The identification of a novel human homologue of the SH3 binding glutamic acid-rich (SH3BGR) gene establishes a new family of highly conserved small proteins related to Thioredoxin Superfamily

The SH3 binding glutamic acid-rich (SH3BGR) gene was cloned in an effort to identify genes located to human chromosome 21, within the congenital heart disease region, and expressed in the developing heart. After the identification of SH3BGR, two human homologous genes, SH3BGRL and SH3BGRL3, were identified and mapped to chromosome Xq13.3 and 1p34.3-35, respectively. SH3BGRL and SH3BGRL3 code for small proteins similar to the N-terminal region of the SH3BGR protein. SH3BGRL3 protein shows a significant similarity to Glutaredoxin 1 of Escherichia coli, and all the three proteins are predicted to belong to Thioredoxin-like protein Superfamily. Here we describe the identification and characterization of an additional human homologue of SH3BGR, named SH3BGRL2. The SH3BGRL2 gene maps to chromosome 6q13-15 and its messenger RNA has a large 3' untranslated region containing several AUUUA repeats. SH3BGRL2 codes for a protein of 107 amino acids, which, like SH3BGRL and SH3BGRL3 proteins, is highly homologous to the N-terminal region of the SH3BGR protein and appears to be related to Glutaredoxins and to PKC-interacting cousin of thioredoxin homology domain. We propose that the identification of SH3BGRL2 establishes a novel family of human genes, coding for highly conserved small proteins belonging to Thioredoxin-like protein Superfamily.     

NMR structure and regulated expression in APL cell of human SH3BGRL3

SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor alpha (TNF-alpha) inhibitory protein, TIP-B1. In this paper, we determined its solution structure by multi-dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX-like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all-trans retinoic acid-induced pathway.     

Crystal structure of the glutaredoxin-like protein SH3BGRL3 at 1.6 Angstrom resolution

We report the 1.6 Angstrom resolution crystal structure of SH3BGRL3, a member of a new mammalian protein family of unknown function. The observed "thioredoxin fold" of SH3BGRL3 matches the tertiary structure of glutaredoxins, even in the N-terminal region where the sequence similarity between the two protein families is negligible. In particular, SH3BGRL3 displays structural modifications at the N-terminal Cys-x-x-Cys loop, responsible for glutathione binding and catalysis in glutaredoxins. The loop hosts a six residue insertion, yielding an extra N-terminal-capped helical turn, first observed here for the thioredoxin fold. This, together with deletion of both Cys residues, results in a substantial reshaping of the neighboring cleft, where glutathione is hosted in glutaredoxins. While not active in redox reaction and glutathione binding, SH3BGRL3 may act as an endogenous modulator of glutaredoxin activities by competing, with its fully conserved thioredoxin fold, for binding to yet unknown target proteins.     

Genome-Wide Survey and Expression Analysis Suggest Diverse Roles of Glutaredoxin Gene Family Members During Development and Response to Various Stimuli in Rice

Glutaredoxins (GRXs) are glutathione-dependent oxidoreductase enzymes involved in a variety of cellular processes. In this study, our analysis revealed the presence of 48 genes encoding GRX proteins in the rice genome. GRX proteins could be classified into four classes, namely CC-, CGFS-, CPYC- and GRL-type, based on phylogenetic analysis. The classification was supported with organization of predicted conserved putative motifs in GRX proteins. We found that expansion of this gene family has occurred largely via whole genome duplication events in a species-specific manner. We explored rice oligonucleotide array data to gain insights into the function of GRX gene family members during various stages of development and in response to environmental stimuli. The comprehensive expression analysis suggested diverse roles of GRX genes during growth and development in rice. Some of the GRX genes were expressed in specific organs/developmental stages only. The expression of many of rice GRX genes was influenced by various phytohormones, abiotic and biotic stress conditions, suggesting an important role of GRX proteins in response to these stimuli. The identification of GRX genes showing differential expression in specific tissues or in response to environmental stimuli provide a new avenue for in-depth characterization of selected genes of importance.     

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.     

Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons

Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein.     

