Tracking a protein following dissociation from a protein–protein complex using a split SNAP-tag system

Protein–protein interactions (PPIs) are important for various biological processes in living cells. Several methods have been developed for the visualization of PPIs in vivo; however, these methods are unsuitable for visualization of post-PPI events such as dissociation and translocation. In this study, we applied a split SNAP-tag system for the visualization of post-PPI events. This method enabled tracking of the protein following dissociation from the protein–protein complex. Thus, the split SNAP-tag system should prove to be a useful tool for visualization of post-PPI events.     

LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis

SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis.     

Tomato SlSnRK1 protein interacts with and phosphorylates βC1, a pathogenesis protein encoded by a geminivirus β-satellite

The βC1 protein of tomato yellow leaf curl China β-satellite functions as a pathogenicity determinant. To better understand the molecular basis of βC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using βC1 as bait. βC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that βC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with βC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the βC1 protein.     

Protein tyrosine phosphatase interacting protein 51—a jack-of-all-trades protein

Protein tyrosine phosphatase interacting protein 51 (PTPIP51) interacts both in vitro and in vivo with PTP1B, a protein tyrosine phosphatase involved in cellular regulation. PTPIP51 is known to be expressed in many different types of tissues. It is involved in cellular processes such as proliferation, differentiation and apoptosis. Nevertheless, the exact cellular function of PTPIP51 is still unknown. The present review summarizes our current knowledge of the PTPIP51 gene and its mRNA and protein structure.     

Is NSF a fusion protein?

N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase required for vesicular transport throughout the constitutive secretory and endocytic pathways. Recently, NSF has also been implicated in regulated exocytosis in synapses--based on SNAP-mediated binding in vitro to a complex of neurotoxin substrates (termed 'SNAREs'). This work has generated an hypothesis in which the interaction of SNAREs (SNAP receptors) on the vesicle membrane with those on the target membrane forms a docking complex to which SNAPs bind, thus allowing NSF to bind and elicit membrane fusion. However, current evidence supports an earlier, pre-fusion role for NSF. We speculate that this role may be as a molecular chaperone for the membrane docking/fusion machinery.     

Are histones, tubulin, and actin derived from a common ancestral protein?

Histones and the cytoskeletal components tubulin and actin all act as thermal ratchets, using the energy present in Brownian motion to do work. All three also bind to nucleotides. Here we suggest that histones, tubulin, and actin derive from a common ancestral protein. There is some sequence similarity between histone 2A and the bacterial tubulin homologue FtsZ. Histones and actin also share some sequence similarity in the nucleotides and at phosphate-binding sites. Thus, actin and tubulin may also be related, although this is not obvious from sequence analysis. Indeed, actin and tubulin are closely functionally related and cooperate in many cellular processes. Interestingly, recent advances in nanotechnology suggest that thermal ratchets may be able to impart lifelike properties; thus, the evolution of the ancestral histone, tubulin, and actin thermal ratchet may have been crucial in the development of complexity in living organisms.     

Is unphosphorylated Rex,as multifunctional protein of HTLV-1, a fully intrinsically disordered protein? An in silico study

Intracellularlocation of a viral unspliced mRNA in host cell is a crucial factor for normal life of the virus. Rex is a neucleo-cytoplasmic shuffling protein of Human T-cell Leukemia Virus-1(HTLV-1)which has important role in active transport of cargo-containing RNA from nucleus to cytoplasm. Therefore, it plays a crucial role in the disease development by the virus. In spite of its importance, the 3d-structurephosphorylated and unphosphorylated of this protein has not been determined. In this study, first we predicted whether Rex protein is an ordered or disordered protein. In second step protein 3Dstructure of Rex was obtained. The content of disorder-promoting amino acids, flexibility, hydrophobicity, short linear motifs (SLiMs) and protein binding regions and probability of Rex crystallization were calculated by various In Silico methods. The3D models of Rex protein were obtained by various In Silico methods, such as homology modeling, threading and ab initio, including; I-TASSER, LOMETS, SPARSKS, ROBBETA and QUARK servers. By comparing and analyzing Qmean, z-scores and energy levels of selected models, the best structures with highest favored region in Ramachandran plot (higher than 90%) was refined with MODREFINER software. In silico analysis of Rex physicochemical properties and also predicted SLiMs and binding regions sites confirms that unphosphorylated Rex protein in HTLV-1 as Rev protin in HIV is wholly disordered protein belongs to the class of intrinsically disordered proteins with extended disorder (native coils, native pre-molten globules).     

Fibronectin: a chromatin-associated protein?

