Identification of a binding site in the disintegrin domain of fertilin required for sperm-egg fusion.

Fertilization and certain later stages in mammalian embryonic development require fusion between membranes of individual cells. The mechanism of eukaryotic cell-cell fusion is unknown, and no surface molecules required for this process have been unequivocally identified. The role of the sperm surface protein fertilin in sperm-egg fusion was tested by using peptide analogues of a potential integrin binding site in the fertilin beta subunit. Peptide analogues that include a TDE sequence from the disintegrin region of fertilin beta are able to bind to the egg plasma membrane and strongly inhibit sperm-egg fusion. These results show that the disintegrin domain of fertilin beta binds to the egg plasma membrane and that this binding is required for membrane fusion.     

Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease

Amyloid beta peptide (Abeta), the principal proteinaceous component of amyloid plaques in brains of Alzheimer's disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative alpha-secretase within the Abeta sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsalpha into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated alpha-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous alpha-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Abeta fragments confirm the correct alpha-secretase cleavage site and demonstrate a dependence on the substrate's conformation. Our results provide evidence that ADAM 10 has alpha-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer's disease.     

The presequence of Euglena LHCPII, a cytoplasmically synthesized chloroplast protein, contains a functional endoplasmic reticulum-targeting domain.

The precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) is unique; it is a polyprotein, synthesized on membrane-bound ribosomes and transported to the Golgi apparatus prior to chloroplast localization. A cDNA corresponding to the 5' end of LHCPII mRNA has been isolated and sequenced. The deduced amino acid sequence of this cDNA indicates that Euglena pLHCPII contains a 141-amino acid N-terminal extension. The N-terminal extension contains three hydrophobic domains and a potential signal peptidase cleavage site at amino acid 35. Cotranslational processing by canine microsomes removed approximately 35 amino acids from an in vitro synthesized 33-kDa pLHCPII composed of a 141-amino acid N-terminal extension and a 180-amino acid partial LHCPII unit truncated at the beginning of the third membrane-spanning hydrophobic domain. Processed pLHCPII was degraded by exogenous protease, indicating that it had not been translocated to the microsomal lumen. Extraction with 0.1 M Na2CO3, pH 11.5, did not remove the processed pLHCPII from the microsomal membrane. A stop-transfer membrane anchor sequence appears to anchor the nascent protein within the membrane, preventing translocation into the lumen. Taken together, these results provide biochemical evidence for a functional cleaved signal sequence within the N-terminal extension of a Euglena cytoplasmically synthesized chloroplast-localized protein.     

The hydrophobic internal region of bovine prion protein shares structural and functional properties with HIV type 1 fusion peptide

The conserved fusion peptide at the N-terminus of HIV-1 envelope glycoprotein gp41 is involved in the virus-cell fusion reaction and in the cytopathic effects promoted by expression in single cells. The conserved bovine prion protein 121KHVAGAAAAGAVVGGLGGYMLGSAMSR147 transmembrane region (BPrP(tm)) contains a sequence rich in Gly residues [i.e., 130GAVVGGLGGYMLGSAMSR147 (BPrP(mi))] that shows homology with HIV-1 fusion peptide. As in the case of the latter peptide, analysis of the BPrP(mi) interfacial hydrophobicity confirms that this stretch bears an intrinsic tendency to partitioning from the aqueous phase into membranes. Experimental analyses of BPrP(mi)-lipid interactions suggest several similarities between this sequence and HIV-1 fusion peptide. Infrared spectroscopy reveals a conformational plasticity of the membrane-associated prion sequence comparable to that displayed by the viral sequence. The adoption of a mainly alpha-helical structure correlates with the formation of lytic pores. This helical structure can be converted into a beta-sheet conformation by addition of calcium, a process that is accompanied by vesicle membrane fusion. In contrast, transmembrane BPrP(tm) associates with membranes in a nonlytic, mainly helical structure but also containing some random coil. Upon addition of calcium, the random coils disappear while the helical conformation remains. In the absence of membranes both prion and HIV-1 sequences form amyloid-type fibers. It is proposed that during the pathological processes induced by secreted PrPSc and its proteolytic fragments, conformational polymorphism displayed by membrane-inserted BPrP(mi) may play a role at perturbing the general architecture of the membrane lipid bilayer and inducing protein-protein aggregation at membrane surfaces. These findings suggest the existence of common mechanisms underlying cytotoxicity by PrP and HIV-1 gp41.     

A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats.

Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.     