Yeast adaptor protein, Nbp2p, is conserved regulator of fungal Ptc1p phosphatases and is involved in multiple signaling pathways

Nbp2p is an Src homology 3 (SH3) domain-containing yeast protein that is involved in a variety of cellular processes. This small adaptor protein binds to a number of different proteins through its SH3 domain, and a region N-terminal to the SH3 domain binds to the protein phosphatase, Ptc1p. Despite its involvement in a large number of physical and genetic interactions, the only well characterized function of Nbp2p is to recruit Ptc1p to the high osmolarity glycerol pathway, which results in down-regulation of this pathway. In this study, we have discovered that Nbp2p orthologues exist in all Ascomycete and Basidiomycete fungal genomes and that all possess an SH3 domain and a conserved novel Ptc1p binding motif. The ubiquitous occurrence of these two features, which we have shown are both critical for Nbp2p function in Saccharomyces cerevisiae, implies that a conserved role of Nbp2p in all of these fungal species is the targeting of Ptc1p to proteins recognized by the SH3 domain. We also show that in a manner analogous to its role in the high osmolarity glycerol pathway, Nbp2p functions in the down-regulation of the cell wall integrity pathway through SH3 domain-mediated interaction with Bck1p, a component kinase of this pathway. Based on functional studies on the Schizosaccharomyces pombe and Neurospora crassa Nbp2p orthologues and the high conservation of the Nbp2p binding site in Bck1p orthologues, this function of Nbp2p appears to be conserved across Ascomycetes. Our results also clearly imply a function for the Nbp2p-Ptc1p complex other cellular processes.     

A novel LIM and SH3 protein (lasp-2) highly expressing in chicken brain

From eluates of F-actin affinity chromatography of chicken brain, we identified a novel actin-binding protein (lasp-2) whose gene was predicted in silico. We cloned cDNA of chicken lasp-2 and analyzed its structure, expression, activity, and localization with lasp-1 (LIM and SH3 protein 1), a previously identified actin-binding protein closely related to lasp-2. Chicken lasp-2 showed high homology to mammalian putative lasp-2. Both chicken lasp-1 and chicken lasp-2 have N-terminal LIM domains, C-terminal SH3 domains, and internal nebulin repeats. However, lasp-2 is greatly different from lasp-1 in the sequence between the second nebulin repeat and a SH3 domain, and the region is conserved in chicken, mouse, and human. As expected from its structural similarity to lasp-1, lasp-2 possessed actin-binding activity and localized with actin filament in filopodia of neuroblastoma. In contrast to lasp-1, which is widely distributed in non-muscle tissues, lasp-2 was highly expressed in brain.     

陆地棉类谷氧还蛋(GhGRL)基因家族生物信息学分析

谷氧还蛋白(glutaredoxin, GRX)是一类小分子氧化还原蛋白,可以调节蛋白质的氧化还原状态从而维持蛋白质的功能,在生物的生长发育及抗氧化反应中起着重要的作用。类谷氧还蛋白蛋白(glutaredoxin-like, GRL)是新划分的GRX类型,本研究为深入探究GRL基因家族在陆地棉中功能,对GhGRL基因家族进行生物信息学及表达分析。研究结果表明,32个GhGRL基因主要定位于细胞核,它们均具有GRX-GRX-Like保守结构域。GhGRL基因所编码的氨基酸的多重序列比对和保守序列分析发现,该家族成员序列相似性约为31.31%,大部分包含4个保守基序,同时这4个保守基序与保守结构域重叠;根据GhGRL基因的系统进化树可将32个GhGRL基因分为3亚组,基因结构分析发现该家族基因大部分无内含子;染色体定位分析显示GhGRL基因分散在19个染色体上,每条染色体上的GhGRL基因数目有很大的差别;表达谱数据分析表明,大部分GhGRL基因在根、茎、雄蕊、雌蕊、子房、叶片和花等7个组织器官中均有表达,并且有差异。以上结果有利于了解棉花GhGRL基因家族的基本情况,为深入研究该基因家族在生物学功能提供基础。     

Molecular cloning of actin filament-associated protein: a putative adaptor in stretch-induced Src activation

