Biochimie最新文献

PA-BJ is a serine protease present in Bothrops jararaca venom that triggers platelet aggregation and granule secretion by activating the protease-activated receptors PAR-1 and PAR-4, without clotting fibrinogen. These receptors also have a relevant role in endothelial cells, however, the interaction of PA-BJ with other membrane-bound or soluble targets is not known. Here we explored the activity of PA-BJ on endothelial cell receptor, cytoskeleton, and coagulation proteins in vitro, and show the degradation of fibrinogen and protein C, and the limited proteolysis of actin, EPCR, PAR-1, and thrombomodulin. Antithrombin, factors XI and XIII and protein S were not cleaved by PA-BJ. Moreover, using surface plasmon resonance PA-BJ was demonstrated to bind to actin, EPCR, fibrinogen, PAR-1, and thrombomodulin, with dissociation constants (K_D) in the micromolar range. Considering that these proteins play critical roles in pathways of blood coagulation and maintenance of endothelium integrity, their binding and cleavage by PA-BJ could contribute to the alterations in hemostasis and cell permeability observed in B. jararaca envenomation process.

Obesity treatment requires an individualized approach, emphasizing the need to identify metabolic pathways of diagnostic relevance. Toll-like receptors (TLRs), particularly TLR2 and TLR4, play a crucial role in metabolic disorders, as receptor deficiencies improves insulin sensitivity and reduces obesity-related inflammation. Additionally, hydrogen sulfide (H₂S) influences lipolysis, adipogenesis, and adipose tissue browning through persulfidation. This study investigates the impact of a high-fat diet (HFD) on low molecular weight sulfur compounds in the visceral adipose tissue (VAT) of C57BL/6 and TLR2-deficient mice. It focuses on key enzymes involved in H₂S metabolism: cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CGL), 3-mercaptopyruvate sulfurtransferase (MPST), and thiosulfate sulfurtransferase (TST). In C57BL/6 mice on HFD, MPST activity decreased, while CBS level increased, potentially compensating for H₂S production. In contrast, TLR2-deficient mice on HFD exhibited higher MPST activity but reduced level of CBS and CGL activity, suggesting that TLR2 deficiency mitigates HFD-induced changes in sulfur metabolism. TST activity was lower in TLR2-deficient mice, indicating an independent regulatory role of TLR2 in TST activity. Elevated oxidative stress, reflected by increased glutathione levels, was observed in wild-type mice. Interestingly, cysteine and cystine were detectable only in the VAT of the C57BL/6 ND group and were absent in all other groups. The capacity for hydrogen sulfide production in tissues from TLR2-/-B6 HFD group was significantly lower than in the C57BL/6 HFD group. In conclusion, TLR2 modulates sulfur metabolism, oxidative stress, and inflammation in obesity. TLR2 deficiency disrupts H₂S production and redox balance, potentially contributing to metabolic dysfunction, highlighting TLR2 as a potential therapeutic target for obesity-related metabolic disorders.

The parasite of the genus Leishmania is the causative agent of diseases that affect humans called leishmaniasis. These diseases affect millions of people worldwide and the currently existing drugs are either very toxic or the parasites acquire resistance. Therefore, new elimination mechanisms need to be elucidated so that new therapeutic strategies can be developed. Much has already been discussed about the role of neutrophils in Leishmania infection, and their participation is still controversial. A recent study showed that receptors present in the neutrophil membrane, the purinergic receptors, can control the infection when activated, but the triggering mechanism has not been elucidated. In this review, we will address the possible participation of purinergic receptors expressed in the neutrophil extracellular membrane that may be participating in the detection of Leishmania infection and their possible effects during parasitism.

Macrolactin A (McA) is a secondary metabolite produced by Bacillus species. It has been known for its antimicrobial properties since the late 1980s, although the exact mechanism of its antibacterial activity remains unknown. In this study, we have found that McA is an inhibitor of protein synthesis in bacteria. Our conclusion is based on the results obtained by in vivo and in vitro bioreporter systems. We demonstrated that the inhibitory activity of McA is independent of bacterial species. However, the concentration of McA required to inhibit protein synthesis in the E. coli cell-free translational model was found to be 50 times lower than the concentration required in the S. aureus cell-free translational model. To investigate the mechanism of McA's inhibitory activity, we conducted a toe-printing assay, sequenced and annotated the genomes of McA-resistant Bacillus pumilus McA^R and its parental strain. The results showed that McA inhibits the initial step of the elongation phase of protein synthesis. We identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu). Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu. A cross-resistance assay was conducted using kirromycin on B. pumilus McA^R. The results confirmed the assumption that McA has a mode of action similar to that of other elfamycin-like antibiotics (targeting EF-Tu). Overall, our study addresses a significant gap in our understanding of the mechanism of action of McA, a representative member of the macrolide antibiotics.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are involved in many cellular processes and possess unequalled catalytic versatility. Rational design through site-directed mutagenesis is a powerful strategy for creating tailor-made enzymes for a wide range of biocatalytic applications. PLP-dependent methionine γ-lyase (MGL), which degrades sulfur-containing amino acids, is an encouraging enzyme for many therapeutic purposes - from combating bacterial resistant strains and fungi to antitumor activity. A two-component biosystem MGL/S-alk(en)yl-l-cysteine sulfoxide (an uncommon substrate for this enzyme) produces antimicrobial thiosulfinates during the β-elimination reaction. SH-groups of the enzyme are modified by the products of this reaction, which leads to the inactivation of the enzyme. Successful and efficient rational stabilization of MGL from Clostridium novyi can be achieved using site-directed mutagenesis. We have managed to obtain an improved version of the enzyme better than the natural one regarding the β-elimination reaction. Cys118, Cys184 and Cys273 of MGL from Clostridium novyi were substituted by His and two Ala, respectively. The resulting Cys-del variant had 2-3-fold improved k_cat/K_m value for conventional β-eliminating substrates and up to 10-fold increase in catalytic efficiency with S-substituted-l-cysteine sulfoxides compared to the wild-type MGL. The Cys-del MGL remained active under the thiosulfinates, which are products of β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides. Moreover, Cys-del variant proved to be more stable during shelf storage. Thus, we have created an effective enzyme component of the biocatalytic system that is capable of generating antimicrobial drugs - thiosulfinates.

