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In the current paper, two presumable mechanisms of protein folding are discussed; one of them, the 'nucleation mechanism', is exemplified by 3β-corner domains containing proteins, while the other, 'structural units mechanism', is exemplified by serine proteases. The analysis of the spatial structure of 3β-corners and of the common features of the amino acid sequences encoding them made it possible to conclude, that 3β-corners are capable of assuming their unique structures on their own and can serve as the nuclei or preformed structural units in the process of protein folding. The high order protein structures may be obtained by the further successive addition of β strands to a 3β-corner acting as a nucleus, according to certain rules and restrictions. On the other hand, 3β-corner may serve as a preformed structural unit, and the association of two 3β-corners may result in the formation of such 3D structures, which can be found in the domains of serine proteases and similar proteins. [ABSTRACT FROM AUTHOR]

Granzymes are a family of serine proteases found in the lytic granules of cytotoxic T lymphocytes and natural killer (NK) cells, which are involved in killing of susceptible target cells. Most information on granzymes and their enzymatic specificities derive from studies in humans and mice. Although granzymes shared by both species show a high level of conservation, the complement of granzyme genes differs between the species. The aim of this study was to identify granzyme genes expressed in cattle, determine their genomic locations and analyse their sequences to predict likely functional specificities. Orthologues of the five granzyme genes found in humans (A, B, H, K and M) were identified, as well a novel gene designated granzyme O, most closely related to granzyme A. An orthologue of granzyme O was found in pigs and a non-function version was detected in the human genome. Use of specific PCRs demonstrated that all of these genes, including granzyme O, are expressed in activated subsets of bovine lymphocytes, with particularly high levels in CD8 T cells. Consistent with findings in humans and mice, the granzyme-encoding genes were located on three distinct genomic loci, which correspond to different proteolytic enzymatic activities, namely trypsin-like, chymotrypsin-like and metase-like. Analysis of amino acid sequences indicated that the granzyme proteins have broadly similar enzymatic specificities to their human and murine counterparts but indicated that granzyme B has a different secondary specificity. These findings provide the basis for further work to examine their role in the cytotoxic activity of bovine CD8 T cells. [ABSTRACT FROM AUTHOR]

Seryl-histidine dipeptide (Ser-His) has been recognized as the shortest peptide with hydrolysis cleavage activity; however, its protein cleavage spectrum has not yet been fully explored. Here, four differently folded proteins were treated with Ser-His, and the digestion products were evaluated with high-resolution mass spectrometry. The cleavage efficiency and cleavage propensity of Ser-His against these protein substrates were calculated at both the primary and secondary sequence levels. The above experiments show that Ser-His cleaves a broad spectrum of substrate proteins of varying secondary structures. Moreover, Ser-His could cleave at all 20 amino acids with different efficiencies according to the protein, which means that Ser-His has the original digestion function of serine proteases. Furthermore, we collected and compared the catalytic sites and cleavage sites of 340 extant serine proteases derived from 17 representative organisms. A consensus motif Ser-[X]-His was identified as the major pattern at the catalytic sites of serine proteases from all of the organisms represented except Danio rerio, which uses Ser-Lys instead. This finding indicates that Ser-His is the core component of the serine protease catalytic site. Moreover, our analysis revealed that the cleavage sites of modern serine proteases have become more specific over the evolutionary history of this family. Based on the above analysis results, it could be found that Ser-His is likely the original serine protease and maybe the evolutionary core of modern serine proteases. [ABSTRACT FROM AUTHOR]

A new member of the genus Nepovirus was isolated from blueberry in Japan. The virus was associated with latent infection of blueberry trees and provisionally named blueberry latent spherical virus (BLSV). BLSV was found to have isometric particles approximately 30 nm in diameter, which were composed of a single coat protein (CP) of 55 kDa. The viral genome consisted of two positive-sense single-stranded RNA species (RNA1 and RNA2), which were 7,960 and 6,344 nucleotides (nt) long, respectively. The organization of RNA1 and RNA2 was similar to that of nepoviruses. The 3′ non-coding regions of RNA1 and RNA2 were 1,379 nt and 1,392 nt long, respectively. The amino acid sequences of the BLSV polymerase and CP shared the highest amino acid sequence similarities with those of the subgroup C nepoviruses (57% and 43%, respectively). Additionally, the BLSV genome, in contrast to other nepovirus genomes, was predicted to encode a serine protease. [ABSTRACT FROM AUTHOR]