Mechanical stretch-induced activation of c-Src is an important step for signal transduction of stretch-induced fetal rat lung cell proliferation. This process appears to be mediated through actin filament-associated protein (AFAP), encoded by a gene originally cloned from the chicken. In the present study, we cloned the rat AFAP gene from fetal rat lungs. Its mRNA and protein are differentially expressed among various tissues. The protein is colocalized with actin filaments in fetal rat lung epithelial cells and fibroblasts. Mechanical stretch increased tyrosine phosphorylation of rat AFAP and its binding to c-Src within the initial several minutes. Src SH2 and SH3 binding motifs are highly conserved in the AFAP proteins (from chicken, rat to human). On the basis of the molecular structure of AFAP protein, we speculate that it is an adaptor in mechanical stretch-induced activation of c-Src. A novel model of mechanoreception is proposed.     

Chromosomal assignment and expression pattern of the murine Lasp-1 gene

The human Lasp-1 (LIM and SH3 protein) gene was previously identified by differential screening of a breast cancer-derived metastatic lymph node cDNA library. It was located on the q12–q21 region of human chromosome 17 and was shown to be amplified and overexpressed in 12% of breast tumours. Lasp-1 defines a new LIM-protein subfamily, as it associates a C-terminal Src homology 3 (SH3) domain to a N-terminal LIM motif. In this study, the isolation and characterization of the cDNA encoding the mouse Lasp-1 protein are described, and it is shown to be highly conserved with its human counterpart. In addition to the LIM and SH3 domains, both human and mouse Lasp-1 contain an actin-binding domain. The mouse gene was mapped by in situ hybridization to the 11C–11D region of chromosome 11. Northern blot analysis shows that this gene is expressed from 7.5 to 17.5 days post-coitum of mouse embryogenesis and in almost all adult tissues.     

Schistosoma haematobium: analysis of eggshell protein genes and their expression

Eggshell protein genes of Schistosoma mansoni that encode a 14 kDa protein have been shown to be highly conserved and expressed in a sex-, tissue-, and temporal-specific manner. To initiate studies on the eggshell protein genes of S. haematobium, a cDNA probe, pSMf 61-46, representing a S. mansoni eggshell protein mRNA was used to screen a S. haematobium genomic library. Of the seven independent recombinant clones isolated, two (lambda SH 2-1 and lambda SH 6-1) were analyzed and compared to those of S. mansoni. lambda SH 2-1 and lambda SH 6-1 each contain a different genomic copy of the gene encoding a 19.8 and 17.6 kDa protein, respectively. This is due to an additional 78 bp present in the coding region of lambda SH 2-1 relative to lambda SH 6-1. The rest of the coding sequences are identical, and the 5' and 3' untranslated regions are nearly identical. The deduced amino acid sequences of S. haematobium eggshell proteins are very rich in glycine (47 and 50%) when compared to 43.5% glycine in the protein encoded by S. mansoni. Long stretches of glycines, as many as 15 in a row, occur in the S. haematobium sequence. DNA comparison of the eggshell protein genes of the two schistosome species yielded an overall homology of 83.1%. The homology is much higher in the 5' and 3' untranslated regions than in the protein-coding regions. Genomic clones of both species contained second open reading frames, which appeared to be kept open as a consequence of the amino acid composition of the other. There are no introns in S. haematobium or S. mansoni eggshell protein genes, and the genomic Southern data indicated a similar arrangement of these genes in the genome of both species. Primer extension experiments and dideoxynucleotide sequencing of the RNA determined the mRNA cap site sequence as ATCAT and ATCAC in lambda SH 2-1 and lambda SH 6-1, respectively. Northern blot analysis determined the size of the mRNA to be about 1.0 kp. Expression of the RNA from these genes appears to be regulated in a manner similar to the corresponding genes in S. mansoni. mRNA is found only in mature females and first appears at 70 days after infection of hamsters. DNA sequence comparisons of the 5' flanking regions of S. haematobium and S. mansoni eggshell protein genes to each other and to those of silkmoth and Drosophila revealed several short sequence elements that are shared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presence of SH2 domains of phospholipase C gamma 1 enhances substrate phosphorylation by increasing the affinity toward the epidermal growth factor receptor.

