Protein and Peptide Letters - Volume 23, Issue 6, 2016

We have studied the effect of guanidinium chloride (Gdn.Cl) and different osmolytes such as betaine, trimethylamine-N-oxide (TMAO) and urea on the rate of chymotrypsin catalyzed reaction. The rates were measured using three synthetic chromogenic substrates, succinyl-ala-ala-pro-arg-pNA (AAPR), succinyl-ala-ala-pro-leu-pNA (AAPL), and succinyl-ala-ala-pro-phe-pNA (AAPF). Qualitatively, the results with the three substrates were identical. Guanidinium chloride and urea produced a linear decrease while TMAO produced a linear increase in the rate with increase in osmolyte concentration. Betaine had practically no effect on the rate of enzyme catalyzed reaction up to a concentration of 1.2 M. However, quantitatively the rate change per molar concentration of osmolyte (or Gdn.Cl) was significantly larger for AAPR that has a polar and cationic reactive site residue than the two substrates (AAPL and AAPF) that have non-polar reactive site residues. These results suggest that the chemical nature of the substrate (and presumably the active site of the enzyme) plays an important role in determining the effect of osmolytes in enzyme catalyzed reactions.

Arginine kinase is an essential enzyme which is closely related to energy metabolism in marine invertebrates. Arginine kinase provides a significant role in quick response to environmental change and stress. In this study, we simulated a tertiary structure of Sepia pharaonis arginine kinase (SPAK) based on the gene sequence and conducted the molecular dynamics simulations between SPAK and Zn2+. Using these results, the Zn2+ binding sites were predicted and the initial effect of Zn2+ on the SPAK structure was elucidated. Subsequently, the experimental kinetic results were compared with the simulation results. Zn2+ markedly inhibited the activity of SPAK in a manner of non-competitive inhibitions for both arginine and ATP. We also found that Zn2+ binding to SPAK resulted in tertiary conformational change accompanying with the hydrophobic residues exposure. These changes caused SPAK aggregation directly. We screened two protectants, glycine and proline, which effectively prevented SPAK aggregation and recovered the structure and activity. Overall, our study suggested the inhibitory effect of Zn2+ on SPAK and Zn2+ can trigger SPAK aggregation after exposing large extent of hydrophobic surface. The protective effects of glycine and proline against Zn2+ on SPAK folding were also demonstrated.

ESCRTs (Endosomal Sorting Complexes Required for Transport) are required for the formation of the intraluminal vesicles in the multivesicular bodies and are involved in other topologically similar processes such as cytokinesis, nuclear envelope sealing and viral egress. The final complex ESCRT-III is disassembled by the recruitment and activation of the AAA-ATPase VPS4 to the endosomal membranes. This recruitment is due to the binding of VPS4 N-terminal MIT with MIM1 and MIM2 domains present in the CHMPs proteins. By analyzing different cellular membrane remodeling events in which VPS4 is involved, here we provide evidence that the K61 residue, mapping within the MIT domain of VPS4B (K59 in VPS4A), is involved in VPS4 functioning. Posttranslational modifications of this residue might modulate MIT-MIM2 binding affinity and, as a consequence, VPS4 functions.

High levels of circulating immunoglobulin G (IgG) and serum albumin (SA) are maintained through recycling by the neonatal Fc receptor (FcRn). FcRn interacts with IgG and SA in a pHdependent manner and rescues them from lysosomal degradation. We have determined the crystal structure of extracellular domain of human FcRn, a heterodimeric complex of α-chain and β2- microglobulin, at pH 4.5. The structure was compared with the previously reported unliganded human FcRn structure at pH 8.5 and complex structures of FcRn bound to SA and/or Fc determined at acidic pHs. Structural differences are more pronounced between the two unliganded FcRn structures at pH 4.5 and pH 8.5 than between unliganded FcRn and the complex structures at acidic pHs. At acidic pH, protonation of H166 induces interactions with E54 and Y60 stabilizing the “WW loop” important for SA binding, and H161 interacts with E165 causing conformational changes of helix 3. These structural changes make the FcRn amenable for binding with SA at acidic pH. The Fc binding surface does not show any major main chain differences between the unliganded structures at pH 8.5 and pH 4.5. Side chain changes upon Fc binding were observed when compared with the complex structures. This suggests that major structural differences observed between unliganded and ligand bound structures are primarily due to pH changes rather than ligand binding.

