Anabel Marina

Differential Association of HLA-B*2705 and B*2709 to Ankylosing Spondylitis Correlates with Limited Peptide Subsets but Not with Altered Cell Surface Stability

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to β2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype.

Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis

The Merluccidae family comprises marine species, some of them of high commercial value and others less appreciated, whose commercialization in Europe under the generic name of “hake” is highly remarkable. The potential of proteomics was employed in this study with the aim of achieving the differential characterization of five different hake species: Merluccius merluccius (European hake), M. australis (Southern hake), M. hubbsi (Argentinian hake), M. gayi (Chilean hake), and M. capensis (Cape hake), some of them very closely related. Species‐specific polypeptides were observed for the five hake species studied in isoelectric focusing (IEF) and/or two‐dimensional electrophoresis (2‐DE) high‐resolution gels. The peptide mass maps of two polypeptide groups, previously selected by 2‐DE analysis as potentially species‐specific, were obtained by “in‐gel” tryptic digestion, followed by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). Analysis of group A polypeptides (with pI in the range of 5.0–5.5 and molecular mass of 17 kDa), allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the peptide mass‐maps from the heat‐resistant parvalbumin fraction (pI below 4.5; molecular mass <12 kDa) allowed the detection of a peptide characteristic of M. australis not present in the other four hake species tested. A specific 17 kDa protein from M. merluccius was also partially sequenced by nanospray‐ion trap‐tandem MS, revealing a high homology with rat nucleoside diphosphate kinase A (NDKA). This work opens the way to the application of proteomics to the differential characterization of commercial hake species at the molecular level.

The Peptide Repertoires of HLA-B27 Subtypes Differentially Associated to Spondyloarthropathy (B*2704 and B*2706) Differ by Specific Changes at Three Anchor Positions

HLA-B*2704 is strongly associated with ankylosing spondylitis. B*2706, which differs from B*2704 by two amino acid changes, is not associated with this disease. A systematic comparison of the B*2704- and B*2706-bound peptide repertoires was carried out to elucidate their overlap and differential features and to correlate them with disease susceptibility. Both subtypes shared about 90% of their peptide repertoires, consisting of peptides with Arg2and C-terminal aliphatic or Phe residues. B*2706 polymorphism influenced specificity at three anchor positions: it favored basic residues at P3 and PΩ-2 and impaired binding of Tyr and Arg at PΩ. Thus, the main structural feature of peptides differentially bound to B*2704 was the presence of C-terminal Tyr or Arg, together with a strong preference for aliphatic/aromatic P3 residues. This is the only known feature of B*2704 and B*2706 that correlates to their differential association with spondyloarthropathy. The concomitant presence of basic P3 and PΩ-2 residues was observed only among peptides differentially bound to B*2706, suggesting that it impairs binding to B*2704. Similarity between peptide overlap and the degree of cross-reaction with alloreactive T lymphocytes suggested that the majority of shared ligands maintain unaltered antigenic features in the context of both subtypes.

Transcription factor AP‐2 activity is modulated by protein kinase A‐mediated phosphorylation

We recently reported that APOE promoter activity is stimulated by cAMP, this effect being mediated by factor AP‐2 [Garcı́a et al. (1996) J. Neurosci. 16, 7550–7556]. Here, we study whether cAMP‐induced phosphorylation modulates the activity of AP‐2. Recombinant AP‐2 was phosphorylated in vitro by protein kinase A (PKA) at Ser239. Mutation of Ser239 to Ala abolished in vitro phosphorylation of AP‐2 by PKA, but not the DNA binding activity of AP‐2. Cotransfection studies showed that PKA stimulated the effect of AP‐2 on the APOE promoter, but not that of the S239A mutant. Therefore, cAMP may modulate AP‐2 activity by PKA‐induced phosphorylation of this factor.

Application of proteomics for fast identification of species-specific peptides from marine species.

In this work, a novel approach based on proteomics is applied for the analysis of the three European marine mussel species: Mytilus edulis (ME), Mytilus galloprovincialis (MG) and Mytilus trossulus (MT), which are of interest in biotechnology and food industry. The proteomes of these species are poorly described in databases, are difficult to diagnose, and have a controversial taxonomy, To characterise species‐specific peptides, we compared 51 matrix‐assisted laser desorption/ioization‐time of flight peptide mass maps generated from 6 random selected prominent spots derived from the two‐dimensional electrophoresis analysis of foot protein extracts from several individuals. Minor species‐specific differences in the peptide maps were detected in only one of the spots, corresponding to tropomyosin. Two peptides were unique to ME and MG individuals, whereas another peptide was present only in MT individuals. The sequence of these peptides was characterised by, nanoelectrospray ionization‐ion trap (nanoESI‐IT) tandem mass spectrometry (MS/MS) analysis followed by database searching and de novo sequence interpretation. We detected a single T to D amino acid substitution in MT tropomyosin. Unambiguous and highly‐specific species identification was then demonstrated by analysing peptide extracts from tropomyosin spots by micro high‐performande liquid chromatography (microHPL) ESI‐IT mass spectrometry using the selected ion monitoring configuration, focused on these peptides, in continuous MS/MS operation. Our results suggest that proteomics may be successfully applied for the identification of species whose proteome is not present in databases.

