Protein and Peptide Letters - Volume 28, Issue 9, 2021

Aim: To identify naturally occurring variants of IAPP capable of inhibiting the aggregation of human IAPP and protecting living cells from the toxic effects of human IAPP. Background: The loss of insulin-producing β-cells and the overall progression of type 2 diabetes appears to be linked to the formation of toxic human IAPP (hIAPP, Islet Amyloid Polypeptide, amylin) amyloid in the pancreas. Inhibiting the initial aggregation of hIAPP has the potential to slow, if not stop entirely, the loss of β-cells and halt the progression of the disease. Objective: To identify and characterize naturally occurring variants of IAPP capable of inhibiting human IAPP aggregation. Methods: Synthetic human IAPP was incubated with synthetic IAPP variants identified from natural sources under conditions known to promote amyloid-based aggregation. To identify IAPP variants capable of inhibiting human IAPP aggregation, Thioflavin T-binding fluorescence, atomic force microscopy, and cell-rescue assays were performed. Results: While most IAPP variants showed little to no ability to inhibit human IAPP aggregation, several variants showed some ability to inhibit aggregation, with two variants showing substantial inhibitory potential. Conclusion: Several naturally occurring IAPP variants capable of inhibiting human IAPP aggregation were identified and characterized.

Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio-function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta- barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

The main role of platelets is to contribute to hemostasis. However, under pathophysiological conditions, platelet activation may lead to thrombotic events of cardiovascular diseases. Thus, anti-thrombotic treatment is important in patients with cardiovascular disease. This review focuses on a platelet receptor, a transmembrane protein, the Multidrug Resistance Protein 4, MRP4, which contributes to platelet activation, by extruding endogenous molecules responsible for their activation and accumulation. The regulation of the intracellular concentration levels of these molecules by MRP4 turned to make the protein suspicious and at the same time an interesting regulatory factor of platelet normal function. Especially, the possible role of MRP4 in the excretion of xenobiotic and antiplatelet drugs such as aspirin is discussed, thus imparting platelet aspirin tolerance and correlating the protein with the ineffectiveness of aspirin antiplatelet therapy. Based on the above, this review finally underlines that the development of a highly selective and targeted strategy for platelet MRP4 inhibition will also lead to inhibition of platelet activation and accumulation.

Serine is ubiquitously synthesized in all living organisms from the glycolysis intermediate 3-phosphoglycerate (PGA) by phosphoserine biosynthetic pathway, consisting of three different enzymes, namely: 3-phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Any functional defect or mutation in these enzymes may cause deliberating conditions, such as colon cancer progression and chemoresistance in humans. Phosphoserine aminotransferase (PSAT) is the second enzyme in this pathway that converts phosphohydroxypyruvate (PHP) to O-phospho-L-serine (OPLS). Humans encode two isoforms of this enzyme: PSAT1 and PSAT2. PSAT1 exists as a functional dimer, where each protomer has a large and a small domain; each large domain contains a Lys residue that covalently binds PLP. The PLP-binding site of human PSAT1 and most of its active site residues are highly conserved in all known PSAT structures except for Cys-80. Interestingly, Two PSAT structures from different organisms show halide binding near their active site. While the human PSAT1 shows a water molecule at this site with different interacting residues, suggesting the inability of halide binding in the human enzyme. Analysis of the human PSAT1 structure showed a big patch of positive charge around the active site, in contrast to the bacterial PSATs. Compared to human PSAT1, the PSAT2 isoform lacks 46 residues at its C-terminal tail. This tail region is present at the opening of the active site as observed in the other PSAT structures. Further structural work on human PSAT2 may reveal the functional importance of these 46 residues.

Background: Chitin, the second most abundant polysaccharide in nature, is a constantly valuable and renewable raw material after cellulose. Due to advancement in technology, industrial interest has grown to take advantage of the chitin. Objective: Now, biomass is being treated with diverse microbial enzymes or cells for the production of desired products under best industrial conditions. Glycosidic bonds in chitin structure are degraded by chitinase enzymes, which are characterized into number of glycoside hydrolase (GHs) families. Methods: Thermophilic microorganisms are remarkable sources of industrially important thermostable enzymes, having ability to survive harsh industrial processing conditions. Thermostable chitinases have an edge over mesophilic chitinases as they can hydrolyse the substrate at relatively high temperatures and exhibit decreased viscosity, significantly reduced contamination risk, thermal and chemical stability and increased solubility. Various methods are employed to purify the enzyme and increase its yield by optimizing various parameters such as temperature, pH, agitation, and by investigating the effect of different chemicals and metal ions etc. Results: Thermostable chitinase enzymes show high specific activity at elevated temperature which distinguish them from mesophiles. Genetic engineering can be used for further improvement of natural chitinases, and unlimited potential for the production of thermophilic chitinases has been highlighted due to advancement in synthetic biological techniques. Thermostable chitinases are then used in different fields such as bioremediation, medicine, agriculture and pharmaceuticals. Conclusion: This review will provide information about chitinases, biotechnological potential of thermostable enzyme and the methods by which they are being produced and optimized for several industrial applications. Some of the applications of thermostable chitinases have also been briefly described.