Mammalian cells are widely used for recombinant protein production in research and biotechnology. Utilization of export signals significantly facilitates production and purification processes. 35 years after the discovery of the mammalian export machinery, there still are obscurities regarding the efficiency of the export signals. The aim of this study was the comparative evaluation of the efficiency of selected export signals using adipocytes as a cell model. Adipocytes have a large capacity for protein secretion including several enzymes, adipokines, and other signaling molecules, providing a valid system for a quantitative evaluation. Constructs that expressed N-terminal fusion export signals were generated to express Enhanced Green Fluorescence Protein (EGFP) as a reporter for quantitative and qualitative evaluation. Furthermore, fluorescent microscopy was used to trace the intracellular traffic of the reporter. The export efficiency of six selected proteins secreted from adipocytes was evaluated. Quantitative comparison of intracellular and exported fractions of the recombinant constructs demonstrated a similar efficiency among the studied sequences with minor variations. The export signal of Retinol Binding Protein (RBP4) exhibited the highest efficiency. This study presents the first quantitative data showing variations among export signals, in adipocytes which will help optimization of recombinant protein distribution.

The influence of buffer composition on the conformational stability of native and calciumdepleted Bacillus licheniformis α-amylase (BLA) was investigated against guanidine hydrochloride (GdnHCl) denaturation using circular dichroism, fluorescence and UV-difference spectroscopy. Differential effect of buffer composition on GdnHCl denaturation of BLA was evident from the magnitude of these spectral signals, which followed the order: sodium phosphate > Tris-HCl > HEPES > MOPS. These effects became more pronounced with calcium-depleted BLA. Sephacryl S-200 gel chromatographic results showed significant BLA aggregation in the presence of 6 M GdnHCl.

In this work, we report the synthesis of a peptide analogue of the KTTKS, termed Ac- Wahx-KTTKS and evaluate its cytotoxicity and role in biosynthesis of collagen for future application in skin aging. The peptide was obtained with purity higher than 97.5%. In the cytotoxicity assay, we observed non-toxic effects for Ac-WAhx-KTTKS at concentrations below 600 μM for HaCaT and 500 μM for HepG2 cells, respectively. After 24 and 48 h it was possible to observe significant changes in collagen synthesis in the groups treated with various concentrations of the peptide. In conclusion, the Ac-Wahx- KTTKS peptide increased collagen synthesis in fibroblasts by 80% and it is a promising candidate for improving skin aging.

Peptidase N (PepN) is a broad specific metallo-peptidase and the sole member of the M1 class encoded by Escherichia coli. Comparative analysis of residues present in the S1 subsite of E. coli PepN with other family members revealed that Tyr-381 is conserved whereas Glu-121, Gln-119 and Tyr-376 are partially conserved. The functional importance of these amino acids was investigated by protein engineering studies. The change in Glu-121 to Gln and Tyr-381 to Phe led to catalytically inactive PepN. At the same time, the change in Gln-119 to His (Q119H) and Tyr-376 to Phe (Y376F) led to alterations in substrate specificity. Kinetic studies revealed that purified PepN variants, Q119H and Y376F, cleaved some substrates (e.g. Arg) similar to wild type PepN. However, these variants displayed lower efficacy with other substrates (e.g. Tyr, AAF and Suc-AAF). Q119H or Y376F, cleave a natural peptide (insulin B chain) and a loosely folded protein (casein) with greatly reduced efficacy. The double mutant, i.e. harboring both Q119H and Y376F, displays greatly reduced catalytic activity with respect to all substrates studied. The in vivo significance was addressed by expressing these variants in ΔpepN during nutritional downshift and high temperature (NDHT) stress. Compared to wild type PepN, the Y376F and Q119H variants display lower intracellular amounts of free N-terminal amino acids and reduction in growth during NDHT stress. Finally, structural modeling, using the crystal structure of E. coli PepN bound to substrates, Arg or Tyr, shed insights into the roles of Q119H and Y376F in determining substrate preferences.

