A map of human protein interactions derived from co-expression of human mRNAs and their orthologs

The human protein interaction network will offer global insights into the molecular organization of cells and provide a framework for modeling human disease, but the network's large scale demands new approaches. We report a set of 7000 physical associations among human proteins inferred from indirect evidence: the comparison of human mRNA co-expression patterns with those of orthologous genes in five other eukaryotes, which we demonstrate identifies proteins in the same physical complexes. To evaluate the accuracy of the predicted physical associations, we apply quantitative mass spectrometry shotgun proteomics to measure elution profiles of 3013 human proteins during native biochemical fractionation, demonstrating systematically that putative interaction partners tend to co-sediment. We further validate uncharacterized proteins implicated by the associations in ribosome biogenesis, including WBSCR20C, associated with Williams–Beuren syndrome. This meta-analysis therefore exploits non-protein-based data, but successfully predicts associations, including 5589 novel human physical protein associations, with measured accuracies of 54±10%, comparable to direct large-scale interaction assays. The new associations' derivation from conserved in vivo phenomena argues strongly for their biological relevance

Longitudinal landscapes of serum antibody repertoires after influenza infection and vaccination

Vaccination is the most effective means of infectious disease prevention. Despite its success, however, we still lack a clear understanding of vaccine responses in humans. For example, influenza vaccines still leave a large fraction of population vulnerable. Over the past decade, single B-cell analysis and next-generation sequencing (NGS) technologies have become invaluable tools for studying the antibody repertoire to influenza. Such studies have led to discoveries of broadly-neutralizing antibodies (bNAbs), which can neutralize across multiple strains of influenza virus, promoting the notion of designing a universal vaccine that will elicit such antibodies. One of such isolated bNAbs, called FI6, showed remarkable ability to neutralize all of the influenza A virus strains through targeting the conserved epitope in the stem of hemagglutinin (HA). However, it remains unclear whether such bNAbs actually play a role in conferring protection against influenza since antibody proteins (not B-cells) need to circulate at physiologically relevant concentrations in serum to have implications in protection. Using high-resolution proteomics coupled with NGS, we quantitatively determined the serological antibody repertoire to CA09 HA (H1) at the individual clonotype-level in a donor (whom FI6 was isolated from) following influenza infection (in 2010 with pandemic CA09) and vaccination across five years (2010-2014 with seasonal flu vaccine). We analyzed the temporal changes of head-targeting and stem-binding antibodies, illustrating the gradual increase of stem-targeting antibodies following repeated exposures to CA09 HA. Following vaccination in 2014, \u3e60% of the repertoire consisted of one single clonotype of stem-binding antibody that was present at very low abundance in 2010. Our data demonstrate that the repetitive exposure to influenza skews the serological repertoire toward antibodies that target conserved epitopes, and these antibodies continue to be boosted every time the same epitopes are encountered. Once elicited, stem-binding antibodies displayed a tendency to persist in serum across multiple years while head-specific antibodies decayed quicker. The differential longevity of stem-binding and head-specific antibodies presented here has direct implications for the design of the future universal vaccine

Molecular understanding of the serum antibody repertoires after seasonal influenza vaccination among different age cohorts

Numerous influenza vaccination studies based on bulk serology have indicated that the antibody responses to the vaccine markedly decrease in the elderly. However, whether such decline results from the changes in the overall quantity or the quality of the circulating antibodies in serum remains unknown. Utilizing novel antibody repertoire profiling technologies, combining tandem mass spectrometry (LC-MS/MS) and high-throughput sequencing, we investigated the influenza-specific serological repertoires of 10 donors ranging from 26 to 70 years old vaccinated with Fluzone® 2013-2014 and/or 2014-2015. In particular, we determined the serum antibodies that are specific to the H1 or H3 component of the vaccine or cross-reactive between the two (H1+H3) and examined their relative quantitative distributions. Our data indicate that the proportion of H1+H3 antibodies significantly increases in the elderly and that the somatic hypermutation rates of the influenza-specific antibodies are higher in the elderly. These results suggest that the repeated exposure to the different virus subtypes could have led to the prolonged selection of H1+H3 antibodies targeting highly conserved epitopes. To evaluate the potency of the antibodies circulating in different age groups, we recombinantly expressed a number of representative monoclonal antibodies isolated from the donors in different age groups for further characterizations. Overall, our analysis suggests that the influenza-specific repertoire in the elderly may converge toward shared epitopes but the quality of the antibodies can be superior in terms of cross-reactivity. However, because the antibody repertoire “shrinks” as we age while targeting more conserved epitopes across different influenza subtypes, it is possible that the elderly is particularly susceptible to significantly altered strains. Collectively, profiling vaccine induced serological repertoires among different age cohorts can provide unprecedented insights regarding humoral immunity associated with age and a potential explanation for the vulnerability of the elderly

Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry

Proteomic analysis of T cells emerging from quiescence identifies dynamic network-level changes in key cellular processes. Disruption of two such processes, ribosome biogenesis and RNA splicing, reveals that the programs controlling cell growth and cell-cycle entry are separable

Expression of Human nPTB Is Limited by Extreme Suboptimal Codon Content

Background: The frequency of synonymous codon usage varies widely between organisms. Suboptimal codon content limits expression of viral, experimental or therapeutic heterologous proteins due to limiting cognate tRNAs. Codon content is therefore often adjusted to match codon bias of the host organism. Codon content also varies between genes within individual mammalian species. However, little attention has been paid to the consequences of codon content upon translation of host proteins. Methodology/Principal Findings: In comparing the splicing repressor activities of transfected human PTB and its two tissue-restricted paralogs–nPTB and ROD1–we found that the three proteins were expressed at widely varying levels. nPTB was expressed at 1–3 % the level of PTB despite similar levels of mRNA expression and 74 % amino acid identity. The low nPTB expression was due to the high proportion of codons with A or U at the third codon position, which are suboptimal in human mRNAs. Optimization of the nPTB codon content, akin to the ‘‘humanization’ ’ of foreign ORFs, allowed efficient translation in vivo and in vitro to levels comparable with PTB. We were then able to demonstrate that all three proteins act as splicing repressors. Conclusions/Significance: Our results provide a striking illustration of the importance of mRNA codon content in determining levels of protein expression, even within cells of the natural host species

High Glucose Suppresses Human Islet Insulin Biosynthesis by Inducing miR-133a Leading to Decreased Polypyrimidine Tract Binding Protein-Expression

BACKGROUND: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates. METHODOLOGY/PRINCIPAL FINDINGS: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose. CONCLUSION: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction

PTBP1 Is Required for Embryonic Development before Gastrulation

Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures

NeuN/Rbfox3 Nuclear and Cytoplasmic Isoforms Differentially Regulate Alternative Splicing and Nonsense-Mediated Decay of Rbfox2

Anti-NeuN (Neuronal Nuclei) is a monoclonal antibody used extensively to specifically detect post-mitotic neurons. Anti-NeuN reactivity is predominantly nuclear; by western it detects multiple bands ranging in molecular weight from 45 kDa to >75 kDa. Expression screening putatively identified R3hdm2 as NeuN; however immunoprecipitation and mass spectrometry of the two major NeuN species at 45–50 kDa identified both as the RNA binding protein Rbfox3 (a member of the Fox family of alternative splicing factors), confirming and extending the identification of the 45 kDa band as Rbfox3 by Kim et al. Mapping of the anti-NeuN reactive epitopes in both R3hdm2 and Rbfox3 reveals a common proline- and glutamine-rich domain that lies at the N-terminus of the Rbfox3 protein. Our data suggests that alternative splicing of the Rbfox3 pre-mRNA itself leads to the production of four protein isoforms that migrate in the 45–50 kDa range, and that one of these splicing choices regulates Rbfox3/NeuN sub-cellular steady-state distribution, through the addition or removal of a short C-terminal extension containing the second half of a bipartite hydrophobic proline-tyrosine nuclear localization signal. Rbfox3 regulates alternative splicing of the Rbfox2 pre-mRNA, producing a message encoding a dominant negative form of the Rbfox2 protein. We show here that nuclear Rbfox3 isoforms can also enhance the inclusion of cryptic exons in the Rbfox2 mRNA, resulting in nonsense-mediated decay of the message, thereby contributing to the negative regulation of Rbfox2 by Rbfox3 through a novel mechanism