Evidence of saxitoxin derivatives as causative agents in the 1997 mass mortality of monk seals in the Cape Blanc Peninsula

Monk seals in Cape Blanc (Western Sahara coast) suffered a mass mortality during May-July 1997 which was attributed to a morbillivirus. High performance liquid chromatography (HPLC) analysis on tissues of seals killed during the outbreak and on related fauna showed peaks with retention times coincident with those of some saxitoxin derivatives but their identity was not proved. Here we present results of further HPLC analyses that unambiguously prove the identity of these toxins by mass spectrometry (MS), supporting the hypothesis that this mortality of monk seals was caused by biotoxins rather than by a morbillivirus.

Molecular characterization of a dual inhibitory and mutagenic activity of 5-fluorouridine triphosphate on viral RNA synthesis. Implications for lethal mutagenesis.

The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.

Mutual regulation between SIAH2 and DYRK2 controls hypoxic and genotoxic signaling pathways

The ubiquitin E3 ligase SIAH2 is an important regulator of the hypoxic response as it leads to the ubiquitin/proteasomal degradation of prolyl hydroxylases such as PHD3, which in turn increases the stability of hypoxia-inducible factor (HIF)-1α. In the present study, we identify the serine/threonine kinase DYRK2 as SIAH2 interaction partner that phosphorylates SIAH2 at five residues (Ser16, Thr26, Ser28, Ser68, and Thr119). Phosphomimetic and phospho-mutant forms of SIAH2 exhibit different subcellular localizations and consequently change in PHD3 degrading activity. Accordingly, phosphorylated SIAH2 is more active than the wild-type E3 ligase and shows an increased ability to trigger the HIF-1α-mediated transcriptional response and angiogenesis. We also found that SIAH2 knockdown increases DYRK2 stability, whereas SIAH2 expression facilitates DYRK2 polyubiquitination and degradation. Hypoxic conditions cause a SIAH2-dependent DYRK2 polyubiquitination and degradation which ultimately also results in an impaired SIAH2 phosphorylation. Similarly, DYRK2-mediated phosphorylation of p53 at Ser46 is impaired under hypoxic conditions, suggesting a molecular mechanism underlying chemotherapy resistance in solid tumors.

Application of highly sensitive saturation labeling to the analysis of differential protein expression in infected ticks from limited samples

BackgroundTicks are vectors of pathogens that affect human and animal health worldwide. Proteomics and genomics studies of infected ticks are required to understand tick-pathogen interactions and identify potential vaccine antigens to control pathogen transmission. One of the limitations for proteomics research in ticks is the amount of protein that can be obtained from these organisms. In the work reported here, individual naturally-infected and uninfected Rhipicephalus spp. ticks were processed using a method that permits simultaneous extraction of DNA, RNA and proteins. This approach allowed using DNA to determine pathogen infection, protein for proteomics studies and RNA to characterize mRNA levels for some of the differentially expressed proteins. Differential protein expression in response to natural infection with different pathogens was characterized by two-dimensional (2-D) differential in gel electrophoresis (DIGE) saturation labeling in combination with mass spectrometry analysis. To our knowledge, this is the first report of the application of DIGE saturation labeling to study tick proteins.ResultsQuesting and feeding Rhipicephalus spp. adult ticks were collected in 27 farms located in different Sicilian regions. From 300 collected ticks, only 16 were found to be infected: R. sanguineus with Rickettsia conorii and Ehrlichia canis; R. bursa with Theileria annulata; and R. turanicus with Anaplasma ovis. The proteomic analysis conducted from a limited amount of proteins allowed the identification of host, pathogen and tick proteins differentially expressed as a consequence of infection.ConclusionThese results showed that DIGE saturation labeling is a powerful technology for proteomics studies in small number of ticks and provided new information about the effect of pathogen infection in ticks.

Lesser protein degradation machinery correlates with higher BM86 tick vaccine efficacy in Rhipicephalus annulatus when compared to Rhipicephalus microplus.

Infestations with cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipicephalus annulatus, economically impact cattle production in tropical and subtropical regions of the world. Vaccines containing the recombinant R. microplus BM86 gut antigen were developed and commercialized to induce an immunological protection in cattle against tick infestations. These vaccines demonstrated that tick control by vaccination is cost-effective, reduces environmental contamination and prevents the selection of drug resistant ticks that result from repeated acaricide applications. The protection elicited by BM86-containing vaccines against tick infestations is mediated by a collaborative action between the complement system and IgG antibodies. The efficacy of the vaccination with BM86 and other tick antigens is always higher for R. annulatus than against R. microplus, suggesting that tick genetic and/or physiological factors may affect tick vaccine efficacy. These factors may be related to BM86 protein levels or tick physiological processes such as feeding and protein degradation that could result in more efficient antibody-antigen interactions and vaccine efficacy. To test this hypothesis, we compared the proteome in R. annulatus and R. microplus female ticks after feeding on BM86-vaccinated and control cattle. The results showed that cattle proteins were under represented in R. annulatus when compared to R. microplus, suggesting that R. annulatus ticks ingested less blood, a difference that increased when feeding on vaccinated cattle, probably reflecting the effect of antibody-BM86 interactions on this process. The results also showed that tick protein degradation machinery was under represented in R. annulatus when compared to R. microplus. BM86 mRNA and protein levels were similar in both tick species, suggesting that lesser protease activity in R. annulatus results in more efficient antibody-antigen interactions and higher vaccine efficacy. These results have important implications for tick vaccine research, indicating that not only genetic differences, but also physiological factors may influence tick vaccine efficacy.