Protein and Peptide Letters - Volume 28, Issue 3, 2021

Background: Orientia tsutsugamushi (Ot) is an obligate, intracellular, gram-negative bacterium causing scrub typhus. Some of its encoded proteins play key roles in the adhesion and internalization of the Ot strain into host cells and are suitable resources for vaccine development and tools for scrub typhus diagnosis. Surface cell antigen (Sca) proteins, classified as autotransporter (AT) proteins, are one of the largest protein families involved in bacterial pathogenesis and can be promising candidates for vaccine development. These proteins are typically large and contain inhibitory domains; therefore, recombinant proteins without such domains have been evaluated for this purpose. However, the expression for recombinant Sca proteins containing the AT domain, which might largely affect their protective role against scrub typhus, has not been analyzed and optimized. Objective: In this study, we optimized expression and purification conditions for individual Ot Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] harboring the AT domain. Methods: To this end, we subcloned sequences of codon-optimized DNA encoding Sca protein fragments into the Escherichia coli expression vector. In addition, the expression condition for individual Sca fragments was optimized, and the proteins were purified using one-step Ni-NTA column method. The purified fractions were re-folded by serial dilution method, followed by BCA quantification and densitometric analysis to estimate the yield and purity of proteins. Results: We prepared platforms for expression of recombinant Sca protein fragments [ScaA (27–1461), ScaC (257–526), ScaD (26–998), and ScaE (35–760)] containing an AT domain without the signal peptide and transmembrane (TM) domain. The protein yield per liter of culture with >70% of purity was ScaC (257–576), ScaE (35–760), ScaD (26-998), and ScaA (27-1461) in order. Conclusion: Our results could be used to develop Sca AT-domain based vaccines and tools for scrub typhus diagnosis with rapid and cost-effective ways.

Background: Hyperandrogenism is a pivotal mediator in the pathogenesis of the polycystic ovary syndrome (PCOS), but the mechanisms of androgen excess in this condition are not fully understood. Angiotensin (Ang)-(1-7) is an active peptide of the renin-angiotensin system (RAS) that stimulates ovarian follicular growth and testosterone release in vitro. Objective: To investigate whether Ang-(1-7), its receptor Mas and angiotensin-converting enzyme 2 (ACE2), the enzyme that converts Ang II into Ang-(1-7), are expressed in rat polycystic ovaries (PCO) and thus if this peptide system might be associated with excess androgen production in PCO. Methods: A rat model that shares some features of PCOS such as disruption of folliculogenesis and multiple ovarian cyst formation was used in the study. Results: We found reduced levels of Ang-(1-7) and Mas receptor in PCO compared to normal ovaries. Also, ACE2 mRNA expression was reduced in PCO compared to ovaries of control rats (p < 0.05). PCO had high levels of estrogen and testosterone and increased mRNA for upstream enzymes of the steroidogenic cascade, but not of P450 aromatase. Conclusion: These findings suggest that the ovarian ACE2-Ang-(1-7)-Mas receptor axis is inhibited and therefore may not be a co-factor of excess testosterone production in rat PCO.

Spider silks have received extensive attention from scientists and industries around the world because of their remarkable mechanical properties, which include high tensile strength and extensibility. It is a leading-edge biomaterial resource, with a wide range of potential applications. Spider silks are composed of silk proteins, which are usually very large molecules, yet many silk proteins still remain largely underexplored. While there are numerous reviews on spider silks from diverse perspectives, here we provide a most up-to-date overview of the spider silk component protein family in terms of its molecular structure, evolution, hydrophobicity, and biomedical applications. Given the confusion regarding spidroin naming, we emphasize the need for coherent and consistent nomenclature for spidroins and provide recommendations for pre-existing spidroin names that are inconsistent with nomenclature. We then review recent advances in the components, identification, and structures of spidroin genes. We next discuss the hydrophobicity of spidroins, with particular attention on the unique aquatic spider silks. Aquatic spider silks are less known but may inspire innovation in biomaterials. Furthermore, we provide new insights into antimicrobial peptides from spider silk glands. Finally, we present possibilities for future uses of spider silks.

