Glutamate receptor-interacting protein 1 protein binds to the microtubule-associated protein

To identify the interaction proteins for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.     

On the relationship between the protein structure and protein dynamics

Recently, we have developed a method (Shih et al., Proteins: Structure, Function, and Bioinformatics 2007;68: 34-38) to compute correlation of fluctuations of proteins. This method, referred to as the protein fixed-point (PFP) model, is based on the positional vectors of atoms issuing from the fixed point, which is the point of the least fluctuations in proteins. One corollary from this model is that atoms lying on the same shell centered at the fixed point will have the same thermal fluctuations. In practice, this model provides a convenient way to compute the average dynamical properties of proteins directly from the geometrical shapes of proteins without the need of any mechanical models, and hence no trajectory integration or sophisticated matrix operations are needed. As a result, it is more efficient than molecular dynamics simulation or normal mode analysis. Though in the previous study the PFP model has been successfully applied to a number of proteins of various folds, it is not clear to what extent this model will be applied. In this article, we have carried out the comprehensive analysis of the PFP model for a dataset comprising 972 high-resolution X-ray structures with pairwise sequence identity or=0.5. Our result shows that the fixed-point model is indeed quite general and will be a useful tool for high throughput analysis of dynamical properties of proteins.     

Dephosphorylation of the deinhibitor protein by the PCSH protein phosphatase

The deinhibitor protein, responsible for the decreased sensitivity of the ATP,Mg-dependent protein phosphatase to inhibitor-1 and the modulator protein, is inactivated by cyclic AMP-dependent protein kinase and reactivated by dephosphorylation. The specificity of this reaction was tested with the ATP,Mg-dependent phosphatase in its activated or spontaneously active form, four different forms of polycation-stimulated phosphatases (PCSH, PCSM, PCSL and PCSC) and calcineurin. Only the high -Mr polycation-stimulated protein phosphatase (PCSH), but not its catalytic subunit (PCSC), shows a high degree of specificity for the deinhibitor protein. Deinhibitor phosphatase activity of PCSH is affected neither by polycations nor by Mn ions.     

Converting the prion protein: what makes the protein infectious

The discovery of prion disease transmission in mammals, as well as a non-Mendelian type of inheritance in yeast, has led to the establishment of a new concept in biology, the prion hypothesis. The prion hypothesis postulates that an abnormal protein conformation propagates itself in an autocatalytic manner via recruitment of the normal isoform of the same protein as a substrate, and thereby acts either as a transmissible agent of disease (in mammals) or as a heritable determinant of phenotype (in yeast and fungus). Although reconstitution of fully infectious PrP(Sc)in vitro from synthetic components has not yet been achieved, numerous lines of evidence indicate that the prion protein is the major and essential component, if not the only one, of the prion infectious agent. This article summarizes our current knowledge about the chemical nature of the prion infectious agent, describes potential strategies and challenges related to the generation of prion infectivity de novo, proposes new hypotheses to explain the apparently low infectivity observed in the first synthetic mammalian prions, and describes plausible effects of chemical modifications on prion conversion.     

Prion protein scrapie and the normal cellular prion protein

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP^C) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.     

Modulation of the protein environment in the hydrophilic pore of the ammonia transporter protein AmtB upon GlnK protein binding

The conduction of ammonia/ammonium (NH3/NH4(+)) through the channel protein AmtB is inhibited by the binding of the signal transduction protein GlnK. In the AmtB-GlnK binding interface, there exists an NH3/NH4(+) binding site--Am6. The calculated pK(a) values at the Am6 sites in both the AmtB-GlnK complex and isolated AmtB implies the dominance of an uncharged NH3 state. The GlnK protein binding causes a significant downshift in the Am6 pK(a) value of the AmtB. However, this downshift is perfectly compensated by the reorientation of the protein backbone (carbonyl group of Cys312 from the AmtB part) upon AmtB-GlnK complex formation.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

Elucidating the protein cold-adaptation: Investigation of the parameters enhancing protein psychrophilicity

To investigate the role of the critical parameters in adaptation of proteins to low temperatures, a comparative systematic analysis was performed. Several parameters were proposed to have contribution to cold adaptation of proteins. Among proposed parameters, total values of residual structure states, secondary structure states and oligomeric states were alike in both psychrophilic and mesophilic proteins. In addition, our results provided new quantitative information about the trends in the substitution preference of Ile, Phe, Tyr, Lys, Arg, His, Glu and Leu with most of amino acids and substitution avoidance of Gly, Thr and Ala with most of amino acids. These findings would help future efforts propose a strategy for designing psychrophilic proteins.     

