The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca2+-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca2+-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2. 相似文献
We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F2Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies. 相似文献
γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex. 相似文献
Overload of intracellular Ca2+ has been implicated in the pathogenesis of neuronal disorders, such as Alzheimer’s disease. Various mechanisms produce abnormalities
in intracellular Ca2+ homeostasis systems. L-type Ca2+ channels have been known to be closely involved in the mechanisms underlying the neurodegenerative properties of amyloid-β
(Aβ) peptides. However, most studies of L-type Ca2+ channels in Aβ-related mechanisms have been limited to CaV1.2, and surprisingly little is known about the involvement of CaV1.3 in Aβ-induced neuronal toxicity. In the present study, we examined the expression patterns of CaV1.3 after Aβ25–35 exposure for 24 h and compared them with the expression patterns of CaV1.2. The expression levels of CaV1.3 were not significantly changed by Aβ25–35 at both the mRNA levels and the total protein level in cultured hippocampal neurons. However, surface protein levels of CaV1.3 were significantly increased by Aβ25–35, but not by Aβ35–25. We next found that acute treatment with Aβ25–35 increased CaV1.3 channel activities in HEK293 cells using whole-cell patch-clamp recordings. Furthermore, using GTP pulldown and co-immunoprecipitation
assays in HEK293 cell lysates, we found that amyloid precursor protein interacts with β3 subunits of Ca2+ channels instead of CaV1.2 or CaV1.3 α1 subunits. These results show that Aβ25–35 chronically or acutely upregulates CaV1.3 in the rat hippocampal and human kidney cells (HEK293). This suggests that CaV1.3 has a potential role along with CaV1.2 in the pathogenesis of Alzheimer’s disease. 相似文献
Protein interactions among RNA polymerase small subunits from the archaeon Methanococcus jannaschii were investigated using affinity pulldown assays in pairwise and higher-order combinations. In the most extensive study of archaeal RNA polymerase subunit interactions to date, including 37 pairs of proteins, 10 ternary combinations, and three quaternary combinations, we found evidence for pairwise interactions of subunit D with subunits L and N, and a ternary complex of subunits D, L and N. No other small subunit interactions occurred. These results are consistent with interactions observed in a crystal structure of eukaryotic RNA polymerase II and support a common archaeal/eukaryal RNA polymerase architecture. We further propose that subunit E" is not an integral member of archaeal RNA polymerases. Finally, we discuss the relative accuracy of the various methods that have been used to predict protein-protein interactions in RNA polymerase. 相似文献
Introduction: The Adhesion and Degranulation promoting Adaptor Protein (ADAP) is phosphorylated upon T cell activation and acts as a scaffold for the formation of a signaling complex that integrates molecular interactions between T cell or chemokine receptors, the actin cytoskeleton, and integrin-mediated cellular adhesion and migration.
Areas covered: This article reviews current knowledge of the functions of the adapter protein ADAP in T cell signaling with a focus on the role of individual phosphotyrosine (pY) motifs for SH2 domain mediated interactions. The data presented was obtained from literature searches (PubMed) as well as the authors own research on the topic.
Expert commentary: ADAP can be regarded as a paradigmatic example of how tyrosine phosphorylation sites serve as dynamic interaction hubs. Molecular crowding at unstructured and redundant sites (pY595, pY651) is contrasted by more specific interactions enabled by the three-dimensional environment of a particular phosphotyrosine motif (pY571). 相似文献
Glutathione-S-transferase (GST)-fusion proteins are used extensively for structural, biochemical, and functional analyses. Although the conformation of the target protein is of critical importance, confirmation of the folded state of the target is often not undertaken or is cumbersome because of the requirement to first remove the GST tag. Here, we demonstrate that it is possible to record conventional (15)N-HSQC NMR spectra of small GST-fusion proteins and that the observed signals arise almost exclusively from the target protein. This approach constitutes a rapid and straightforward means of assessing the conformation of a GST-fusion protein without having to cleave the GST and should prove valuable, both to biochemists seeking to check the conformation of their proteins prior to functional studies and to structural biologists screening protein constructs for suitability as targets for structural studies. 相似文献