339 research outputs found
a Minor Fraction of Molecules Retains the Decoy Epitope, a Presumed Molecular Cause for Viral Persistence
Porcine reproductive and respiratory syndrome virus (PRRSV) is the major
pathogen in the pig industry. Variability of the antigens and persistence are
the biggest challenges for successful control and elimination of the disease.
GP5, the major glycoprotein of PRRSV, is considered an important target of
neutralizing antibodies, which however appear only late in infection. This was
attributed to the presence of a "decoy epitope" located near a hypervariable
region of GP5. This region also harbors the predicted signal peptide cleavage
sites and (dependent on the virus strain) a variable number of potential
N-glycosylation sites. Molecular processing of GP5 has not been addressed
experimentally so far: whether and where the signal peptide is cleaved and (as
a consequence) whether the "decoy epitope" is present in virus particles. We
show that the signal peptide of GP5 from the American type 2 reference strain
VR-2332 is cleaved, both during in vitro translation in the presence of
microsomes and in transfected cells. This was found to be independent of
neighboring glycosylation sites and occurred in a variety of porcine cells for
GP5 sequences derived from various type 2 strains. The exact signal peptide
cleavage site was elucidated by mass spectrometry of virus-derived and
recombinant GP5. The results revealed that the signal peptide of GP5 is
cleaved at two sites. As a result, a mixture of GP5 proteins exists in virus
particles, some of which still contain the "decoy epitope" sequence.
Heterogeneity was also observed for the use of glycosylation sites in the
hypervariable region. Lastly, GP5 mutants were engineered where one of the
signal peptide cleavage sites was blocked. Wildtype GP5 exhibited exactly the
same SDS-PAGE mobility as the mutant that is cleavable at site 2 only. This
indicates that the overwhelming majority of all GP5 molecules does not contain
the "decoy epitope"
Quantitative analysis of the human T cell palmitome
Palmitoylation is a reversible post-translational modification used to
inducibly compartmentalize proteins in cellular membranes, affecting the
function of receptors and intracellular signaling proteins. The identification
of protein “palmitomes” in several cell lines raises the question to what
extent this modification is conserved in primary cells. Here we use primary T
cells with acyl-biotin exchange and quantitative mass spectrometry to identify
a pool of proteins previously unreported as palmitoylated in vivo
The complex co-translational processing of glycoprotein GP5 of type 1 porcine reproductive and respiratory syndrome virus
GP5 and M, the major membrane proteins of porcine reproductive and respiratory syndrome virus (PRRSV), are the driving force for virus budding and a target for antibodies. We studied co-translational processing of GP5 from an European PRRSV-1 strain. Using mass spectrometry, we show that in virus particles of a Lelystad variant, the signal peptide of GP5 was absent due to cleavage between glycine-34 and asparagine-35. This cleavage site removes an epitope for a neutralizing monoclonal antibody, but leaves intact another epitope recognized by neutralizing pig sera. Upon ectopic expression of this GP5 in cells, signal peptide cleavage was however inefficient. Complete cleavage occurred when cysteine-24 was changed to proline or an unused glycosylation site involving asparagine-35 was mutated. Insertion of proline at position 24 also caused carbohydrate attachment to asparagine-35. Glycosylation sites introduced downstream of residue 35 were used, but did not inhibit signal peptide processing. Co-expression of the M protein rescued this processing defect in GP5, suggesting a novel function of M towards GP5. We speculate that a complex interplay of the co-translational modifications of GP5 affect the N-terminal structure of the mature proteins and hence its antigenicity
A strategy for enrichment of claudins based on their affinity to Clostridium perfringens enterotoxin
<p>Abstract</p> <p>Background</p> <p>Claudins, a family of protein localized in tight junctions, are essential for the control of paracellular permeation in epithelia and endothelia. The interaction of several claudins with <it>Clostridium perfringens </it>enterotoxin (CPE) has been exploited for an affinity-based enrichment of CPE-binding claudins from lysates of normal rat cholangiocytes.</p> <p>Results</p> <p>Immunoblotting and mass spectrometry (MS) experiments demonstrate strong enrichment of the CPE-binding claudins -3, -4 and -7, indicating specific association with glutathione-S-transferase (GST)-CPE<sub>116–319 </sub>fusion protein. In parallel, the co-elution of (non-CPE-binding) claudin-1 and claudin-5 was observed. The complete set of co-enriched proteins was identified by MS after electrophoretic separation. Relative mass spectrometric protein quantification with stable isotope labeling with amino acids in cell culture (SILAC) made it possible to discriminate specific binding from non-specific association to GST and/or matrix material.</p> <p>Conclusion</p> <p>CPE<sub>116–319 </sub>provides an efficient tool for single step enrichment of different claudins from cell lysates. Numerous proteins were shown to be co-enriched with the CPE-binding claudins, but there are no indications (except for claudins -1 and -5) for an association with tight junctions.</p
Plasma proteomic analysis of active and torpid greater mouse-eared bats (Myotis myotis)
Hibernation is a physiological adaptation to overcome extreme environmental
conditions. It is characterized by prolonged periods of torpor interrupted by
temporary arousals during winter. During torpor, body functions are suppressed
and restored rapidly to almost pre-hibernation levels during arousal. Although
molecular studies have been performed on hibernating rodents and bears, it is
unclear how generalizable the results are among hibernating species with
different physiology such as bats. As targeted blood proteomic analysis are
lacking in small hibernators, we investigated the general plasma proteomic
profile of European Myotis myotis and hibernation associated changes between
torpid and active individuals by two-dimensional gel electrophoresis. Results
revealed an alternation of proteins involved in transport, fuel switching,
innate immunity and blood coagulation between the two physiological states.
