Protein and Peptide Letters - Volume 21, Issue 7, 2014

Aminoacyl-tRNA protein transferases post-translationally conjugate an amino acid from an aminoacyl-tRNA onto the N-terminus of a target polypeptide. The eubacterial aminoacyl-tRNA protein transferase, L/F transferase, utilizes both leucyl-tRNALeu and phenylalanyl-tRNAPhe as substrates. X-ray crystal structures with substrate analogues, the minimal substrate phenylalanyl adenosine (rA-Phe) and inhibitor puromycin, have been used to characterize tRNA recognition by L/F transferase. However analyses of these two X-ray crystal structures reveal significant differences in binding. Through structural analyses, mutagenesis, and enzymatic activity assays, we rationalize and demonstrate that the substrate analogues bind to L/F transferase with similar binding affinities using a series of different interactions by the various chemical groups of the analogues. Our data also demonstrates that enlarging the hydrophobic pocket of L/F transferase selectively enhances puromycin inhibition and may aid in the development of improved inhibitors for this class of enzymes.

Geranylgeranyl diphosphate synthase (GGPP synthase or GGPPS) is among the short-chain prenyltransferases, catalyzing the formation of the acyclic precursor GGPP for the biosynthesis of a variety of isoprenoids. GGPPSs have been extensively studied in plants, eubacteria, archaebacteria, yeast and mammals, but up to now information about an insect GGPPS is still scarce. Here we cloned the cDNA encoding an insect GGPPS from the cotton aphid (designated as AgGGPPS). AgGGPPS had an open reading frame of 930 bp, coding for 309 amino acids, with a theoretical pI/Mw of 6.21/35.7kDa. Sequence analysis showed that the amino acid sequence of AgGGPPS included the conserved aspartaterich motifs characterized by all prenyltransferases known to date, and could be classified as type-III GGPPS. Phylogenetic analysis showed that all animal GGPPSs were placed in a large group next to fungal GGPPS, and plant and bacterial GGPPSs formed another large group. AgGGPPS was over-expressed in Escherichia coli, and recombinant protein was purified by affinity chromatography. In vitro enzymatic activity assay coupled with product identification by gas chromatography- mass spectrometry demonstrated that AgGGPPS could catalyzed the formation of GGPP with DMAPP, GPP or FPP as the allylic cosubstrates in the presence of IPP, suggesting that AgGGPPS accepted not only FPP but also GPP and DMAPP as the allylic cosubstrate, different from other type-III GGPPSs which accepted only FPP as the allylic cosubstrate. This is the first report that a novel catalytic property exists in the type-III animal GGPPSs.

Ubiquitin carboxyl hydrolase L1 (UCH-L1) is an abundant multifunctional neuron protein. It plays an important role in maintaining the ubiquitin proteasome system (UPS), vital for recognizing and degrading dysfunctional proteins in organisms. In recent decades, UCH-L1 has been implicated in the pathogenesis of many diseases, including neurodegenerative disorders, cancer and diabetes. However, the mechanisms of UCH-L1 involvement have yet to be revealed in detail. Since UCH-L1 contributes many different functions to cell metabolism, its role and regulation might be more complex than previously thought and it has become a research target in many laboratories. In this review, we summarize recent findings related to the actions of UCH-L1 in several human diseases.

Hatching enzyme (HE) is secreted from the blastula stage during fertilization and can cleave the egg membrane. The structural identification and proteolytic effects on the collagen and fibrinogen were investigated in this study. Approximate 20 kDa of Asn-linked oligosaccharides were attached to the HE. Five peptide fragments of the starfish HE were homogenous to those of the coat matrix protein of starfish Patiria pectinifera. Amino acids of the starfish HE consisted of mainly Leu (10.0%), Asp (12.5%), and Glu (12.8%). Collagenolytic and fibrinolytic activities of the starfish HE were weaker than those of collagenase and α-chymotrypsin. The degree values of hydrolysis for collagenase and α- chymotrypsin were significantly higher than those of HE in a dose- and time-dependent manner. The peptide mappings of the starfish HE on the collagenolysis (110.7, 84.7, and 20.8 kDa) and fibrinogenolysis (34, 30, and 29 kDa) were different from those of collagenase and α-chymotrypsin. Based on the proteolytic effects on the collagen and fibrinogen, the starfish hatching enzyme might have the potential application to remove the matrix composition in scar or keloid tissue.

Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBSCT180; C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGI α, PKG-Iα1-159 play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBSCT180 prevents an atomic level understanding of the MBS–PKG contractile complex. MBSCT180 is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBSCT180 (Sharma et al, Prot Expr Purif 2012). Here we report 1H, 13C, 15N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

Lethal Toxin Neutralizing Factor (LTNF) isolated from Opossum (Didephis virginiana) has been shown to exhibit anti-venom and anti-allergic property. The small synthetic peptide- LT10 derived from N-terminal of LTNF also showed this property in vivo. We applied molecular modeling, docking and molecular dynamic (MD) simulation techniques to compute the interaction of LT10 peptide with few snake venom enzymes, namely PLA2 from Naja naja and Atrolysin –C from Crotalus atrox. Our in silico interaction analyses provides molecular basis that might account for antivenom activity of LT10 peptide towards these two snake venom toxins. The understanding of such key molecular interactions could be useful for design and development of better anti-snake venom therapies.

