Biochimie最新文献

We discuss the diverse functions of proteases in the context of their biotechnological and medical significance, as well as analytical approaches used to determine the functional activity of these enzymes. An insight into modern approaches to studying the kinetics and specificity of proteases, based on spectral (absorption, fluorescence), mass spectrometric, immunological, calorimetric, and electrochemical methods of analysis is given. We also examine in detail electrochemical systems for determining the activity and specificity of proteases. Particular attention is given to exploring innovative electrochemical systems based on the detection of the electrochemical oxidation signal of amino acid residues, thereby eliminating the need for extra redox labels in the process of peptide synthesis. In the review, we highlight the main prospects for the further development of electrochemical systems for the study of biotechnologically and medically significant proteases, which will enable the miniaturization of the analytical process for determining the catalytic activity of these enzymes.

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

The incidence of breast cancer is perpetually growing globally, and it remains a major public health problem and the leading cause of mortality in women. Though the aberrant activities of the Hippo pathway have been reported to be associated with cancer, constructive knowledge of the pathway connecting the various elements of breast cancer remains to be elucidated. The Hippo transducers, yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ), are reported to be either tumor suppressors, oncogenes, or independent prognostic markers in breast cancer. Thus, there is further need for an explicative evaluation of the dilemma with this molecular contribution of Hippo transducers in modulating breast malignancy. In this review, we summarize the intricate crosstalk of the Hippo pathway in different aspects of breast malignancy, including stem-likeness, cellular signaling, metabolic adaptations, tumor microenvironment, and immune responses. The collective data shows that Hippo transducers play an indispensable role in mammary tumor formation, progression, and dissemination. However, the cellular functions of YAP/TAZ in tumorigenesis might be largely dependent on the mechanical and biophysical cues they interact with, as well as on the cell phenotype. This review provides a glimpse into the plausible biological contributions of the cascade to the inward progression of breast carcinoma and suggests potential therapeutic prospects.

PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of phosphomannomutase-1 to enhance intracellular glucose-1,6-bisphosphate have been proposed as therapeutical approaches.

We used Recombinant Bacterial Thermal Shift Assay to assess the binding of a substrate analog to phosphomannomutase-2 and the specific binding to phosphomannomutase-1 of an FDA-approved drug - clodronate. We also deepened the clodronate binding by enzyme activity assays and in silico docking. Our results confirmed the selective binding of clodronate to phosphomannomutase-1 and shed light on such binding.

Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides.

The mitochondrial translocator protein (TSPO) is an outer mitochondrial protein membrane with high affinity for cholesterol. It is expressed in most tissues but is more particularly enriched in steroidogenic tissues. TSPO is involved in various biological mechanisms and TSPO regulation has been related to several diseases. However, despite a considerable number of published studies interested in TSPO over the past forty years, the precise function of the protein remains obscure. Most of the functions attributed to TSPO have been identified using pharmacological ligands of this protein, leading to much debate about the accuracy of these findings. In addition, research on the physiological role of TSPO has been hampered by the lack of in vivo deletion models. Studies to perform genetic deletion of Tspo in animal models have long been unsuccessful, which led to the conclusions that the deletion was deleterious and the gene essential to life. During the last decades, thanks to the significant technical advances allowing genome modification, several models of animal genetically modified for TSPO have been developed. These models have modified our view regarding TSPO and profoundly improved our fundamental knowledge on this protein. However, to date, they did not allow to elucidate the precise molecular function of TSPO and numerous questions persist concerning the physiological role of TSPO and its future as a therapeutic target. This article chronologically reviews the development of deletion and induction models of TSPO.

