Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

NaCl对大麦幼苗根系蛋白质和游离氨基酸含量的影响

大麦幼苗在２００ｍｍｏｌ／ＬＮａＣｌ处理６ｄ过程中,根中蛋白水麦活性下降,可溶性蛋白含量在处理１、２、４ｄ下降,６ｄ时有所提高。多数游离氨基酸相对含量及其总量呈上升势,胁迫１、２、６ｄ时,总量较对照分别上升６４．２０％、５６．６９％和１．６９％,其中Ｐｒｏ、Ｇｌｕ、Ａｌａ和Ｔｈｒ较为突出,盐胁迫下根系质膜和液泡膜结合慢白含量上升。可溶性蛋经ＳＤＳ－ＰＡＧＥ电泳和扫描分析,在盐胁迫１、２、６ｄ中２５     

Viral Membrane Protein Topology Is Dictated by Multiple Determinants in Its Sequence

The targeting, insertion, and topology of membrane proteins have been extensively studied in both prokaryotes and eukaryotes. However, the mechanisms used by viral membrane proteins to generate the correct topology within cellular membranes are less well understood. Here, the effect of flanking charges and the hydrophobicity of the N-terminal hydrophobic segment on viral membrane protein topogenesis are examined systematically. Experimental data reveal that the classical topological determinants have only a minor effect on the overall topology of p9, a plant viral movement protein. Since only a few individual sequence alterations cause an inversion of p9 topology, its topological stability is robust. This result further indicates that the protein has multiple, and perhaps redundant, structural features that ensure that it always adopts the same topology. These critical topogenic sequences appear to be recognized and acted upon from the initial stages of protein biosynthesis, even before the ribosome ends protein translation.     

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

The Influence of water salinity on the free amino acid concentration in muscle and hepatopancreas of adult shrimps, Penaeus japonicus

Variations of the total free amino acid (FAA) pool and the content of specific amino acids have been measured in the muscle and hepatopancreas of adult shrimps, Penaeus japonicus, acclimatized at five water salinities: 38, 32, 26, 20 and 14%‰ The FAA content is always higher in muscle than in hepatopancreas at all tested salinites. On the other hand, the hepatopancreas exhibits the highest concentrations of essential amino acids. Two steps in the evolution of FAA content can be observed, the first one regarding decrease in salinity from 38 to 20%‰ and the second one, when salinity goes below 20%_°. The first step can be characterized by a 16% decrease of total FAA content in the muscle and a 36% increase in the hepatopancreas. In muscle, the variations are mainly due to changes in non-essential FAA content, whereas in the hepatopancreas, they are linked to variations in essential FAA content. The other step is characterized by a drastic increase in moisture and decrease in FAA content in both studied organs when water salinity is 14%‰ The total FAA content is about 40% lower in shrimps at 14%_° compared to 38%‰ seawater salinity. During adaptation, the FAA pool (mainly NEFAAs) of muscle seems to be directly related to osmoregulation, whereas in the hepatopancreas, its evolution seems to be linked with energy expenditure and protein synthesis. The results are evaluated in order to elucidate the role of FAA in intracellular osmoregulation and in relation to animal ecology.     

The extension reached by the minimization of the polarity distances during the evolution of the genetic code

Summary The level reached by the optimization of the polarity distances during the evolution of the genetic code was investigated. The results, although not conclusive, indicate that this optimization level is higher than the data reported in the literature. The results seem compatible with the reaching of an evolutionary minimum, with respect to the optimization of the polarity distances, by the genetic code during its formation.     

Revisited and large-scale synthesis and purification of the mutated and wild type neu/erbB-2 membrane-spanning segment.

Solid-phase syntheses of the hydrophobic peptides Neu(TM35) ((1)EQRASPVTFIIATVVGVLLFLILVVVVGILIKRRR(35)) and Neu*(TM35) ((1)EQRASPVTFIIATVEGVLLFLILVVVVGILIKRRR(35)), corresponding to the native and mutated (V15E) transmembrane domain of the neu/erbB-2 tyrosine kinase receptor, respectively, were accomplished using Fmoc chemistry. The use of a new resin and cleavage and purification conditions led to large increases in yields and peptide purity. Two (15)N-labelled versions of both wild type and mutated peptides were also synthesized. Approximately 20-40 mg of peptide was obtained using a small-scale synthesis, whereas ca 100 mg of pure peptide was collected on a medium scale. Peptide purity, as monitored by HPLC and mass spectrometry, ranged from 95 to 98% for the six peptides synthesized. Secondary structure as determined by UV circular dichroism (CD) in trifluoroethanol (TFE) showed ca 74% alpha-helical content for the native peptide and ca 63% for that bearing the mutation. Secondary structure of Neu(TM35) was retained in DMPC (dimyristoylphosphatidylcholine)/DCPC (dicaproylphosphatidylcholine) membrane bicelles, and evidences for dimers/oligomers in the lipid bilayer were found.     

The Amino Acid Sequence of Wood Duck Lysozyme

The amino acid sequence of wood duck (Aix sponsa) lysozyme was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had the highest similarity to duck III lysozyme with four amino acid substitutions, and had eighteen amino acid substitutions from chicken lysozyme. The valine at position 75 was newly detected in chicken-type lysozymes. In the active site, Tyr34 and Glu57 were found at subsites F and D, respectively, when compared with chicken lysozyme.