Protein and Peptide Letters - Volume 29, Issue 4, 2022

Background: Deltamethrin (DLM) is a commercial insecticide of the synthetic pyrethroid family that is used to control disease-causing insects and vectors. When humans are exposed to the fumes or aerosols of DLM, it enters the body via cuticular absorption and reacts with proteins and other biomolecules. Objective: Alpha-2-macroglobulin (α2M) is a serum proteinase inhibitor that also carries out receptor- mediated endocytosis of extracellular substances. This study was done to decipher the structural and functional alterations of α2M by DLM. Methods: Various spectroscopic techniques, including UV absorption and fluorescence spectroscopy, binding studies, and molecular docking, were used to characterize the interaction of DLM with α2M. The affinity constant was calculated from the Stern-Volmer equation using fluorescence data. Results: The UV-Vis and fluorescence spectral studies indicated the formation of a complex between α2M and DLM. Thermodynamically, the interaction was found to be spontaneous with ΔG = -4.23 kcal/mol. CD spectra suggested a change in the secondary structure of the protein from β to α helical content with increasing concentration of DLM. The molecular docking study by Autodock Vina established the interaction of DLM with Glu-926, Ala-1103, Ala-1108, Val-1116, Asn-1159, Glu-1220, Leu-1261, Thr-1272, Ile-1390, Pro-1391, Lys-1393, Val-1396, Lys-1397, Thr-1408, Glu-1409, Val-1410, Ser-1411, Ser-1412, and Asn-1413 with an improved docking score of -6.191 kcal/mol. The binding was carried out in the vicinity of the receptor-binding domain at the C-terminal of α2M. Conclusion: The decrease in the functional activity and structural changes of protein after binding with DLM has a significant effect on human α2M. The information may be useful for exploring the role of DLM in a clinical chemistry laboratory.

Background and Objectives: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) and its PEGylated form (PEG-GCSF) are used in cancer therapy. Thus, developing a more cost-effectively method for expressing rhG-CSF and the PEGylation optimization of rhG-CSF by reaction engineering and subsequent purification strategy is necessary. Methods: RhG-CSF expression in Escherichia coli BL21 (DE3) was carried out by auto-induction batch fermentation and improved for maximizing rhG-CSF productivity. After that, purified rhGCSF was PEGylated using methoxy polyethylene glycol propionaldehydes (mPEG20-ALD). The various conditions effect of extraction and purification of rhG-CSF and PEG-GCSF were assayed. Results: The assessment results revealed that the auto-induction batch cultivation strategy had maximum productivity, and rhG-CSF purity was more than 99%. The obtained data of rhG-CSF PEGylation displayed that the optimized conditions of rhG-CSF PEGylation and purification enhanced homogeneity PEG-GCSF and managed reaction toward optimal yield of PEG-GCSF (70%) and purity of 99.9%. Findings from FTIR, CD, fluorescence spectroscopy, and bioassay revealed that PEGylation was executed exactly in the rhG-CSF N-terminus, and products maintained their conformation properties. Conclusion: Overall, the developed approach expanded strategies for high yield rhG-CSF by simplified auto-induction batch fermentation system and rhG-CSF PEGylation, which are simple and timesaving, economical, and high efficiency.

Background: Protein sequence and structure analyses have been essential components of bioinformatics and structural biology. They provide a deeper insight into the physicochemical properties, structure, and subsequent functions of a protein. Advanced computational approaches and bioinformatics utilities help solve several issues related to protein analysis. Still, beginners and non-professional may struggle when encountering a wide variety of computational tools and the sheer number of input parameter variables required by each tool. Methods: We introduce a free-to-access graphical user interface (GUI) named PyPAn 'Python-based Protein Analysis' for varieties of protein sequence/structure analyses. PyPAn serves as a universal platform to analyze protein sequences, structure, and their properties. PyPAn facilitates onboard analysis of each task in just a single click. It can be used to calculate the physicochemical properties, including instability index and molar extinction coefficient, for a protein. PyPAn is one of the few computational tools that allow users to generate a Ramachandran plot and calculate solvent accessibility and the radius of gyration (Rg) of proteins at once. In addition, it can refine the protein model along with computation and minimization of its energy. Results: PyPAn can generate a recommendation for an appropriate structure modelling method to employ for a query protein sequence. PyPAn is one of the few, if not the only, Python-based computational GUI tools with an array of options for the user to employ as they see fit. Conclusion: PyPAn aims to unify many successful academically significant proteomic applications and is freely available for academic and industrial research uses at https://hassanlab.org/pypan.