Serine proteinases and Kunitz-type inhibitors are widely represented in the venoms of snakes belonging to different genera. During the studies of the venoms of snakes inhabiting Russia, we have cloned cDNAs coding for novel proteins of these families. A novel serine proteinase that we named nikobin was identified in the venom gland of the Nikolsky viper. The amino acid sequence of nikobin deduced from the cDNA sequence slightly differs from those of the serine proteinases found in other snakes, displaying 15 unique amino acid substitutions. This is the first serine proteinase from a viper of the Vipera genus for which the complete amino acid sequence has been determined. A cDNA coding for a Kunitz-type inhibitor has also been cloned. The deduced amino acid sequence of the inhibitor displays overall homology to the already known sequences of analogous proteins from vipers of the Vipera genus. However, several unusual amino acid substitutions that can cause a change of the inhibitor activity have been detected. [ABSTRACT FROM AUTHOR]

Abstract  A serine protease from Bothrops atrox (Peruvian specimen’s venom) was isolated in two chromatographic steps in LC molecular exclusion and reverse phase-HPLC. This protein was denominated Ba III-4 (33,080.265 Da determinated by MALDI-TOF mass spectrometry) and showed pI of 5.06, Km 0.2 � 10−1  M and the V m�x 4.1 � 10−1 nmoles p-NA/lt/min on the synthetic substrate BapNA. Ba III-4 also showed ability to coagulate bovine fibrinogen. The serine protease was inhibited by soyben trypsin inhibitor and DA2II, which is an anti-hemorrhagic factor isolated from the opossum specie Didelphis albiventris. The primary structure of Ba III-4 showed the presence of His(44), Asp(94) and Ser(193) residues in the corresponding positions to the catalytic triad established in the serine proteases and Ser(193) are inhibited by phenylmethylsulfonylfluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro, as well as 12 half-cysteine residues. Ba III-4 contained 293 amino acid residues and the primary structure of VIGGDECDIN EHPFLAFMYY SPRYFCGMTL INQEWVLTAA HCRYFCGMTL IHLGVHRESE KANYDEVRRF PKEKYFIFCD NNFTDDEVDK DIMLIRLDKP VSNSEHIAPL SLPSNPPSVG SVCRIMGWGQ TTTSPIDVLS PDEPHCANIN LFDNTVCHTA HPQVANTRTS TDTLCAGDLQ GGRDTCNGDS GGPLICNEQL HGILSWGGDP CAQPNKPAFY TKVYYFDHPW IKSIIAGNKK TVNFTCPPLR SDAKDDSTTY INQEWDWVLT AEHCDRTHMR NSFYDYSSIN SDS. Titration experiments did not show the presence of free sulfhydryl groups after 4 h incubation, nor were differences found in relation to titration kinetics in the presence of nondenaturating buffer. The isolation of this protein, Ba III-4, is of potential interest for the understanding of the pathomechanism of the snake venom action and for the identification of new blood coagulation enzymes of natural sources. [ABSTRACT FROM AUTHOR]

A trypsin-type serine proteinase (WSP) was purified previously from the sarcoplasmic fraction of skeletal muscle of white croaker ( Argyrosomus argentatus) by Yanagihara et al. ((1991) Nippon Suisan Gakaishi, 57, 133–142). However, further research on WSP was not carried out. In the present study, we determined the N-terminal amino acid sequence of this enzyme (27 amino acid residues), which revealed relatively high identity in the conserved region to other trypsin-type serine proteinases. Degradation action of WSP on neuropeptides is also reported in this manuscript. The results show that WSP only cleaves at the carboxyl side of Arg or Lys residue of the peptides, especially between dibasic amino acid residues such as Arg—Arg and Arg—Lys. [ABSTRACT FROM AUTHOR]