A New Way of Preserving Large Number of Samples Simply and Economically for Proteomics and Biomarker Study

OPINIO

Medication Needs to be Considered in Biomarker Discovery

OPINIO

Molecular characterization and ligand binding specificity of the PDZ domain-containing protein GIPC3 from Schistosoma japonicum

BACKGROUND: Schistosomiasis is a serious global health problem that afflicts more than 230 million people in 77 countries. Long-term mass treatments with the only available drug, praziquantel, have caused growing concerns about drug resistance. PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins are recognized as potential targets for the next generation of drug development. However, the PDZ domain-containing protein family in parasites has largely been unexplored. METHODS: We present the molecular characteristics of a PDZ domain-containing protein, GIPC3, from Schistosoma japonicum (SjGIPC3) according to bioinformatics analysis and experimental approaches. The ligand binding specificity of the PDZ domain of SjGIPC3 was confirmed by screening an arbitrary peptide library in yeast two-hybrid (Y2H) assays. The native ligand candidates were predicted by Tailfit software based on the C-terminal binding specificity, and further validated by Y2H assays. RESULTS: SjGIPC3 is a single PDZ domain-containing protein comprised of 328 amino acid residues. Structural prediction revealed that a conserved PDZ domain was presented in the middle region of the protein. Phylogenetic analysis revealed that SjGIPC3 and other trematode orthologues clustered into a well-defined cluster but were distinguishable from those of other phyla. Transcriptional analysis by quantitative RT-PCR revealed that the SjGIPC3 gene was relatively highly expressed in the stages within the host, especially in male adult worms. By using Y2H assays to screen an arbitrary peptide library, we confirmed the C-terminal binding specificity of the SjGIPC3-PDZ domain, which could be deduced as a consensus sequence, -[SDEC]-[STIL]-[HSNQDE]-[VIL]*. Furthermore, six proteins were predicted to be native ligand candidates of SjGIPC3 based on the C-terminal binding properties and other biological information; four of these were confirmed to be potential ligands using the Y2H system. CONCLUSIONS: In this study, we first characterized a PDZ domain-containing protein GIPC3 in S. japonicum. The SjGIPC3-PDZ domain is able to bind both type I and II ligand C-terminal motifs. The identification of native ligand will help reveal the potential biological function of SjGIPC3. These data will facilitate the identification of novel drug targets against S. japonicum infections

Novel Nonphosphorylated Peptides with Conserved Sequences Selectively Bind to Grb7 SH2 Domain with Affinity Comparable to Its Phosphorylated Ligand

The Grb7 (growth factor receptor-bound 7) protein, a member of the Grb7 protein family, is found to be highly expressed in such metastatic tumors as breast cancer, esophageal cancer, liver cancer, etc. The src-homology 2 (SH2) domain in the C-terminus is reported to be mainly involved in Grb7 signaling pathways. Using the random peptide library, we identified a series of Grb7 SH2 domain-binding nonphosphorylated peptides in the yeast two-hybrid system. These peptides have a conserved GIPT/K/N sequence at the N-terminus and G/WD/IP at the C-terminus, and the region between the N-and C-terminus contains fifteen amino acids enriched with serines, threonines and prolines. The association between the nonphosphorylated peptides and the Grb7 SH2 domain occurred in vitro and ex vivo. When competing for binding to the Grb7 SH2 domain in a complex, one synthesized nonphosphorylated ligand, containing the twenty-two amino acid-motif sequence, showed at least comparable affinity to the phosphorylated ligand of ErbB3 in vitro, and its overexpression inhibited the proliferation of SK-BR-3 cells. Such nonphosphorylated peptides may be useful for rational design of drugs targeted against cancers that express high levels of Grb7 protein

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

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.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.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

Myelin basic protein is a sequence-specific RNA-binding protein

Myelin basic protein (MBP) accounts for 30% to 40% of the protein in CNS myelin. MBP has been localized immunocytochemically to the major dense line of myelin, which corresponds to the apposed cytoplasmic leaflets of the myelin membrane. It is believed that MBP play a role in mediating close membrane apposition during myelin compaction.^ In this work we show that: (1) MBP binds RNA both on nitrocellulose and in solution. (2) Recombinant 18.5kD mouse MBP binds RNA with higher affinity than other MBP isoforms. Insertion of exon 2, or deletion of exon 6, in 21.5kD, 17kD and 14kD MBP isoforms reduces the binding affinity. (3) UV crosslinking and tryptic peptide mapping indicates that three tryptic peptides in 18.5kD MBP contact RNA. (4) Systematic Evolution of Ligands by EXponential enrichment (SELEX) analysis indicates that MBP has higher affinity to selected RNA sequences than to a pool of random sequences. Selected sequences have significantly higher GC content than the pool of random sequence (t $\u3c$ 0.0005). Many selected RNA sequences contain one or more copies of the sequence, CAGUGU, or related sequences. The selected RNA sequence with the highest affinity for MBP contains three copies of CAGUGU. The dissociation constant (Kd) of 18.5kD MBP for these sequences in 250mM NaCl, pH7.2 is 10\sp{-7} to 10\sp{-8}M. Homologous sequences are found in connexin 32 (Cx32) and MBP mRNA. (5) Computer modeling, using neural network secondary structure prediction and homology modeling reveal that the 18.5kD MBP could adopt a characteristic alpha-beta sandwich fold, which is found in U1A and other RNA-binding proteins.^ MBP-RNA interactions may be involved in Cx32 and/or MBP gene regulation, or may play a role in transport and/or localization of their mRNAs.