Background: Age-related macular degeneration (AMD) can be characterised by degeneration of retinal pigment epithelial (RPE) cells and the accumulation, in retinal drusen deposits, of amyloid beta-peptides proteolytically derived, by secretases, from the amyloid precursor protein (APP). Ultraviolet (UV) light exposure is a risk factor for the development of AMD. Objectives: In the current study, we investigated whether APP and/or its proteolysis are linked to the UVA resistance or proliferation of ARPE-19 human RPE cells. Methods: Cell viability was determined, following UVA exposure, with prior small interfering RNA-mediated APP depletion or secretase inhibitor treatments. APP levels/proteolysis were analysed by immunoblotting. Cells were also grown in the presence/absence of secretase inhibitors to assess their effects on longer-term culture growth. Finally, the effects of APP proteolytic fragments on ARPE-19 cell proliferation were monitored following co-culture with human embryonic kidney cells stably over-expressing these fragments. Results: Endogenous APP was depleted following UVA irradiation and β-secretase, but not α- secretase, the processing of the protein was reduced. Experimental APP depletion or γ-secretase (but not α- or β-secretase) inhibition ablated the detrimental effect of UVA on cell viability. In contrast, α-secretase, and possibly γ-secretase but not β-secretase activity, appeared to promote the longerterm proliferation of ARPE-19 cells in the absence of UVA irradiation. Conclusion: There are clear but differential links between APP expression/proteolysis and the proliferation and UVA resistance of ARPE-19 cells indicating that the protein should be investigated further in relation to the identification of possible drug targets for the treatment of AMD.

Background: B-cell epitope prediction for antipeptide antibody responses enables peptide-based vaccine design and related translational applications. This entails estimating epitopeparatope binding free-energy changes from antigen sequence; but attempts to do so assuming uniform epitope length (e.g., of hexapeptide sequences, each spanning a typical paratope diameter when fully extended) have neglected empirically established variation in epitope length. Objective: This work aimed to develop a sequence-based physicochemical approach to variablelength B-cell epitope prediction for antipeptide paratopes recognizing flexibly disordered targets. Methods: Said approach was developed by analogy between epitope-paratope binding and protein folding modeled as polymer collapse, treating paratope structure implicitly. Epitope-paratope binding was thus conceptually resolved into processes of epitope compaction, collapse and contact, with epitope collapse presenting the main entropic barrier limiting epitope length among nonpolyproline sequences. The resulting algorithm was implemented as a computer program, namely the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), which is freely accessible via an online interface (http://badong.freeshell.org/haptic.htm). This was used in conjunction with published data on representative known peptide immunogens. Results: HAPTIC predicted immunodominant epitope sequences with lengths limited by penalties for both compaction and collapse, consistent with known paratope-bound structures of flexibly disordered epitopes. In most cases, the predicted association constant was greater than its experimentally determined counterpart but below the predicted upper bound for affinity maturation in vivo. Conclusion: HAPTIC provides a physicochemically plausible means for estimating the affinity of antipeptide paratopes for sterically accessible and flexibly disordered peptidic antigen sequences by explicitly considering candidate B-cell epitopes of variable length.