Background: Heat shock proteins (HSPs) represent a group of important proteins which are produced by all kinds of organisms especially under stressful conditions. DnaK, an Hsp70 homolog in prokaryotes, has indispensable roles when microbes was confronted with stress conditions. However, few data on DnaK from Rhodococcus sp. were available in the literature. In a previous study, we reported that toluene and phenol stress gave rise to a 29.87-fold and 3.93-fold increase for the expression of DnaK from R. ruber SD3, respectively. Thus, we deduced DnaK was in correlation with the organic solvent tolerance of R. ruber SD3. Objective: To elucidate the role of DnaK in the organic solvent tolerance of R. ruber SD3, expression, purification and functional analysis of Dnak from R. ruber SD3 were performed in the present paper. Methods: In this article, DnaK from R. ruber SD3 was heterologously expressed in E. coli BL21(DE3) and purified by affinity chromatography. Functional analysis of DnaK was performed using determination of kinetics, docking, assay of chaperone activity and microbial growth. Results: The recombinant DnaK was rapidly purified by affinity chromatography with the purification fold of 1.9 and the recovery rate of 57.9%. Km, Vmax and Kcat for Dnak from R. ruber SD3 were 80.8 μM, 58.1 nmol/min and 374.3 S^-1, respectively. The recombinant protein formed trimer in vitro, with the calculated molecular weight of 214 kDa. According to in-silico analysis, DnaK interacted with other molecular chaperones and some important proteins in the metabolism. The specific activity of catalase in the presence of recombinant DnaK was 1.85 times or 2.00 times that in the presence of BSA or Tris-HCl buffer after exposure to 54 °C for 1h. E. coli transformant with pET28-dnak showed higher growth than E. coli transformant with pET28 at 43°C and in the presence of phenol, respectively. Conclusion: The biochemical properties and the interaction analysis of DnaK from R. ruber SD3 deepened our understanding of DnaK function. DnaK played an important role in microbial growth when R. ruber was subjected to various stress such as heating and organic solvent.

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

Background: Thermophilic fungi have recently emerged as a promising source of thermostable enzymes. Superoxide dismutases are key antioxidant metalloenzymes with promising therapeutic effects in various diseases, both acute and chronic. However, structural heterogeneity and low thermostability limit their therapeutic efficacy. Objective: Although several studies from hypethermophilic superoxide dismutases (SODs) have been reported, information about Cu,Zn-SODs from thermophilic fungi is scarce. Chaetomium thermophilum is a thermophilic fungus that could provide proteins with thermophilic properties. Methods: The enzyme was expressed in Pichia pastoris cells and crystallized using the vapor-diffusion method. X-ray data were collected, and the structure was determined and refined to 1.56 Å resolution. Structural analysis and comparisons were carried out. Results: The presence of 8 molecules (A through H) in the asymmetric unit resulted in four different interfaces. Molecules A and F form the typical homodimer which is also found in other Cu,Zn- SODs. Zinc was present in all subunits of the structure while copper was found in only four subunits with reduced occupancy (C, D, E and F). Conclusion: The ability of the enzyme to form oligomers and the elevated Thr:Ser ratio may be contributing factors to its thermal stability. Two hydrophobic residues that participate in interface formation and are not present in other CuZn-SODs may play a role in the formation of new interfaces and the oligomerization process. The CtSOD crystal structure reported here is the first Cu,Zn-SOD structure from a thermophilic fungus.

Background: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent. Objective: This study aimed to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide. Methods: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A) were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain. Results: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and it exhibited larvicidal activity against Aedes aegypti. Conclusion: B. subtilis was successfully developed as a host for the production of secreted Mtx2, and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.

Background: Cancers of cervix, head and neck regions have been found to be associated with Human Papilloma Virus (HPV) infection. E1 protein makes an important papillomavirus replication factor. Among the ORFs of papillomaviruses, the most conserved sequence is that of the E1 ORF. It is the viral helicase with being a member of class of ATPases associated with diverse cellular activities (AAA+) helicases. The interactions of E1 with human DNA and proteins occurs in the presence of short linear peptide motifs on E1 identical to those on human proteins. Methods: Different Motifs were identified on HPV16 E1 by using ELMs. Elastic network models were generated by using 3D structures of E1. Their dynamic fluctuations were analyzed on the basis of B factors, correlation analysis and deformation energies. Results: 3 motifs were identified on E1 which can interact with Cdk and Cyclin domains of human proteins. 11 motifs identified on E1 have their CDs of Pkinase on human proteins. LIG_MYND_2 has been identified as involved in stabilizing interaction of E1 with Hsp40 and Hsp70. These motifs and amino acids comprising these motifs play a major role in maintaining interactions with human proteins, ultimately causing infections leading to cancers. Conclusion: Our study identified various motifs on E1 which interact with specific counter domains found in human proteins, already reported having the interactions with E1. We also validated the involvement of these specific motifs containing regions of E1 by modeling elastic networks of E1. These motif involving interactions could be used as drug targets.

Background: Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation. Objective: A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario. Methods: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein. Results: The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested. Conclusion: The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.