The results suggest that metabolic changes during hibernation are associated
with plasma proteomic changes. Further characterization of the proteomic
plasma profile identified transport proteins, coagulation proteins and
complement factors and detected a high abundance of alpha-fetoprotein. We were
able to establish for the first time a basic myotid bat plasma proteomic
profile and further demonstrated a modulated protein expression during torpor
in Myotis myotis, indicating both novel physiological pathways in bats in
general, and during hibernation in particular
Proteome analysis of the HIV-1 Gag interactome
AbstractHuman immunodeficiency virus Gag drives assembly of virions in infected cells and interacts with host factors which facilitate or restrict viral replication. Although several Gag-binding proteins have been characterized, understanding of virus–host interactions remains incomplete. In a series of six affinity purification screens, we have identified protein candidates for interaction with HIV-1 Gag. Proteins previously found in virions or identified in siRNA screens for host factors influencing HIV-1 replication were recovered. Helicases, translation factors, cytoskeletal and motor proteins, factors involved in RNA degradation and RNA interference were enriched in the interaction data. Cellular networks of cytoskeleton, SR proteins and tRNA synthetases were identified. Most prominently, components of cytoplasmic RNA transport granules were co-purified with Gag. This study provides a survey of known Gag–host interactions and identifies novel Gag binding candidates. These factors are associated with distinct molecular functions and cellular pathways relevant in host–pathogen interactions
Splicing-accessible coding 3′UTRs control protein stability and interaction networks
Background
3′-Untranslated regions (3′UTRs) play crucial roles in mRNA metabolism, such as by controlling mRNA stability, translation efficiency, and localization. Intriguingly, in some genes the 3′UTR is longer than their coding regions, pointing to additional, unknown functions. Here, we describe a protein-coding function of 3′UTRs upon frameshift-inducing alternative splicing in more than 10% of human and mouse protein-coding genes.
Results
3′UTR-encoded amino acid sequences show an enrichment of PxxP motifs and lead to interactome rewiring. Furthermore, an elevated proline content increases protein disorder and reduces protein stability, thus allowing splicing-controlled regulation of protein half-life. This could also act as a surveillance mechanism for erroneous skipping of penultimate exons resulting in transcripts that escape nonsense mediated decay. The impact of frameshift-inducing alternative splicing on disease development is emphasized by a retinitis pigmentosa-causing mutation leading to translation of a 3′UTR-encoded, proline-rich, destabilized frameshift-protein with altered protein-protein interactions.
Conclusions
We describe a widespread, evolutionarily conserved mechanism that enriches the mammalian proteome, controls protein expression and protein-protein interactions, and has important implications for the discovery of novel, potentially disease-relevant protein variants
No personality without experience? A test on Rana dalmatina tadpoles
While the number of studies reporting the presence of individual behavioral consistency (animal personality, behavioral syndrome) has boomed in the recent years, there is still much controversy about the proximate and ultimate mechanisms resulting in the phenomenon. For instance, direct environmental effects during ontogeny (phenotypic plasticity) as the proximate mechanism behind the emergence of consistent individual differences in behavior are usually overlooked compared to environmental effects operating across generations (genetic adaptation). Here, we tested the effects of sociality and perceived predation risk during ontogeny on the strength of behavioral consistency in agile frog (Rana dalmatina) tadpoles in a factorial common garden experiment. Tadpoles reared alone and without predatory cues showed zero repeatability within (i.e., lack of personality) and zero correlation between (i.e., lack of syndrome) activity and risk‐taking. On the other hand, cues from predators alone induced both activity and risk‐taking personalities, while cues from predators and conspecifics together resulted in an activity – risk‐taking behavioral syndrome. Our results show that individual experience has an unequivocal role in the emergence of behavioral consistency. In this particular case, the development of behavioral consistency was most likely the result of genotype × environment interactions, or with other words, individual variation in behavioral plasticity
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