4 research outputs found
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Drosophila Protein interaction Map (DPiM): A paradigm for metazoan protein complex interactions
Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein—especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex “map” provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map
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A Conserved CCCH-type Zinc Finger Protein Regulates mRNA Nuclear Adenylation and Export
Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export
Dominance of the CD4+ T helper cell response during acute resolving hepatitis A virus infection
Hepatitis A virus (HAV) infection typically resolves within 4–7 wk but symptomatic relapse occurs in up to 20% of cases. Immune mechanisms that terminate acute HAV infection, and prevent a relapse of virus replication and liver disease, are unknown. Here, patterns of T cell immunity, virus replication, and hepatocellular injury were studied in two HAV-infected chimpanzees. HAV-specific CD8(+) T cells were either not detected in the blood or failed to display effector function until after viremia and hepatitis began to subside. The function of CD8(+) T cells improved slowly as the cells acquired a memory phenotype but was largely restricted to production of IFN-γ. In contrast, CD4(+) T cells produced multiple cytokines when viremia first declined. Moreover, only CD4(+) T cells responded during a transient resurgence of fecal HAV shedding. This helper response then contracted slowly over several months as HAV genomes were eliminated from liver. The findings indicate a dominant role for CD4(+) T cells in the termination of HAV infection and, possibly, surveillance of an intrahepatic reservoir of HAV genomes that decays slowly. Rapid contraction or failure to sustain such a CD4(+) T cell response after resolution of symptoms could increase the risk of relapsing hepatitis A