Falcipain-2 (FP2) is an important hemoglobinase from the malaria parasite Plasmodium falciparum and a suitable target for the development of an antimalarial chemotherapy. Many reports have indicated that radical nitrogen species (RNS) including nitric oxide (NO) are inhibitors of P. falciparum growth and promoters of recovery from malaria symptoms. In this scenario, FP2 emerges as a potential target of RNS, since its inhibition partially hinders the parasite growth. We report that in vitro FP2 did not undergo S-nitrosylation when exposed to the NO-donor GSNO. However, it was modified by a combined mechanism of methionine oxidation and tyrosine nitration in response to SIN-1, and NaNO2- H2O2 treatment. The treatments with the nitrating agents caused a pronounced decrease in protease activity most likely induced by a disruption on the secondary and tertiary structure of FP2. Our data also demonstrate that at least four tyrosine residues were nitrated and found on the surface of the enzyme, partially or completely exposed to the solvent. Although performed in vitro, these results suggest that falcipain-2 may be a target of RNS activity and its inhibition could explain the hindering of the parasite growth when exposed to these radicals. The understanding of the molecular mechanisms involving free radicals and its inhibition activity towards FP2 may be effective in the development of antimalarial therapies.

The high protein concentration, unique composition and complex geometry of the lens makes it transparent. α-, β-, and γ-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (ζ)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10 % of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions. ζ -Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of -crystallin in the eye lens is not well understood, however, truncated ζ-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of ζ-crystallin was reported. In this study, recombinant camel ζ-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant ζ-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the ζ-crystallin conformation, we have used far-UV and near-UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 50% of the of ζ-crystallin activity was lost at 50 μM DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of ζ-crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).

Hepatitis C virus (HCV) triggers coordinated innate and adaptive response in host cell. HCV genome and proteins of the replicating virus are recognized as non-self-antigens by host cell to activate Toll Like Receptors (TLRs). Activated TLRs ultimately express cytokines, which can clear virus either by activating interferon (IFN), protein kinase C (PKC) and RNA Lase system or through activation of cytotoxic T-lymphocytes. Interleukin-12 (IL-12) is a potent antiviral cytokine, capable of clearing HCV by bridging both innate and adaptive antiviral immune response. Activation of TLR-4 on macrophages surface induces expression of IL-12 via NF-ΚB and AP-1 transcriptional pathway. After expression, IL- 12 releases IFN-γ, which activates anti-HCV cytotoxic lymphocytes. Conversely, in chronic HCV infection downregulation of IL-12 has been reported instead of by number of studies. Keeping in view of the above mentioned facts, this study was designed to evaluate HCV-core mediated down-regulation of IL-12 transcriptional pathway by employing a logical modeling approach based on the Ren´e Thomas formalism. The logical parameters of entities were estimated by using SMBioNet. The Logical model represents all possible dynamics of protein expression involved during course of HCV pathology. Results demonstrated that at chronic stage of infection, though TLR-4 was constantly active but yet it failed to express the NF-ΚB, AP-1, IL-12 and IFN-. This mechanism was indicative of incorporation of core mediated changes in IL-12 regulatory pathway. Moreover, results also indicate that HCV adopts different trajectories to accomplish the persistence of chronic phase of infection. It also implicated that human immune system tries to clear HCV but core is capable of inducing system oscillations to evade the immunity.