Current Protein and Peptide Science - Volume 25, Issue 2, 2024

Cyanobacteria have emerged as a microbial cell factory to produce a variety of bioproducts, including peptides and proteins. Cyanobacteria stand out among other organisms due to their photoautotrophic metabolism and ability to produce a wide range of metabolites. As photoautotrophic hosts can produce industrial compounds and proteins by using minimal resources such as sunlight, atmospheric carbon dioxide, and fewer nutrients, cyanobacteria are cost-effective industrial hosts. Therefore, the use of protein engineering tools for rational protein design, and the desired modification of enzyme activity has become a desirable undertaking in cyanobacterial biology. Protein engineering can improve their biological functions as well as the stability of their intracellular proteins. This review aims to highlight the success of protein engineering in the direction of cyanobacterial biotechnology and outlines the emerging technologies, current challenges, and prospects of protein engineering in cyanobacterial biotechnology.

With global climate changes and the increased demand for food due to expected world population growth, genetic improvement programs have aimed at producing crops with increased yield and tolerance to environmental stresses, such as drought, salinity, and pathogens. On the other hand, genetic improvement programs via biotechnology require candidate genes that confer traits of interest to be incorporated into improved crops. In this regard, genes encoding transcription factors (TFs) can be promising since they are proteins that transcriptionally regulate the expression of target genes related to the most diverse roles in the plant, including defense against stresses. Among TFs, bZIP (basic leucine zipper) proteins regulate many developmental and physiological processes in the plant, such as seed formation, fruit ripening, nutrient assimilation, and defense response to abiotic and biotic stresses. In this review, we aim to highlight the main advances in the potential use of bZIP TFs in the genetic improvement of crops. We address this potential mainly regarding crop tolerance to stresses and other agricultural traits, such as increased yield and fruit features.

Membrane protein human concentrative nucleoside transporter 3 (hCNT3) can not only transport extracellular nucleosides into the cell but also transport various nucleoside-derived anticancer drugs to the focus of infection for therapeutic effects. Typical nucleoside anticancer drugs, including fludarabine, cladabine, decitabine, and clofarabine, are recognized by hCNT3 and then delivered to the lesion site for their therapeutic effects. hCNT3 is highly conserved during the evolution from lower to higher vertebrates, which contains scaffold and transport domains in structure and delivers substrates by coupling with Na⁺ and H⁺ ions in function. In the process of substrate delivery, the transport domain rises from the lower side of transmembrane 9 (TM9) in the inward conformation to the upper side of the outward conformation, accompanied by the collaborative motion of TM7b/ TM4b and hairpin 1b (HP1b)/ HP2b. With the report of a series of three-dimensional structures of homologous CNTs, the structural characteristics and biological functions of hCNT3 have attracted increasing attention from pharmacists and biologists. Our research group has also recently designed an anticancer lead compound with high hCNT3 transport potential based on the structure of 5-fluorouracil. In this work, the sequence evolution, conservation, molecular structure, cationic chelation, substrate recognition, elevator motion pattern and nucleoside derivative drugs of hCNT3 were reviewed, and the differences in hCNT3 transport mode and nucleoside anticancer drug modification were summarized, aiming to provide theoretical guidance for the subsequent molecular design of novel anticancer drugs targeting hCNT3.

ARL15 is a member of the RAS superfamily of small GTPases and is associated with several metabolic traits, including increased risk of diabetes, rheumatoid arthritis and lipid metabolism disorders. The ARL15 gene encodes for an uncharacterized small GTP binding protein. Its precise role in human physiology remains unknown, but several genetic association studies have recognized different variants in this gene to be statistically associated with numerous traits and complex diseases. Here, we provided the unique features of ARL15 small G protein, its association with varied metabolic and lifestyle diseases, its function in vesicular and lipid trafficking, and its binding partners. We outlined this protein as a promising and emerging therapeutic target to combat metabolic disorders like cardiovascular diseases, diabetes and rheumatoid arthritis. The review provides a comprehensive description of the current advancements in ARL15 research with a perspective that focused research will position this small GTPase as a viable target for the treatment of rheumatoid arthritis.

In recent years, the CRISPR/Cas9 system has become a rapidly advancing gene editing technology with significant advantages in various fields, particularly biomedicine. Liver cancer is a severe malignancy that threatens human health and is primarily treated with surgery, radiotherapy, and chemotherapy. However, surgery may not be suitable for advanced cases of liver cancer with distant metastases. Moreover, radiotherapy and chemotherapy have low specificity and numerous side effects that limit their effectiveness; therefore, more effective and safer treatments are required. With the advancement of the biomolecular mechanism of cancer, CRISPR/Cas9 gene editing technology has been widely used in the study of liver cancer to gain insights into gene functions, establish tumor models, screen tumor phenotype-related genes, and perform gene therapy. This review outlines the research progress of CRISPR/Cas9 gene editing technology in the treatment of liver cancer and provides a relevant theoretical basis for its research and application in the treatment of liver cancer.

The structural ensembles of intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) cannot be easily characterized using conventional experimental techniques. Computational techniques complement experiments and provide useful insights into the structural ensembles of IDPs and proteins with IDRs. Herein, we discuss computational techniques such as homology modeling, molecular dynamics simulations, machine learning with molecular dynamics, and quantum computing that can be applied to the studies of IDPs and hybrid proteins with IDRs. We also provide useful future perspectives for computational techniques that can be applied to IDPs and hybrid proteins containing ordered domains and IDRs.

Introduction: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. Objectives: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. Methods: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. Results: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10^-6 M) and Ki (1.05 x 10^-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 μM, prolonging clotting time by 2.6 times. Conclusion: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.

Background: Melasma is a skin hyperpigmentary disorder that develops over time. Genetic factors, oxidative stress, female sex hormones, and UV light may all play a role in the disorder's progression. Aims: To compare the levels of oxidative stress and tyrosinase activity in melasma patients with healthy volunteers. Methods: After written consent, 130 patients were enrolled in a case–control study. 65 cases were of melasma disorder, and 65 were served as control. Homogenized skin tissues were taken and used to estimate superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx) (antioxidants), malondialdehyde (MDA) and tyrosine hydroxylase (TH). Results: Melasma patients had lower basal levels of systemic antioxidants than healthy subjects. Tyrosinase activity was shown to be greater in lesional skin than in non-lesional skin. In controls, there was a good positive relationship between TH and MDA and an excellent negative relationship between GPx and GSH. In melasma patients, there were significant associations between CAT, GPx, SOD and MDA. Conclusions: Increased oxidative stress may affect tyrosinase activity and eumelanin synthesis via the anabolic pathway of melanin synthesis, according to our findings. In conclusion, we discovered a negative relationship between antioxidants and tyrosinase activity.