Eli Song

Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes.

Rab10 activation promotes GLUT4 storage vesicle recruitment to the plasma membrane in response to insulin and coordinates with myosin-Va to mediate vesicle fusion.

An Attempt to Understand Kidney's Protein Handling Function by Comparing Plasma and Urine Proteomes

Background With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form “reference profiles” of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology. Methodology/Principal Findings After removing secreted proteins downstream of the kidney, 2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched. Conclusion/Significance The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma) and output (urine), it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It can also be anticipated that there will be more applications for proteomics in organ function research.

A High Efficiency Strategy for Binding Property Characterization of Peptide-binding Domains

A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale.

Proteomics Strategy to Identify Substrates of LNX, a PDZ Domain- containing E3 Ubiquitin Ligase

Ubiquitin ligases (E3s) confer specificity to ubiquitination by recognizing target substrates. However, the substrates of most E3s have not been extensively discovered, and new methods are needed to efficiently and comprehensively identify these substrates. Mostly, E3s specifically recognize substrates via their protein interaction domains. We developed a novel integrated strategy to identify substrates of E3s containing protein interaction domains on a proteomic scale. The binding properties of the protein interaction domains were characterized by screening a random peptide library using a yeast two-hybrid system. Artificial degrons, consisting of a preferential ubiquitination sequence and particular interaction domain-binding motifs, were tested as potential substrates by in vitro ubiquitination assays. Using this strategy, not only substrates but also nonsubstrate regulators can be discovered. The detailed substrate recognition mechanisms, which are useful for drug discovery, can also be characterized. We used the Ligand of Numb protein X (LNX) family of E3s, a group of PDZ domain-containing RING-type E3 ubiquitin ligases, to demonstrate the feasibility of this strategy. Many potential substrates of LNX E3s were identified. Eight of the nine selected candidates were ubiquitinated in vitro, and two novel endogenous substrates, PDZ-binding kinase (PBK) and breakpoint cluster region protein (BCR), were confirmed in vivo. We further revealed that the LNX1-mediated ubiquitination and degradation of PBK inhibited cell proliferation and enhanced sensitivity to doxorubicin-induced apoptosis. The substrate recognition mechanism of LNX E3s was also characterized; this process involves the recognition of substrates via their specific PDZ domains by binding to the C-termini of the target proteins. This strategy can potentially be extended to a variety of E3s that contain protein interaction domain(s), thereby serving as a powerful tool for the comprehensive identification of their substrates on a proteomic scale.

HID-1 is a novel player in the regulation of neuropeptide sorting

Peptide hormones and neuropeptides are packaged and stored in a specialized intracellular organelle called the dense core vesicle. It remains elusive how peptide cargoes are correctly sorted. In the present study, we show that a highly conserved Golgi-localized protein named HID-1 acts to prevent mis-sorting of peptide cargoes to lysosomes for degradation via a PtdIns3P-dependent trafficking pathway. Epistasis analysis suggests that rab-2 is epistatic to hid-1.