去整合素金属蛋白酶10在糖尿病冠状动脉支架内再狭窄中作用的研究

目的:探讨去整合素金属蛋白酶10(ADAM10)在糖尿病冠状动脉(冠脉)支架内再狭窄(ISR)中的作用。方法:在17头糖尿病猪和10头正常猪的冠脉内置入雷帕霉素洗脱支架,6个月后行冠脉造影,留取发生和未发生ISR的冠脉组织,Western blot检测ADAM10表达水平。在人主动脉平滑肌细胞(HASMC)中感染ADAM10的过表达和敲减病毒,BrdU检测细胞增殖,划痕实验检测细胞迁移能力。分别用低糖培养基、高糖培养基、晚期糖基化终末产物-牛血清白蛋白(AGE-BSA)、AGE-BSA+AGE受体(RAGE)抗体培养HASMC,实时定量RT-PCR和Western blot检测ADAM10表达水平。结果:在糖尿病组中,未发生ISR和发生ISR的冠脉组织中ADAM10的表达均高于非糖尿病组(3.36±1.27对2.11±2.05,10.48±4.72对6.72±1.36,P均0.01)。在非糖尿病组和糖尿病组,发生ISR的冠脉组织中ADAM10的表达均高于未发生ISR的冠脉组织(P均0.01)。BrdU实验显示,在低糖培养基和高糖培养基中,ADAM10过表达的HASMC增殖均明显高于转染空载体的HASMC(2.25±0.07对1.87±0.08,2.47±0.10对2.07±0.10,P均0.05);而ADAM10敲减的HASMC增殖均明显低于转染空载体的HASMC(1.34±0.10对1.87±0.08,1.46±0.09对2.07±0.10,P均0.05);ADAM10过表达和ADAM10敲减的HASMC在高糖培养基中的增殖均明显高于低糖培养基(P均0.05)。细胞划痕实验显示,在低糖培养基和高糖培养基中,ADAM10过表达的HASMC迁移距离均明显大于转染空载体的HASMC[(1.02±0.12)mm对(0.65±0.04)mm,(1.26±0.06)mm对(0.78±0.06)mm,P均0.05)],而ADAM10敲减的HASMC迁移距离均明显小于转染空载体的HASMC[(0.26±0.06)mm对(90.65±0.04)mm,(0.43±0.14)mm对(0.78±0.06)mm,P均0.05)];ADAM10过表达和ADAM10敲减的HASMC在高糖培养基中的迁移距离均明显大于低糖培养基(P均0.05)。与低糖培养基相比,高糖培养基和AGE-BSA中HASMC ADAM10 mRNA和蛋白的相对表达水平均明显升高(P均0.05);与AGE-BSA相比,AGE-BSA+RAGE抗体中HASMC ADAM10 mRNA和蛋白的相对表达水平均明显降低(P均0.05)。结论:ADAM10在糖尿病发生ISR的冠脉中表达显著升高,ADAM10高表达促进动脉平滑肌细胞增殖和迁移,高糖环境及AGE均可促进ADAM10的表达,ADAM10可能参与了糖尿病冠脉ISR的发生与发展。     

TolA: a membrane protein involved in colicin uptake contains an extended helical region.

The group A colicins and the DNA of many single-stranded filamentous bacteriophage are able to use combinations of the Tol proteins to gain entrance into or across the membrane of Escherichia coli. The TolA protein is a 421-amino acid residue integral membrane protein composed of three domains. Domain I, consisting of the amino-terminal 47 amino acids, contains a 21-residue hydrophobic segment that anchors the protein in the inner membrane. The remaining 374 amino acids, containing the other two domains, reside in the periplasmic space. Domain III, consisting of the carboxyl-terminal 120 residues, is considered to be the functional domain based on the location of the tolA592 deletion mutation. The internal 262 amino acids comprise domain II, which connects domains I and III together via short regions of polyglycine. It contains a large number of 3- to 5-residue polyalanine stretches, many of which have a repeat of the sequence Lys-Ala-Ala-Ala-(Glu/Asp). Circular dichroism analysis of different portions of TolA show domain II to be predominantly alpha-helical in structure while domain III contains approximately 10% helical structure.     

Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications.