Background: Mild hypothermia, i.e. maintenance of organ temperature by up to 8°C lower than body temperature, is a critical strategy for exerting some functions of the cells and organs normally, and is an useful therapy for recovering properly from some diseases, including myocardial infarction, cardiac arrest, brain injury, and ischemic stroke. Nevertheless, there were no focusses so far on organ temperature and potential responses of gene expression to organ temperature in organs of homeothermic animals that survive under normal conditions. Objective: The present study aimed to assess organ temperature in homeothermic animals and evaluate the effect of their organ temperature on the expression of the cold shock protein RNA binding motif protein 3 (RBM3), and to gain insights into the organ temperature-mediated regulation of RBM3 gene transcription via Nuclear factor β-light-chain-enhancer of activated B cells (NF-ΚB) p65, which had been identified as a transcription factor that is activated by undergoing the Ser276 phosphorylation and promotes the RBM3 gene expression during mild hypothermia. Methods: We measured the temperature of several organs, where RBM3 expression was examined, in female and male mice. Next, in male mice, we tested NF-ΚB p65 expression and its Ser276 phosphorylation in organs that have their lower temperature than body temperature and compared them with those in organs that have their temperature near body temperature. Results: Organ temperature was around 32°C in the brain and reproductive organs, which is lower than the body temperature, and around 37°C in the heart, liver, and kidney, which is comparable to the body temperature. The expression of RBM3 was detected greatly in the brain and reproductive organs with their organ temperature of around 32°C, and poorly in the heart, liver, and kidney with their organ temperature of around 37°C. In accordance with the changes in the RBM3 expression, the NF-ΚB p65 Ser276 phosphorylation was detected more greatly in the testis and brain with their organ temperature of around 32°C, than in the heart, liver, and kidney with their organ temperature of around 37°C, although the NF-ΚB p65 expression was unchanged among all the organs tested. Discussion: Our data suggested that organ temperature lower than body temperature causes the expression of RBM3 in the brain and reproductive organs of mice, and that lower organ temperature causes the NF-ΚB p65 activation through the Ser276 phosphorylation, resulting in an increase in the RBM3 gene transcription, in the brain and reproductive organs of mice. Conclusion: The study may present the possibility that organ temperature-induced alterations in gene expression are organ specific in homeotherms and the possibility that organ temperature-induced alterations in gene expression are transcriptionally regulated in some organs of homeotherms.

Background: Although glucocorticoids (GCs) are characterized as powerful agents to treat inflammatory afflictions, they are accompanied by metabolic side effects which limit their usage. β-Sitosterol, as a minor component found in extraction of vegetable oil, was reported to have anti-inflammatory effects in RAW 264.7 cells. Objective: To test whether β-sitosterol has an effect to dissociate transrepression from transactivation as a selective novel GR binder, this work evaluated the dissociated characteristics of β-sitosterol. Methods: The probable binding interaction between β-sitosterol and GR was explored by molecular docking. The GR transcriptional activity of β-sitosterol was assessed in the reporter gene assay. The ability of β-sitosterol to modulate the transactivation and transrepression of GR was evaluated by real-time quantitative PCR analysis. Results and Discussion: In the present study, β-sitosterol treatment cannot induce GR-mediated transactivation. β-Sitosterol exerted a potential to inhibited the expression of GR target transrepressed gene without activating the expression of GR transactivation dependent gene. Molecular docking demonstrated that β-Sitosterol was able to bind the ligand binding domain of GR but unable to induce GR activation. Conclusion: This work offers evidence that β-sitosterol may serve as a selective GR modulator.