src homology region 2 and 3 (SH2 and SH3) domains are conserved noncatalytic regions originally described in cytoplasmic tyrosine kinases and subsequently identified in phospholipase C gamma 1 (PLC gamma 1), GTPase-activating protein of ras, and other signaling proteins. Although numerous studies indicate that SH2 domains promote protein-protein interactions by specific binding to tyrosine phosphorylated proteins, the function of SH3 domains is not known. The SH2 domain of PLC gamma 1 binds to certain tyrosine-phosphorylated growth factor receptors, and following phosphorylation on Tyr783 the enzymatic activity of PLC gamma 1 is enhanced, leading to phosphatidylinositol hydrolysis. To determine the functional role of the SH2 domain(s) on substrate phosphorylation in quantitative terms, we have expressed in Escherichia coli PLC gamma 1 constructs encoding the region containing Tyr783 and Tyr771, their two flanking SH2 domains and the SH3 domain, and five different deletion mutants of this region. These six proteins were purified and subjected to quantitative phosphorylation by the epidermal growth factor receptor (EGFR). Analysis of the kinetics of substrate phosphorylation revealed similar Vmax for the phosphorylation of the various mutant proteins. However, the affinity was enhanced for substrates containing SH2 domains: from S0.5 (average apparent Km) of 110 microM to S0.5 of 20 microM with the addition of a single SH2 domain and S0.5 of 3-4 microM for mutants containing two SH2 domains. The presence of the SH3 domain did not influence the apparent Km of substrate phosphorylation. These results demonstrate that the presence of the SH2 domain in PLC gamma 1 lowers the apparent Km (increases the affinity) of substrate phosphorylation by the EGFR, thereby facilitating PLC gamma 1 phosphorylation and activation.     

Involvement of glutaredoxin-1 and thioredoxin-1 in beta-amyloid toxicity and Alzheimer's disease

Strong evidence indicates oxidative stress in the pathogenesis of Alzheimer's disease (AD). Amyloid beta (Abeta) has been implicated in both oxidative stress mechanisms and in neuronal apoptosis. Glutaredoxin-1 (GRX1) and thioredoxin-1 (TRX1) are antioxidants that can inhibit apoptosis signal-regulating kinase (ASK1). We examined levels of GRX1 and TRX1 in AD brain as well as their effects on Abeta neurotoxicity. We show an increase in GRX1 and a decrease in neuronal TRX1 in AD brains. Using SH-SY5Y cells, we demonstrate that Abeta causes an oxidation of both GRX1 and TRX1, and nuclear export of Daxx, a protein downstream of ASK1. Abeta toxicity was inhibited by insulin-like growth factor-I (IGF-I) and by overexpressing GRX1 or TRX1. Thus, Abeta neurotoxicity might be mediated by oxidation of GRX1 or TRX1 and subsequent activation of the ASK1 cascade. Deregulation of GRX1 and TRX1 antioxidant systems could be important events in AD pathogenesis.     

Localization of glutaredoxin (thioltransferase) in the rat brain and possible functional implications during focal ischemia.

We investigated the distribution of glutaredoxin (GRX, thioltransferase) in the rat brain using the in situ hybridization and immunohistochemical methods. GRX mRNA and GRX were expressed widely in the rat brain. The endothelial cell, tanycyte and ependymal cell expressed GRX mRNA and GRX protein. Neurons in various regions also showed GRX mRNA and GRX. Among them, pyramidal neurons in hippocampal CA3 region expressed a higher level of GRX mRNA. In addition, GRX mRNA signals were reduced after middle cerebral artery occlusion. Immunohistochemical analysis for GRX also revealed that GRX was reduced after ischemia. Northern blot analysis also showed that GRX mRNA from ischemic hemispheres decreased after ischemia. This reduction was parallel with the neuronal damage. This observation indicated that the maintenance of GRX and the redox regulating system was important for neuronal survival against oxidative stress.