Structural characterization of HBXIP: the protein that interacts with the anti-apoptotic protein survivin and the oncogenic viral protein HBx

Hepatitis B X-interacting protein (HBXIP) is a ubiquitous protein that was originally identified as a binding partner of the hepatitis B viral protein HBx. HBXIP is also thought to serve as an anti-apoptotic cofactor of survivin, promoting the suppression of pro-caspase-9 activation. Here we report the crystal structure of the shortest isoform of HBXIP (91 aa long, ∼ 11 kDa) at 1.5 Å resolution. HBXIP crystal shows a monomer per asymmetric unit, with a profilin-like fold which is common to a superfamily of proteins, the Roadblock/LC7 domain family involved in protein-protein interactions. Based on this fold, we propose that HBXIP can form a dimer that can indeed be found in the crystal when symmetric molecules are generated around the asymmetric unit. This dimer shows an extended β-sheet area formed by 10 anti-parallel β-strands from both subunits. Another interesting aspect of the proposed HBXIP dimer interface is the presence of a small leucine zipper between the two α2 helices of each monomer. In solution, the scattering curve obtained by small-angle X-ray scattering for the sample used for crystallization indicates that the protein is predominantly in dimeric form in solution. The fit between the experimental small-angle X-ray scattering curve and the backcalculated curves for two potential crystal dimers shows a significant preference for the Roadblock/LC7 fold dimer model. Moreover, the HBXIP crystal structure represents a step towards understanding the cellular role of HBXIP.     

When protein folding is simplified to protein coiling: the continuum of solenoid protein structures

Solenoid proteins contain repeating structural units that form a continuous superhelix. This category of proteins conveys the least complicated relationship between a sequence and the corresponding three-dimensional structure. Although solenoid proteins are divided into different classes according to commonly used classification schemes, they share many structural and functional properties.     

Characterization of a GTPase-activating protein for the Ras-related Ral protein

We have demonstrated the presence of a GTPase-activating protein (GAP) for the Ras-related Ral A protein in the cytosolic fraction of brain and testis. This protein, designated Ral-GAP, was distinguished from Ras-GAP by its behavior in two chromatography systems and by the fact that the two GAP proteins did not stimulate the GTPase activity of each others target GTP binding proteins. The lack of effect of Ral-GAP on Ras GTPase activity also distinguished it from the product of the neurofibromatosis gene NF-1. Ral-GAP also differed from Rho-GAP and Rap-GAP by virtue of its elution from a gel filtration column with proteins of Mr greater than 10(6). This was likely an overestimate of the protein's molecular mass, however, since it sedimented in sucrose gradients between standard proteins of 150 and 443 kDa. Ral-GAP failed to promote the GTPase activity of mutant Ral proteins containing amino acid substitutions that in Ras lead to GAP-insensitive proteins.     

The DING protein: an autocrine growth-stimulatory protein related to the human synovial stimulatory protein

A synovial stimulating protein (SSP) has previously been isolated from rheumatoid arthritis synovial fluid and from the culture fluid of rheumatoid arthritis synovial fibroblasts. We have previously isolated, from skin fibroblast cultures, a 40 kDa hirudin-binding protein, which had amino acid sequence homology with the SSP. We sought to clarify the relationship, if any, between the SSP and the hirudin-binding protein. We show that the hirudin-binding protein is immunologically cross-reactive with a protein identical with, or very similar to, the SSP. This hirudin-binding protein is produced by normal and rheumatoid arthritis fibroblasts in culture, and also by cervical carcinoma cells. Traces of an SSP-like protein, and of proteins intermediate in size between the SSP and the hirudin-binding protein, suggest that the hirudin-binding protein may be proteolytically derived from the SSP. An SSP-like protein of about 200 kDa is present in all synovial fluid samples, arthritic and normal, indicating that its presence is not a primary cause of rheumatoid arthritis. There is no evidence for the existence of smaller fragments of the SSP-like protein in synovial fluid. A cDNA sequence, coding for part of the 40 kDa protein, has been obtained. The derived amino acid sequence indicates that a domain, previously identified in the dishevelled gene from Drosophila melanogaster, is present in this protein. Peptides predicted from the cDNA sequence were used to raise antisera, which recognise both the 40 kDa protein and the SSP-like protein. One of the antibody preparations is a good inhibitor of fibroblast proliferation, which confirms the autocrine growth-stimulatory role originally proposed for these proteins.     