Background: Myoglobin is an oxygen binding protein and its dysfunction has been associated with the pathology of several human disorders. This study was undertaken to investigation the role of hydrogen peroxide (H2O2) in the formation of met-myoglobin and the protective potential of four different reductants such as uric acid, folic acid, glutathione and ascorbic acid were also tested against met-myoglobin formation. Methods: Human myoglobin was treated with H2O2 in-vitro in order to prepare met-myoglobin. The generation of met-myoglobin was confirmed by UV-visible spectroscopy and its stability was analysed by the treatment of human myoglobin with H2O2 at varying pH or time. High performance liquid chromatography (HPLC) was used to determine the oxidatively modified heme products in met-myoglobin. Spectroscopic analysis was used to identify the protective potential of uric acid, folic acid, glutathione and ascorbic acid against the formation of met-myoglobin. Results: The novel data of this study showed that H2O2 induced extensive damage of myoglobin but the treatment with uric acid, folic acid, glutathione or ascorbic acid provides protection of myoglobin against H2O2 induced oxidative damaged. The study apparently proved the protective potential of all these compounds against the toxicity produced by H2O2. Conclusion: This is the first study that shows uric acid, folic acid, glutathione and ascorbic acid provide protection against the generation of toxic met-myoglobin and might be used therapeutically to modify the blood conditions in order to prevent the progression of human disorders associated with myoglobin dysfunction.

Background: Interactions of drugs with DNA and proteins may modify their biological activities and conformations, which effect transport and biological metabolism of drugs. Objective: In this study the interaction of anticancer drug regorafenib (REG) with calf thymus-DNA (ct-DNA) and human serum albumin (HSA) has been investigated Methods: Hence, for the first time, it was discovered interaction between REG with DNA and HSA using multi-spectroscopic, zeta potential measurements and molecular docking method. Results and Discussion: DNA displacement studies showed that REG does not have any effect on acridine orange and methylene blue bound DNA, though it was substantiated by displacement studies with Hoechst (as groove binder). Furthermore, the different concentrations of REG induce slight changes in the viscosity of ct-DNA. Zeta potential parameters indicated that hydrophobic interaction plays a major role in the DNA-REG complex. Results obtained from molecular docking demonstrate that the REG prefers to bind on the minor groove of DNAs than that of the major groove. Binding properties of HSA reveal that intrinsic fluorescence of HSA could be quenched by REG in a static mode. The competitive experiments in the presence of warfarin and ibuprofen (as site markers) suggested that the binding site of REG to HSA was most probably located in the subdomain IIA. Measurements of the zeta potential indicated that REG bound to HSA mainly by both electrostatic and hydrophobic interactions. It was found on docking procedures that REG could fit well into HSA subdomain IIA, which confirmed the experimental results. Conclusion: In conclusion, REG can be delivered by HSA in a circulatory system and affect DNA as potential target.

Background: In individuals with ovarian cancer, an increase in the circulating level of the epidermal growth factor (EGF) is readily apparent. Ovarian cancer cells exhibit signaling pathway of the epidermal growth factor (EGFR) and respond to the EGF. Annona muricata (AM) has been shown to decrease ovarian cell proliferation however, role of AM in regulating EGF actions is not yet to be reported. Objective: In this study, we proposed that the fractionated compound acetogenin can inhibit the activation of EGFR-regulated signaling cascades such as MAPK7 / PI3K-Akt / mTOR / STAT upon EGF stimulation. Methods: Ethanolic extract was prepared for the whole AM plant and Thin Layer Chromatography (TLC) was performed to characterize the secondary metabolites and each fraction was assessed using kedde reagent for the presence of acetogenin. The effects of acetogenins were then tested on the survival of PA-1 ovarian cancer cells under basal and EGF stimulated conditions. To delineate the role of acetogenin in EGFR signaling cascades, the in silico docking studies were conducted. Results: The fraction of acetogenin decreased the viability of EGF induced PA-1 ovarian cancer cells that indicating the EGF inhibitory effects of acetogenin. The docking studies specifically illustrated that when the acetogenin binding with tyrosine kinase (TK) and regulatory unit (RU) which subsequently resulted in a reduction in EGF induced the survival of PA-1 ovarian cancer cells. Discussion: The vital regulatory role of acetogenin reported in this study indicate significant anticancer activities of acetogenin from AM. The in silico study of the acetogenin function predicted that it binds specifically to Asp837 (phosphor-acceptor site) of EGFR, essential for phosphorylation of substrates in the TK domain and RU which promote downstream signaling. Conclusion: Acetogenin isolated from AM effectively inhibited the survival of PA-1 ovarian cancer cells through impaired EGF signaling.