Aims: The anticancer potential of a purified seed protein from Mallotus philippensis is scientifically evaluated and reported here. Background: Seeds of Mallotus philippensis are used to treat various diseases in the indigenous systems of medicine in India. Objectives: The present study deals with the isolation, purification, identification, and screening of protein of interest that exhibit maximum activity against lung cancer cells from the seed crude protein of Mallotus philippensis. Methods: Size-exclusion with HPLC was used to purify crude protein (15 mg) from M. philippensis seeds. Protein of interest was identified using the LC-MS/MS method and analyzed by in vitro (A549 cell lines) in vivo (B16-F10 cells from melanoma cancer-induced Wistar rats) to estimate anticancer activity. Results: SDS-PAGE was applied to isolate and purify elution III (480 μg/ml). Elution III LCMS/ MS data were used to search the UniProt database and were eventually matched with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). MTT assay of GAPDH-treated A549 cells exhibited an IC50 of 3.03 ± 0.39 μg (24 h) and 1.93 ± 0.19 μg (48 h). AO/EtBr staining showed early and late apoptotic characteristics such as cell membrane blebbing, chromatin condensation, and the formation of apoptotic bodies. Hoechst staining confirmed the death of cells by exhibiting bright blue fluorescent, condensed, and fragmented nuclei. GAPDH-treated rats by 10 and 20 mg/kg bw significantly increased body weight by 29.50 ± 3.06 and 31.33 ± 2.69, respectively, and decreased melanoma metastasis in the lungs by 66.79% and 86.57%, respectively. Further, GAPDH treatment significantly increased the levels of SOD, CAT, and GPx and reduced GST and GSH. Histopathological analysis confirmed nuclear alteration in the lung tissue of the treated groups only. Conclusion: Apoptotic potential of GAPDH against lung carcinoma has been confirmed in the present investigation.

Background: CRM197, a non-toxic diphtheria toxin variant, is widely used as a polysaccharide carrier in a variety of conjugate vaccines and also exhibits antitumor activity. CRM197 commercial production is limited due to the low yield of Corynebacterium diphtheriae C7 (197) tox-. Developing an efficient method for recombinant CRM197 production reduces production costs and is critical for expanding the application coverage of related medical products and basic research. Escherichia coli is a frequently used host for heterologous protein synthesis. However, the primary limitation of this system is the inclusion body formation and the low yield of active protein recovery. Objective: As a result, we attempted to produce CRM197 in the soluble form in E. coli using a small ubiquitin-related modifier (SUMO) tag fusion and an expression strategy optimized for protein production. Methods: CRM197 was expressed intracellularly in E. coli BL21 (DE3) with its N-terminus fused to a SUMO tag preceded by a histidine tag (HSCRM197). To improve the solubility of HSCRM197 in E. coli, a response surface method (RSM) experimental design was used based on three factors: expression temperature, inducer concentration, and sorbitol inclusion in the culture medium. Metal affinity chromatography was used to purify HSCRM197, and the SUMO tag was removed using the SUMO protease's catalytic domain. After adsorbing the SUMO tag on a Ni-NTA column, CRM197 was obtained. DNA degradation activity was determined for both HSCRM197 and CRM197. Results: When HSCRM197 was expressed in E. coli under common expression conditions (37ºC, 1000 μM inducer), 15.4% of the protein was found in the cellular soluble fraction. However, when the RSM-derived expression conditions were used (30ºC, 510 μM inducer, and 200 mM sorbitol), the obtained HSCRM197 was almost completely soluble (96.5% solubility), and the system productivity was 32.67 μg ml^-1 h^-1. HSCRM197 and CRM197 both exhibited nuclease activity. However, the activity of CRM197 was greater than that of HSCRM197. Conclusion: These findings established the utility of the method developed in this study to produce CRM197 for medical applications.

Background: A series of mutants of Rhizomucor miehei lipase (RML) screened through four rounds of directed evolution were studied. Mutants' triglyceride hydrolysis activity was assessed, and their genes were sequenced. Results showed that mutations in the propeptide can improve the activity of RML during evolution. Two parts of propeptide (wild-type and mutant) and mature region were connected by molecular simulation technology. Methods: The spatial structure of the most positive mutants containing the mutations in the propeptide was mainly characterized by the increase in the opening angle of the lid structure in the mature region of RML, the enhancement of the hydrophobicity of the active center, and the triad of the active center shifted outward. Results: The three indexes above explain the mechanism of propeptide mutations on the activity change of the target protein. In addition, statistical analysis of all the mutants screened in directed evolution showed that: (1) most of the mutants with increased activity contained mutations of the propeptide, (2) in the later stage of directed evolution, the number of active mutants decreased gradually, and the mutations of inactivated protein mainly occurred in the mature region, and (3) in the last round of directed evolution, the mutations distributed in the propeptide improved the mutant activity further. The results showed that the propeptide reduced RML's evolutionary pressure and delayed the emergence of the evolutionary platform. Conclusion: These findings reveal the role of propeptide in the evolution of RML and provide strategies for the molecular transformation of other lipases.