BAG3 is a universal adapter protein involved in various cellular processes, including the regulation of apoptosis, chaperone-assisted selective autophagy, and heat shock protein function. The interaction between small heat shock proteins (sHsps) and their α-crystallin domains (Acds) with full-length BAG3 protein and its IPV domain was analyzed using size-exclusion chromatography, native gel electrophoresis, and chemical cross-linking. HspB7 and the 3D mutant of HspB1 (which mimics phosphorylation) showed no interaction, HspB6 weakly interacted, and HspB8 strongly interacted with full-length BAG3. In contrast to the full-length sHsps, their α-crystallin domains (AcdB1, AcdB5, and AcdB6) were able to interact with BAG3, with AcdB8 again being the strongest interactor. Among all the full-length sHsps analyzed, only HspB8 bound to the IPV domain of BAG3. AcdB1, AcdB5, AcdB6, and AcdB8 interacted with the IPV domain of BAG3, with AcdB8 displaying the highest binding efficiency. The stoichiometry of crosslinked complexes formed by HspB8 (or its Acd) and the IPV domain of BAG3 was 2:1, whereas for the other sHsps and their Acds, it was 1:1. These findings suggest that while the IPV domain of BAG3 and the Acds of sHsps play an important role in binding, other structural regions significantly contribute to this interaction. The unique binding efficiency between BAG3 and HspB8 may be attributed to the intrinsic disorder and simple oligomeric structure of HspB8.

This study focuses on the quaternary structure of the viper-secreted phospholipase A₂ (PLA₂), a central toxin in viper envenomation. PLA₂ enzymes catalyze the hydrolysis of the sn-2 ester bond of membrane phospholipids. Small-molecule inhibitors that act as snakebite antidotes, such as varespladib, are currently in clinical trials. These inhibitors likely bind to the enzyme in the aqueous cytosol prior to membrane-binding. Thus, understanding its controversial solution structure is key for drug design. Crystal structures of PLA₂ in the PDB show at least four different dimeric conformations, the most well-known being "extended" and "compact". This variability among enzymes with >50 % sequence identity raises questions about their transferability to aqueous solution. Therefore, we performed extensive molecular dynamics (MD) simulations of several PLA₂ enzymes in water to determine their quaternary structure under physiological conditions. The MD simulations strongly indicate that PLA₂ enzymes adopt a "semi-compact" conformation in cytosol, a hybrid between extended and compact conformations. To our knowledge, this is the first study that determines the most favorable dimeric conformation of PLA₂ enzymes in solution, providing a basis for advancements in snakebite envenoming treatment. Recognizing snakebite envenoming as a neglected tropical disease has driven the search for efficient, affordable alternatives to the current antivenoms. Therefore, understanding the main drug targets within snake venom is crucial to this achievement.

Kingella kingae, an emerging pediatric pathogen, secretes the pore-forming toxin RtxA, which has been implicated in the development of various invasive infections. RtxA is synthesized as a protoxin (proRtxA), which gains its biological activity by fatty acylation of two lysine residues (K558 and K689) by the acyltransferase RtxC. The low acylation level of RtxA at K558 (2-23 %) suggests that the complete acylation at K689 is crucial for toxin activity. Using a bacterial two-hybrid system, we show that substitutions of K558, but not K689, partially reduce the interaction of proRtxA with RtxC and that the acyltransferase interacts independently with each acylated site in vivo. While substitutions of K558 had no effect on the acylation of K689, substitutions of K689 resulted in an average 40 % increase in the acylation of K558. RtxA mutants monoacylated at either K558 or K689 irreversibly bound to erythrocyte membranes, with binding efficiency corresponding to the extent of lysine acylation. However, these mutants lysed erythrocytes with similarly low efficiency as nonacylated proRtxA and showed only residual overall membrane activity in planar lipid bilayers. Interestingly, despite forming fewer pores, the monoacylated mutants exhibited single-pore characteristics, such as conductance and lifetime, similar to those of intact RtxA. These findings indicate that the acylation at either K558 or K689 is sufficient for the irreversible insertion of RtxA into the membrane, but not for the efficient formation of membrane pores. Alternatively, K558 and K689 per se may play a crucial structural role in pore formation, regardless of their acylation status.

During transcription initiation, the holoenzyme of bacterial RNA polymerase (RNAP) specifically recognizes promoters using a dedicated σ factor. During transcription elongation, the core enzyme of RNAP interacts with nucleic acids mainly nonspecifically, by stably locking the DNA template and RNA transcript inside the main cleft. Here, we present a synthetic DNA aptamer that is specifically recognized by both core and holoenzyme RNAPs from extremophilic bacteria of the Deinococcus-Thermus lineage. The aptamer binds RNAP with subnanomolar affinities, forming extremely stable complexes even at high ionic strength conditions, blocks RNAP interactions with the DNA template and inhibits RNAP activity during transcription elongation. We propose that the aptamer binds at a conserved site within the downstream DNA-binding cleft of RNAP and traps it in an inactive conformation. The aptamer can potentially be used for structural studies to reveal RNAP conformational states, affinity binding of RNAP and associated factors, and screening of transcriptional inhibitors.