A computer analysis of 2328 protein sequences comprising about 60% of the Escherichia coli gene products was performed using methods for database screening with individual sequences and alignment blocks. A high fraction of E. coli proteins--86%--shows significant sequence similarity to other proteins in current databases; about 70% show conservation at least at the level of distantly related bacteria, and about 40% contain ancient conserved regions (ACRs) shared with eukaryotic or Archaeal proteins. For > 90% of the E. coli proteins, either functional information or sequence similarity, or both, are available. Forty-six percent of the E. coli proteins belong to 299 clusters of paralogs (intraspecies homologs) defined on the basis of pairwise similarity. Another 10% could be included in 70 superclusters using motif detection methods. The majority of the clusters contain only two to four members. In contrast, nearly 25% of all E. coli proteins belong to the four largest superclusters--namely, permeases, ATPases and GTPases with the conserved "Walker-type" motif, helix-turn-helix regulatory proteins, and NAD(FAD)-binding proteins. We conclude that bacterial protein sequences generally are highly conserved in evolution, with about 50% of all ACR-containing protein families represented among the E. coli gene products. With the current sequence databases and methods of their screening, computer analysis yields useful information on the functions and evolutionary relationships of the vast majority of genes in a bacterial genome. Sequence similarity with E. coli proteins allows the prediction of functions for a number of important eukaryotic genes, including several whose products are implicated in human diseases.     

The Escherichia coli hflA locus encodes a putative GTP-binding protein and two membrane proteins, one of which contains a protease-like domain.

The hflA (high frequency of lysogenization) locus of Escherichia coli governs the lysis-lysogeny decision of bacteriophage lambda by controlling stability of the phage cII protein. hflA contains three genes, hflX, hflK, and hflC, encoding polypeptides of 50, 46, and 37 kDa, respectively. We have determined the nucleotide sequence of 3843 base pairs containing hflA and have found three large open reading frames corresponding to hflX, hflK, and hflC. HflX contains the three sequence motifs typical of GTP-binding proteins and appears to be a member of a distinct family of putative GTPases. HflC and HflK appear to be integral membrane proteins which show some similarity to each other and to a human membrane protein. The C-terminal region of HflC contains a domain resembling the catalytic domain of ClpP, a bacterial ATP-dependent protease. We hypothesize that HflK and HflC constitute a distinct membrane-bound protease whose activity may be modulated by HflX GTPase.     

The ovarian reserve in mammals: a functional and evolutionary perspective

The constitution and the control of the ovarian reserve is of importance in mammals and women. In particular, the number of primordial follicles at puberty is positively correlated with the number of growing follicles and their response to gonadotropin treatments. The size of this ovarian reserve depends on genes involved in germ cell proliferation and differentiation, sexual differentiation, meiosis, germ cell degeneration, formation of primordial follicles, and on a potential mechanism of self-renewal of germ stem cells. In this review, we present the state of the art of the knowledge of genes and factors involved in all these processes. We first focus on the almost 70 genes identified mainly by mouse invalidation models, then we discuss the most plausible hypothesis concerning the possibility of the existence of germ cell self-renewal by neo-oogenesis in animal species and human, with a special interest for the role of corresponding genes in evolutionary distinct model species. All of the genes pointed out here are candidates susceptible to explain fertility defects such as the premature ovarian failure in human.     

Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid.

Primary sequences of cholinesterases and related proteins have been systematically compared. The cholinesterase-like domain of these proteins, about 500 amino acids, may fulfill a catalytic and a structural function. We identified an aspartic acid residue that is conserved among esterases and lipases (Asp-397 in Torpedo acetylcholinesterase) but that had not been considered to be involved in the catalytic mechanism. Site-directed mutagenesis demonstrated that this residue is necessary for activity. Analysis of evolutionary relationships shows that the noncatalytic members of the family do not constitute a separate subgroup, suggesting that loss of catalytic activity occurred independently on several occasions, probably from bifunctional molecules. Cholinesterases may thus be involved in cell-cell interactions in addition to the hydrolysis of acetylcholine. This would explain their specific expression in well-defined territories during embryogenesis before the formation of cholinergic synapses and their presence in noncholinergic tissues.     

Characterization of an amyloid beta precursor protein that binds heparin and contains tyrosine sulfate.

A secreted form of the amyloid beta protein precursor was isolated from the growth conditioned medium of the PC12 sympathetic nerve-like cell line. This protein is recognized by an antiserum that detects a protein of 140 kDa and a less abundant species of 115 kDa on NaDodSO4/acrylamide gels. The amyloid precursor proteins contain O-linked sugars and tyrosine sulfate and bind to the glycosaminoglycan heparin. These results suggest a role for extracellular sulfated glycoproteins in the pathogenesis of Alzheimer disease.     

ADAMTS-1: A cellular disintegrin and metalloprotease with thrombospondin motifs is a target for parathyroid hormone in bone

PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.