We have previously reported that chromatin preparations from human cultured fibroblasts contain a single homologous serum protein. In this paper we present evidence, based on immunological identity and physicochemical properties, that this serum protein is fibronectin. Furthermore, using a radioimmunoassay system, we have estimated that fibronectin represents about 0.7% of the total protein in both chromatin preparations and whole fibroblasts. Using a nitrocellulose filter assay system, we also show that fibronectin is a DNA-binding protein having an equilibrium constant of 4.6 x 10(-6) M. Equilibrium competition experiments have demonstrated that fibronectin has the ability to differentiate among nucleotides, indicating that fibronectin-DNA interaction is at least partially specific, and that a minimum polymer length of 12-18 nucleotides is required for effective binding to occur. Fibronectin has been isolated readily from plasma using DNA-affinity chromatography. We do not have direct evidence that fibronectin is an actual nonhistone chromosomal protein, but fibronectin is a DNA-binding protein (at least under in vitro assay conditions) and appears to be a normal constituent of chromatin as chromatin is currently isolated from cell nuclei.     

Resonance assignments for Oncostatin M, a 24-kDa α-helical protein

Summary Oncostatin M (OM) is a cytokine that shares a structural and functional relationship with interleukin-6, leukemia inhibitory factor, and granulocyte-colony stimulating factor, which regulate the proliferation and differentiation of a variety of cell types. A mutant version of human OM in which two N-linked glycosylation sites and an unpaired cysteine have been mutated to alanine (N76A/C81A/N193A) has been expressed and shown to be active. The triple mutant has been doubly isotope-labeled with ¹³C and ¹⁵N in order to utilize heteronuclear multidimensional NMR techniques for structure determination. Approximately 90% of the backbone resonances were assigned from a combination of triple-resonance data (HNCA, HNCO, CBCACONH, HBHACONH, HNHA and HCACO), intraresidue and sequential NOEs (3D ¹⁵N-NOESY-HMQC and ¹³C-HSQC-NOESY) and side-chain information obtained from the CCONH and HCCONH experiments. Preliminary analysis of the NOE pattern in the ¹⁵N-NOESY-HMQC spectrum and the ¹³C secondary chemical shifts predicts a secondary structure for OM consisting of four -helices with three intervening helical regions, consistent with the four-helix-bundle motif found for this cytokine family. As a 203-residue protein with a molecular weight of 24 kDa, Oncostatin M is the largest -helical protein yet assigned.To whom correspondence should be addressed.     

The nuclear protein HMGB1, a new kind of chemokine?

The chromosomal protein HMGB1 is now regarded as a proinflammatory cytokine. Importantly, HMGB1 has chemotactic activity suggesting its involvement in the early and late events of the inflammatory reaction. Therefore, HMGB1 has all the hallmarks of a chemokine (chemotactic cytokine). We propose to classify HMGB1 into a new group of proteins unrelated structurally to chemokines but having chemokine-like functions, and to name this class CLF (chemokine-like functions). The CLF class should include other unrelated molecules such as urokinase and its receptor, cytokines macrophage migration inhibitory factor (MIF) and interleukin (IL)-6, anaphylatoxin C5a, ribosomal protein S19, and thioredoxin that have similar chemokine-like activities. This innovative concept may lead to the identification of new therapeutic targets.     

Over-producing soluble protein complex and validating protein–protein interaction through a new bacterial co-expression system

Many proteins exert their functions through a protein complex and protein–protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein–protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein–protein interaction of interest in a wide range of biological organisms.     

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer β-sheet

The crystal structure of outer surface protein A (OspA) from Borrelia burgdorferi contains a single-layer -sheet connecting the N- and C-terminal globular domains. The central -sheet consists largely of polar amino acids and it is solvent-exposed on both faces, which so far appears to be unique among known protein structures. We have accomplished nearly complete backbone H, C and N and C^;/H assignments of OspA (28 kDa) using standard triple resonance techniques without perdeuteration. This was made possible by recording spectra at a high temperature (45 °C ). The chemical shift index and ¹⁵N T₁/T₂ ratios show that both the secondary structure and the global conformation of OspA in solution are similar to the crystal structure, suggesting that the unique central -sheet is fairly rigid.     

Characterization of a cDNA encoding a protein with limited similarity to β1, 3-N-acetylglucosaminyltransferase

Glycosyltransferases constitute a large group of enzymes that are involved in a wide range of functions in all living organisms. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a novel human putative glycosyltransferase gene named beta3GnTL1. Its cDNA is 1372 base pair in length, encoding a predicted protein with 361 amino acid residues. The human beta3GnTL1 is located to chromosome 17q25.3 by comparison of its cDNA with human gemome database. RT-PCR result shows the beta3GnTL1 is expressed at various levels in most of tissues examined.     

Obg/CtgA, a signaling protein that controls replication, translation, and morphological development?

The recent finding that the ObgE GTPase acts as a replication checkpoint protein in Escherichia coli has important implications. It reveals the existence of a new pathway of replication control by the nucleotide pool and suggests unsuspected links between replication, proteins synthesis, and cellular differentiation.     

Protein microarrays as a discovery tool for studying protein–protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein–protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein–protein interaction studies.