Modulation of brain protein phosphorylation by the S-100 protein

The effects of the nervous system specific protein, S-100, on protein phosphorylation in rat brain is examined. The S-100 protein inhibits the phosphorylation of several soluble brain proteins in a calcium dependent fashion. The most potent effect exhibited by S-100 was on the phosphorylation of a protein having a molecular weight of 73,000. The data suggest that the calcium binding S-100 protein, for which a function has not yet been assigned, may modulate calcium dependent phosphorylation of selected brain proteins.     

Regulation of protein compartmentalization expands the diversity of protein function

Proteins destined for the secretory pathway are translocated into the endoplasmic reticulum (ER) by signal sequences that vary widely in their functional properties. We have investigated whether differences in signal sequence function have been exploited for cellular benefit. A cytosolic form of the ER chaperone calreticulin was found to arise by an aborted translocation mechanism dependent on its signal sequence and factors in the ER lumen and membrane. A signal sequence that functions independently of these accessory translocation factors selectively eliminated cytosolic calreticulin. In vivo replacement of endogenous calreticulin with a constitutively translocated form influenced glucocorticoid receptor-mediated gene activation without compromising chaperone activity in the ER. Thus, in addition to its well-established ER lumenal functions, calreticulin has an independent role in the cytosol that depends critically on its inefficient compartmentalization. We propose that regulation of protein translocation represents a potentially general mechanism for generating diversity of protein function.     

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed.     

Regulation of protein kinase C by the cytoskeletal protein calponin

Previous studies from this laboratory have shown that, upon agonist activation, calponin co-immunoprecipitates and co-localizes with protein kinase Cepsilon (PKCepsilon) in vascular smooth muscle cells. In the present study we demonstrate that calponin binds directly to the regulatory domain of PKC both in overlay assays and, under native conditions, by sedimentation with lipid vesicles. Calponin was found to bind to the C2 region of both PKCepsilon and PKCalpha with possible involvement of C1B. The C2 region of PKCepsilon binds to the calponin repeats with a requirement for the region between amino acids 160 and 182. We have also found that calponin can directly activate PKC autophosphorylation. By using anti-phosphoantibodies to residue Ser-660 of PKCbetaII, we found that calponin, in a lipid-independent manner, increased auto-phosphorylation of PKCalpha, -epsilon, and -betaII severalfold compared with control conditions. Similarly, calponin was found to increase the amount of (32)P-labeled phosphate incorporated into PKC from [gamma-(32)P]ATP. We also observed that calponin addition strongly increased the incorporation of radiolabeled phosphate into an exogenous PKC peptide substrate, suggesting an activation of enzyme activity. Thus, these results raise the possibility that calponin may function in smooth muscle to regulate PKC activity by facilitating the phosphorylation of PKC.     

利用基因共表达网络分析蛋白质的相互作用

基于Hom in的基因共表达网络的比较分析,发现人类基因共表达网络和蛋白质相互作用数据之间存在一定的相关性。采用基因本体论对这两个网络重叠区域进行基因分类后发现,这些编码的蛋白质主要集中在对刺激物的应答途径之中。通过对该途径中的蛋白质相互作用网络作图,获得了两个独立的功能模块。通过对模块中的基因分类和关键基因分析得出两者分别对应于内外源刺激物的应答功能。本研究对于利用不断丰富的核酸公共数据信息挖掘蛋白质相互作用的研究具有积极的促进作用。     

Appraisal of the protein efficiency ratio of diverse protein concentrates

In this report the biological value of new protein concentrates has been estimated. In growing rats the protein efficiency ratio, together with the load of liver and serum cholesterol, lipids and proteins, has been determined. It has been concluded that all the protein samples have a nutritional value at least like the older products made with proteins of milk.