Background: Aldicarb is a carbamate pesticide commercially used in potato crop production. Once it enters human body, it interacts with diverse proteins and other substances. Objective: Aldicarb is toxic to human health and it is also a cholinesterase inhibitor, which prevents the breakdown of acetylcholine in synapse. Human alpha-2-macroglobulin (α2M), is a large tetrameric glycoprotein of 720 kDa with antiproteinase activity, found abundantly in plasma. Methods: In the present study, the interaction of aldicarb with alpha-2-macroglobulin was explored utilizing various spectroscopic techniques and molecular docking studies. Results: UV-vis and fluorescence spectroscopy suggests the formation of a complex between aldicarb and α2M apparent by increased absorbance and decreased fluorescence with static quenching mode. CD spectroscopy indicates a slight change in the structure of alpha-2-macroglobulin. Docking studies confirm the interaction of aldicarb with Pro- 1391, Leu-1392, Lys-1393, Val-1396, Lys- 1397, Thr-1408, Glu-1409, Val-1410, Asp-282 and Glu-281 in the receptor binding domain at the C-terminal of the alpha 2 macroglobulin. Discussion: In this work, aldicarb is shown to bind with alpha 2-macroglobulin at receptor binding domain which is the binding site for various extracellular and intracellular ligand too. Also, affecting the functional activity of the protein may lead to further physiological consequences. Conclusion: It is possible that aldicarb binds and compromises antiproteinase activity of α2M and binding properties by inducing changes in the secondary structure of the protein.

Background: Chickpea is a widely grown legume in India, Australia, Canada, and Mediterranean regions. Seeds of chickpea are good source of protein for both human and animals. Wild relatives of chickpea (Cicer arietinum) are the potential gene pool for crop improvement; however, very little information is available on the seed proteome of these wild chickpeas. Objective: We aimed to analyze the seed proteome profiles of three wild relatives of chickpea, Cicer bijugum, Cicer judaicum and Cicer microphyllum along with two cultivated varieties JG11 and DCP 92/3. Methods: Total seed proteins were extracted using various extraction buffers for 2-D gel electrophoresis. Protein separated in a 2-D gels were subjected to image analyses, differentially expressed proteins were extracted from the gels and identified by the MALDI TOF/TOF. Seed protease inhibitors were analysed biochemically. Results: We have standardized the 2-D gel electrophoresis method and separated seed proteins using the modified method. We identified a large number (400) of protein proteins which were differentially expressed in cultivated and wild type species of chickpea. A comparative analysis between C. bijugum and JG 11 revealed the presence of 9 over-expressed and 22 under-expressed proteins, while the comparison between C. bijugum with DCP 92/3 showed 8 over-expressed and 18 under-- expressed proteins. Similarly, comparative analysis between C. microphyllum with DCP 92/3 showed 8 over-expressed proteins along with 22 under-expressed proteins, while the comparative study of C. microphyllum with JG11 displayed 9 over-expressed and 24 under-expressed proteins. We also compared C. judaicum with DCP 92/3 which revealed 15 overexpressed and 11 under-expressed proteins. On the other hand, the comparative analysis of C. judaicum with JG11 showed 10 over-expressed proteins, while the numbers of under-expressed proteins were 14. Among the differentially expressed protein proteins, 19 proteins were analyzed by the MS/MS, and peptides were identified using the MASCOT search engine. In the wild relatives the differentially expressed proteins are phosphatidylinositol 4-phosphate 5- kinase, β-1-6 galactosyltransferase, RNA helicase, phenyl alanine ammonia lyase 2, flavone 3’-0-methyl transferase, Argonaute 2, Myb related protein, Tubulin beta-2 chain and others. The most important one was legumin having α- amylase inhibition activity which was up regulated in C. bijugum. We also studied the activity of protease inhibitor (trypsin and α- amylase inhibitors) in these seed lines which showed differential activity of protease inhibitors. The highest trypsin and α- amylase inhibition was observed in C. judaicum and C. bijugum, respectively. Conclusion: The differentially expressed proteins of wild relatives of chickpea appeared to be involved in various metabolic pathways. The study provides us information about the differences in the seed proteome of these wild species and cultivated varieties for the first time.