Targeted disruption of Rab10 causes early embryonic lethality

Intracellular trafficking is a basis of cellular activities, including cell migration, immune response, and development (Lebreton et al., 2003; Lučin et al., 2014; Ulrich and Heisenberg, 2009). In healthy tissue, intracellular trafficking is highly regulated, controlling the form and function of cells (Furthauer and GonzalezGaitan, 2009).Work carried out in animals andplants highlights that regulated intracellular trafficking is important for the development ofmulticellular organisms (Fürthauer andGonzálezGaitán, 2009; Kolotuev et al., 2009; Richter et al., 2009). As a family of Ras-related small GTPase, Rabs function as coordinators for diverse aspects of intracellular trafficking including vesicle budding and formation, cargo sorting and transport to target membranes, and recruitment of key molecules (Stenmark, 2009; Zerial and McBride, 2001). Rab10, widely distributed in intracellular membranes, is highly conserved from Caenorhabditis elegans (C. elegans) to humans. In the C. elegans intestine, Rab10 coordinated the transport of clathrin-independent cargo between early endosomes and recycling endosomes (Chen et al., 2006; Chen et al., 2014). Recent work in Drosophila follicle cells suggested that Rab10 is related to polarized basement membrane secretion during organ morphogenesis (Lerner et al., 2013). In mammalian systems, Rab10 has been implicated in mediating membrane targeting of plasmalemmal precursor vesicles (PPVs) during axon development (Xu et al., 2014). In addition, a large body of evidence has identify Rab10 as an essential component in basolateral recycling in Madin-Darby canine kidney (MDCK) cells and in insulin stimulated GLUT4 recycling in adipocytes (Babbey et al., 2006; Chen et al., 2012). These accumulating results strongly suggest that Rab10 is crucial and has extensive functions in cell biological processes; however, the physiological functions of Rab10 in vivo remain unclear. In this study, we attempted to generate Rab10-deficient mice via homologous recombination in mouse embryonic stem cells to determine the contribution of Rab10 in vivo. Mouse Rab10 is located on chromosome 12 and is composed of 6 exons with the translational start codon ATG in exon 1. In our targeting strategy, we constructed a plasmid that replaced exons 3, 4, and 5 with a neomycin resistance gene cassette resulting in a frame shift in exon 6 (Fig. 1A). The targeting plasmid was transfected into ES cells by electroporation. After screening with G418, positive ES clones were identified via PCR to amplify a 5.8 kb long arm and a 4.0 kb short arm (Fig. 1B). Four out of 102 cell clones displayed the desirable target, approximately 3.9%. After confirmation using Southern blot analysis, the targeted ES clones were injected into C57BL/6 blastocysts to generate chimeric mice which were backcrossed with C57BL/6 mice to produce heterozygous Rab10 mice. Rab10 mice were sequentially backcrossed with C57BL/6 for more than 10 generations to purify their background. Then Rab10 mice were confirmed by Southern blot (Fig. 1C) and the offspring’s genotypes were detected by PCR using genomic tail DNA as a template (Fig. 1D). Heterozygous Rab10 mice displayed no obvious abnormality in weight, diet or fertility during a 12-month observation period. To generate Rab10-null mice, heterozygous mutants were intercrossed. Three weeks after birth, the offspring’s genotypes were identified. Although Rab10 parents showed no obvious abnormality, none of the 82 offspring were found to be homozygous mutants (Rab10) (Fig. 1F) and no increased neonatal mortality was observed in the initial 3 weeks after birth. The ratio between wild-type and heterozygote mice was 0.52, in accordance with Mendel’s law. These results suggest that one functional Rab10 allele is enough for murine embryo development and adults’ survival, but that a double mutant leads to embryonic lethality. To assess the period of developmental failure, embryos from heterozygote hybridization at embryonic day 12.5 (E12.5), 10.5, and 9.5 were collected and genotyped by PCR. As shown in Fig. 1F and 1G, none of the Rab10 embryos was found after E9.5, suggesting that the time of embryonic lethality was before E9.5. Therefore, we continued to analyze earlier embryos at E7.5 and E8.5. In total, out of 35 E7.5 embryos, 7 (20.0%) were homozygous mutants and 1 (2.9%) was a resorbed embryo; out of 71 E8.5 embryos, 8 (11.3%) were homozygous mutants and 10 (14.1%) were resorbed embryos (Fig. 1G). To further confirm these results, E7.5 embryos were collected and analyzed by Western blot with anti-Rab10 antibody. As shown in Fig. 1E, Rab10 protein was totally deleted in Rab10-null mice. Rab10 mutants showed obvious differences from wildtype embryos (Fig. 1I). Mutant embryos at E7.5 were much

HID-1 is a peripheral membrane protein primarily associated with the medial- and trans- Golgi apparatus.

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans- Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.

HID-1 is required for homotypic fusion of immature secretory granules during maturation

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules. DOI: http://dx.doi.org/10.7554/eLife.18134.001

Systematic Analysis of a Simple Adaptor Protein PDZK1: Ligand Identification, Interaction and Functional Prediction of Complex

PDZK1 is a simple adaptor protein with four protein interaction PDZ domains, but without any other known functional domains. Here, we used yeast two-hybrid screening of a random peptide library and high-throughput validation screening of a specialized PDZ ligand candidate library to systematically and comprehensively identify PDZK1 ligands. The potential functional associations of the ligands were predicted by functional annotations from a MILANO literature search and subcellular localizations. The ligands were considered more likely to be functionally associated if they had similar patterns of functions or closely related functions. For some functionally associated ligand pairs, interaction with one ligand was found to be influenced by another ligand in a yeast three-hybrid system. Many G-protein signaling pathway-related proteins were found to interact with PDZK1, and they were likely to be functionally associated with transporters based on their closely related functions. This strategy can be extended to the study of other adaptor proteins that contain peptide-binding domains.

An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells

Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.