A comparison between three-dimensional structures of a wild-type xylanase AoXyn11A and a hybrid xylanase AEx11A revealed that a disulfide bridge (Cys5-Cys32) and an N-glycosylation site (Asn42) were imported into AEx11A by N-terminal substitution of AoXyn11A with EvXyn11TS. Two mutant genes AEx11A C5T and AEx11A N42Q were constructed by mutating Cys5- and Asn42-encoding codons of AEx11A into Thr5- and Gln42-encoding ones, and heterologously expressed in Pichia pastoris GS115, respectively. The temperature optimum of the recombinant AEx11AC5T (reAEx11AC5T) was decreased to 60°C from 80°C of reAEx11A, while its thermal inactivation half-lives at 70 and 80°C shortened to 3 and 1 min from 197 and 25 min of reAEx11A, respectively. However, there was no obvious alteration between reAEx11A and reAEx11AC5T in pH characteristics and kinetic parameters. Furthermore, both reAEx11AN42Q and reAEx11A displayed no significant difference in all enzymatic properties tested, except for the apparent molecular weight. We concluded based on this study that the disulfide bridge of AEx11A was vital to its high thermostability, but the Nglycosylation had no effect on.

Aspergillus fumigatus is a saprophytic fungus as well as a so-called opportunist pathogen. Its biochemical potential and enzyme production justify intensive studies about biomolecules secreted by this microorganism. We describe the alkaline serine peptidase production, with optimum activity at 50°C and a pH of 7.5 and a reduction in proteolytic activity in the presence of the Al+3 ions. When using intramolecularly quenched fluorogenic substrates, the highest catalytic efficiency was observed with the amino acid leucine on subsite S’3 (60,000 mM-1s-1) and preference to non-polar amino acids on subsite S3. In general, however, the peptidase shows non-specificity on other subsites studied. According to the biochemical characteristics, this peptidase may be an important biocatalyst for the hydrolysis of an enormous variety of proteins and can constitute an essential molecule for the saprophytic lifestyle or invasive action of the opportunistic pathogen. The peptidase described herein exhibits an estimated molecular mass of 33 kDa. Mass spectrometry analysis identified the sequence GAPWGLGSISHK displaying similarities to that of serine peptidase from Aspergillus fumigatus. These data may lead to a greater understanding of the advantageous biochemical potential, biotechnological interest, and trends of this fungus in spite of being an opportunist pathogen.

The objective of this study was to design and synthesize a new CPP-PNA conjugate that would be able to penetrate endothelial cells, bind STAT1 mRNA and thereby block the activity of STAT1 (the Signal Transducer and Activator of Transcription 1), which is important in cases of vessel inflammation. In the course of the study, the TAMRA-PTD-4- Hal(traziole-Gly-PNA)-conjugate was successfully synthesized using a specific 1,3-dipolar Huisgen cycloaddition reaction known as a “click reaction”. The hybridization properties of the conjugate to complementary hSTAT1 mRNA and hSTAT1 ssDNA fragments was verified by capillary electrophoresis (CE). Studies have shown that the attachment of a fluorescence-labeled peptide to a PNA sequence via a 1,2,3-triazole ring did not alter the binding properties of the PNA to the complementary hSTAT1 mRNA or hSTAT1 ssDNA fragments maintaining similar binding affinity. Furthermore, the data obtained suggest that the use of such a conjugate to modulate the activity and expression of STAT1 could provide a new therapeutic strategy for atherosclerosis treatment.

RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous doublestranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein’s ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA’s pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase- inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

Identifying protein complexes in protein-protein interaction (PPI) networks is a fundamental problem in computational biology. High-throughput experimental techniques have generated large, experimentally detected PPI datasets. These interactions represent a rich source of data that can be used to detect protein complexes; however, such interactions contain much noise. Therefore, these interactions should be validated before they could be applied to detect protein complexes. We propose an efficient measure to estimate PPI reliability (PPIR) and reduce noise level in two different yeast PPI networks. PPIRU, which is a new protein complex clustering algorithm based on PPIR, is introduced. Experiments demonstrated that interactome graph weighting methods incorporating PPIR clearly improve the results of several clustering algorithms. PPIR also outperforms other PPI graph weighting schemes in most cases. We compare PPIRU with several efficient, existing clustering algorithms and reveal that the accuracy values of PPIRU clusters are much higher than those of other algorithms.

A 64-kDa dimeric lectin was purified from Phaseolus vulgaris cv. Japanese mottled beans. The purification protocol involved ion exchange chromatography with Q-Sepharose and SP-Sepharose and size exclusion chromatography on Superdex 75. The lectin was adsorbed on both Q-Sepharose and SP-Sepharose columns. Finally, the lectin gave a sharp absorbance peak which corresponded to 64 kDa based on results of size exclusion chromatography. Sodium dodecyl sulphate- polyacrylamide gel electrophoresis displayed a single band at around 32 kDa, indicating that the protein was dimeric. The hemagglutination inhibition assay indicated that the lectin showed specificity toward galactose. The lectin preserved hemagglutinating activity below 70 °C and at a pH range 3 – 12. The lectin was able to inhibit proliferation of MCF-7 cells and Hep G2 cells and possessed antifungal activity toward Mycosphaeralla arachidicola with an IC50 value of 3.9 µM. The activity of HIV-1 reverse transcriptase was reduced by 61.9 % in the presence of the lectin at 6.25 µM concentration.