Background: The prevalence of the chronic metabolic disorder Type 2 diabetes mellitus (T2DM) is increasing steadily, and has even turned into an epidemic in some countries. T2DM results from defective responses to insulin and obesity is a major factor behind insulin resistance in T2DM. Insulin receptor substrate (IRS) proteins are adaptor proteins in the insulin receptor signalling pathway. The insulin signalling is controlled through tyrosine phosphorylation of IRS-1 and IRS-2, and dysregulation of IRS proteins signalling may lead to glucose intolerance and eventually insulin resistance. Objective: In this work, we suggest that both glycosylation (O-GlcNAc modification) and phosphorylation of IRS-1 and -2 are involved in the pathogenesis of T2DM. Methods: Phosphorylation and O-GlcNAc modifications (Ser1101 in IRS-1 and Ser1149 in IRS-2) proteins were determined experimentally by sandwich ELISA with specific antibodies and with bioinformatics tools. Results: When IRS-1 (on Ser1101) and IRS-2 (Ser1149) become glycosylated following an increase in UDP-GlcNAc pools, it may contribute to insulin resistance. Whereas when the same (IRS-1 on Ser1101 and IRS-2 on Ser1149) are phosphorylated, the insulin signalling is inhibited. Discussion: In this work OGlcNAc-modified proteins were specifically detected using O-Glc- NAc-specific antibodies, suggesting that elevated levels of O-GlcNAc-modified proteins are found, independently of their possible involvement in Advanced Glycation End products (AGEs). Conclusion: This study suggests a mechanism, which is controlled by posttranslational modifications, and may contribute to the pathogenesis of type II diabetes.

Background: Oesophgeal adenocarcinoma (OAC) is the most frequent cause of cancer death. POLO-like kinase 1 (PLK1) is overexpressed in broad spectrum of tumors and has prognostic value in many cancers including esophageal cancer, suggesting its potential as a therapeutic target. p53, the guardian of genome is the most important tumor suppressors that represses the promoter of PLK1, whereas tumor cells with inactive p53 are arrested in mitosis due to DNA damage. PLK1 expression has been linked to the elevated p53 expression and has been shown to act as a biomarker that predicts poor prognosis in OAC. Objectives: The aim of the present study was identification of PLK1 associated phosphorylation targets in p53 mutant and p53 normal cells to explore the downstream signaling evets. Methods: Here we develop a proof-of-concept phospho-proteomics approach to identify possible biomarkers that can be used to identify mutant p53 or wild-type p53 pathways. We treated PLK1 asynchronously followed by mass spectrometry data analysis. Protein networking and motif analysis tools were used to identify the significant clusters and potential biomarkers. Results: We investigated approximately 1300 potential PLK1-dependent phosphopeptides by LCMS/ MS. In total, 2216 and 1155 high confidence phosphosites were identified in CP-A (p53+) and OE33 (p53-) cell lines owing to PLK1 inhibition. Further clustering and motif assessment uncovered many significant biomarkers with known and novel link to PLK1. Conclusion: Taken together, our study suggests that PLK1 may serve as a potential therapeutic target in human OAC. The data highlight the efficacy and specificity of small molecule PLK1 kinase inhibitors to identify novel signaling pathways in vivo.

The authors are regretful for submitting and approving the publication of incorrect Figure 4 in this article. Below is the corrected version along with the revised caption. The electronic version of the article has already been corrected.