isoforms and their regulation by RNA-binding proteins during rat heart development

Alternative splicing (AS) contributes to the diversity of the proteome by producing multiple isoforms from a single gene. Although short-read RNA-sequencing methods have been the gold standard for determining AS patterns of genes, they have a difficulty in defining full-length mRNA isoforms assembled using different exon combinations. Tropomyosin 1 (TPM1) is an actin-binding protein required for cytoskeletal functions in non-muscle cells and for contraction in muscle cells. Tpm1 undergoes AS regulation to generate muscle versus non-muscle TPM1 protein isoforms with distinct physiological functions. It is unclear which full-length Tpm1 isoforms are produced via AS and how they are regulated during heart development. To address these, we utilized nanopore long-read cDNA sequencing without gene-specific PCR amplification. In rat hearts, we identified full-length Tpm1 isoforms composed of distinct exons with specific exon linkages. We showed that Tpm1 undergoes AS transitions during embryonic heart development such that muscle-specific exons are connected generating predominantly muscle-specific Tpm1 isoforms in adult hearts. We found that the RNA-binding protein RBFOX2 controls AS of rat Tpm1 exon 6a, which is important for cooperative actin binding. Furthermore, RBFOX2 regulates Tpm1 AS of exon 6a antagonistically to the RNA-binding protein PTBP1. In sum, we defined full-length Tpm1 isoforms with different exon combinations that are tightly regulated during cardiac development and provided insights into the regulation of Tpm1 AS by RNA-binding proteins. Our results demonstrate that nanopore sequencing is an excellent tool to determine full-length AS variants of muscle-enriched genes.     

Extractive bioconversions in aqueous two-phase systems: enzymatic hydrolysis of casein proteins

Partition coefficients in poly(ethylene glycol)/dextran aqueous two-phase systems are reported for mixed-casein and its components, alpha, beta and kappa casein. Rates of casein proteolysis by alpha-chymotrypsin and by trypsin are reported in single-phase and aqueous two-phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.     

Novel way to study the chronological changes in yeast outer-wall proteins

The time course of changes in the capacity of synchronized yeast cells to be covalently immobilized was determined and interpreted as an indication of chronological changes in the cell's outer-wall proteins. Corroborative evidence was obtained indicating that these transient changes are dependent on protein synthesis and connected with the progression of the cell cycle through the late S and/or early G2 phases.The author is with the Department of Fermentation Chemistry and Bioengineering, Institute of Chemical Technology, 166 28 Prague 6, Czech Republic     

Using peptide strategy for study functions and structure of signal proteins with enzymatic activity

The peptide strategy, a new direction of molecular endocrinology, includes the synthesis of peptides corresponding to functional regions of signal proteins, the use of the peptides for study of the molecular mechanisms of transduction of hormonal signal into cell ant the development of selective regulators of hormonal signaling systems on the basis of these peptides. The peptide strategy is used for study a wide spectrum of the proteins, components of signal systems, the proteins possessing the catalytic activity in particular, such as tyrosine kinases receptors, the enzymes generating the second messengers, serine/threonine protein kinase, phosphatases. In the first time in the review the data concerning the synthetic peptides, derivatives of the primary structure of proteins with the enzymatic activity, their application for study of the structural-functional organization and the molecular mechanisms of action of signal proteins, and the construction of regulators of fundamental cell processes on the basis of these peptides are analyzed and summarized.