Three glucose-6-phosphatase catalytic subunits, that hydrolyze glucose-6-phosphate (G6P) to glucose and inorganic phosphate, have been identified, designated G6PC1-3, but only G6PC1 and G6PC2 have been implicated in the regulation of fasting blood glucose (FBG). Elevated FBG has been associated with multiple adverse clinical outcomes, including increased risk for type 2 diabetes and various cancers. Therefore, G6PC1 and G6PC2 inhibitors that lower FBG may be of prophylactic value for the prevention of multiple conditions. The studies described here characterize a G6PC2 inhibitor, designated VU0945627, previously identified as Compound 3. We show that VU0945627 preferentially inhibits human G6PC2 versus human G6PC1 but activates human G6PC3. VU0945627 is a mixed G6PC2 inhibitor, increasing the Km but reducing the Vmax for G6P hydrolysis. PyRx virtual docking to an AlphaFold2-derived G6PC2 structural model suggests VU0945627 binds two sites in human G6PC2. Mutation of residues in these sites reduces the inhibitory effect of VU0945627. VU0945627 does not inhibit mouse G6PC2 despite its 84% sequence identity with human G6PC2. Mutagenesis studies suggest this lack of inhibition of mouse G6PC2 is due, in part, to a change in residue 318 from histidine in human G6PC2 to proline in mouse G6PC2. Surprisingly, VU0945627 still inhibited glucose cycling in the mouse islet-derived βTC-3 cell line. Studies using intact mouse liver microsomes and PyRx docking suggest that this observation can be explained by an ability of VU0945627 to also inhibit the G6P transporter SLC37A4. These data will inform future computational modeling studies designed to identify G6PC isoform-specific inhibitors.

A structural homolog of the mammalian TSPO has been identified in the human pathogen Bacillus cereus. BcTSPO, in its recombinant form, has previously been shown to bind and degrade porphyrins. In this study, we generated a ΔtspO mutant strain in B. cereus ATCC 14579 and assessed the impact of the absence of BcTSPO on cellular proteomics and physiological characteristics. The proteomic analysis revealed correlations between the lack of BcTSPO and the observed growth defects, increased oxygen consumption, ATP deficiency, heightened tryptophan catabolism, reduced motility, and impaired biofilm formation in the ΔtspO mutant strain. Our results also suggested that BcTSPO plays a crucial role in regulating intracellular levels of metabolites from the coproporphyrin-dependent branch of the heme biosynthetic pathway. This regulation potentially underlies alterations in the metabolic landscape, emphasizing the pivotal role of BcTSPO in B. cereus aerobic metabolism. Notably, our study unveils, for the first time, the involvement of TSPO in tryptophan metabolism. These findings underscore the multifaceted role of TSPO, not only in metabolic pathways but also potentially in the microorganism's virulence mechanisms.

Rutin, a phenolic compound, exhibits a diverse range of biological properties, including antioxidant, anti-inflammatory, and antimicrobial effects. In this study, we aimed to investigate the potential of rutin, a naturally occurring plant bioactive molecule, to mitigate the neurotoxic effects induced by aluminum chloride (AlCl₃). Over a period of 6 weeks, rats were intraperitoneally injected with AlCl₃ at a weekly dose of 60 mg/kg, while rutin treatment was administered orally via gavage at a daily dose of 30 mg/kg. AlCl₃ exposure resulted in a significant increase lipid peroxidation (LPO) by 316.24%, nitrate levels by 504.14%, and tumor necrosis factor-alpha (TNF-α) levels by 93.82% in brain mitochondria. Additionally, AlCl₃ exposure led to a reduction in glutathione levels and the activity of antioxidant enzymes, including superoxide dismutase (SOD) by 19.74%, glutathione peroxidase (GPx) by 44.76%, and catalase by 50.50%. There was also a significant decline in the activity of mitochondrial complex enzymes. In contrast, rutin treatment significantly enhanced the activity of antioxidant enzymes while concurrently reducing lipid peroxidation levels in rats. Specifically, rutin administration exerted a modulatory effect on the inflammatory response triggered by aluminum exposure, effectively suppressing the excessive production of nitrate and TNF-α. These findings highlight the potential of rutin as an effective therapeutic strategy in mitigating and combating neuro-inflammation and oxidative stress associated with aluminum-induced toxicity, thereby effectively restoring mitochondrial function.