Biochimie最新文献

Leishmania orientalis, previously called L. siamensis, is a new species characterized as causing cutaneous leishmaniasis in Thailand. This study solves the crystal structure of the L. orientalis triosephosphate isomerase (LoTIM) in apo form at 1.88 Å resolution by using molecular replacement method. Tyrosine118 presents in the LoTIM protein sequence, whereas L. mexicana and Trypanosoma cruzi TIMs have a relative Cys118, which plays a major role in their specific ligand binding. Sulfur atom of the Cys57 thiol group is covalently bound to an arsenic (As) atom present in the precipitating solution. Although the electron density of loop-6 (Gly174-Tyr175-Gly176-Lys177-Val178) is missing in the structure due to this region lacking rigidity, the biological assembly of the two monomers of the LoTIM crystal structures are like that of L. mexicana and T. cruzi. 3D molecular protein-ligand docking was performed using the dimeric interfacial pocket of the enzyme as a ligand-binding receptor to identify its specific inhibitors. Five potential inhibiting compounds, including NSC639174, NSC606498, NSC110039, NSC58446, and NSC345647, were obtained with their IC₅₀ 2.79 ± 0.10, 3.28 ± 0.80, 3.67 ± 0.11, 4.59 ± 0.87 and 15.44 ± 0.14 μM, respectively. However, specific inhibition assays against TIMs from L. orientalis and rabbit muscle indicate that NSC639174 and NSC110039 are the most potent inhibitors for LoTIM, whereas NSC58446 inhibits well both the parasitic and rabbit enzymes.

The aim of this work was to revisit the connection between prolyl oligopeptidase (PREP) and α-synuclein (aSyn) by presenting novel data from cell free and cellular assays and to discuss the results in a contemporary context. The aSyn aggregation process was studied using fluorescence correlation spectroscopy and thioflavin-T fluorescence. Binding sites for PREP on the aSyn sequence were determined using peptide arrays. Subcellular localisation of PREP and stress markers were studied using double staining immunofluorescence microscopy in SH-SY5Y cells with and without overexpression of aSyn and PREP, before and after differentiation, and with or without proteolytic stress induced by proteasome inhibition. The interaction between PREP and aSyn was found to be weak and transient. It promotes the early phases of aggregation but does not affect the rate of β-fibril formation. Moreover, this interaction is not dependent upon the C-terminal prolines of aSyn, but is affected by PREP inhibitors and interferes with PREP substrate binding. Although present in the same cellular compartments, there is little evidence for a strong physical association of PREP with aggresomes and stress markers. Instead, there is colocalization with aSyn in the cell periphery and neurites. There is evidence for a binding site for peptides much longer than the usual PREP substrates. The modular assembly of molecular machines and the observation that PREP's protein-protein interactions are tuneable by active site inhibitors, lead to the hypothesis that this binding site features in the cross-talk between autophagy and neuron-specific pathways involving vesicle transport and protein secretion.

Translocator protein (TSPO, 18 kDa), previously known as peripheral-type benzodiazepine receptor, is an evolutionarily conserved membrane protein involved in various physiological processes and patho-physiological conditions. The endogeneous TSPO ligand is a polypeptide of 9 kDa, but dipeptides with biological activity have been previously synthesized and characterized. Herein, we synthesized a phenyl alanine derived ligand with a ¹⁹F labelling which opens prospective for ¹⁹F-MRI and potential ¹⁸F-PET applications. We characterized the coexistence of two conformers that are not equally sensitive to the media used for membrane protein studies. Interaction studies with the recombinant mouse TSPO (mTSPO) in different membrane-mimicking environments are presented using ¹⁹F NMR enabling structure/function characterizations. A change in the mTSPO environment from pure detergent to lipid/detergent mixture reveals different exchange rates between bound and free ligand forms. Competition experiments with the high-affinity drug ligand (R)-PK 11195 suggests that phenyl alanine derived ligand binds in the same protein cavity.

The role of glycogen and lipid metabolism in the testes of Daurian ground squirrels (Spermophilus dauricus) during different stages of the hibernation cycle and their influence on reproductive function remain poorly understood. This study examined testicular morphology across hibernation stages and investigated potential molecular mechanisms. Results showed that: (1) Spermatocyte density was reduced in the torpor group compared to the pre-hibernation (PRE) group, suggesting a suppression of spermatogenesis during torpor. In the post-hibernation (POST) group, reduced spermatocyte density was speculated to correspond to the initial phase of spermatocyte maturation into spermatozoa. (2) Glycogen content was lower during interbout arousal (IBA), while glycogen phosphorylase (GP) activity was significantly elevated compared to the other stages. Sertoli cell density was higher in the IBA group relative to the torpor group, suggesting that elevated GP activity facilitates glycogen breakdown, providing glycolytic substrates for Sertoli cells during this phase. (3) During torpor, triglyceride and fatty acid levels, along with fatty acid synthase and acetyl-CoA carboxylase activities, remained consistent with PRE levels. These findings suggest that fatty acids are crucial for maintaining testicular reproductive function during torpor. In contrast, lipid metabolism indicators declined during the near post-hibernation (NP) and POST stages, likely supporting the rapid reactivation of reproductive processes required for the upcoming breeding season. In summary, this study highlights a dynamic interplay between lipid and glycogen metabolism across hibernation stages, with the transition from lipid-based metabolism during torpor to glycogen utilization during IBA playing a pivotal role in sustaining testicular homeostasis in Daurian ground squirrels.

Enrichment of the habitat of captive rodents Heterocephalus glaber (naked mole rats) to implement their innate behavioral pattern of digging dense soil in search of food, paradoxically led to the appearance of unusual animals in the colony. They showed signs of cachexia, distinguished from other animals by a lower temperature (from 31 °C to 26 °C) and body mass index with decreasing proportion of subcutaneous fat. This animal demonstrated aggressive feeding behavior, but didn't gain weight even after finishing experiment with intensive physical activity. In histochemical analysis of tissues from cachexic and normal animals of the same age and sex liver hyperlipofuscinosis was revealed and indicated that animals when being in a habitat with an increased oxygen content for them (21 % versus 8 % in their natural underground habitat), experienced severe oxidative stress during physical exercise. Stress led to a disruption of the body's regulatory systems, a sharp increase in metabolism even at rest, an overload of the cardiovascular and respiratory systems and damage to organs and tissues. To clarify the pathogenetic mechanism of the observed phenomenon, microRNA was extracted from the animal tissues and sequenced. Then bioinformatics analysis of differential expression of microRNAs between groups of healthy animals and animals with idiopathic cachexia was performed.

The glioma hallmark includes reprogramming metabolism to support biosynthetic and bioenergetic demands, as well as to maintain their redox equilibrium. It has been suggested that the pentose phosphate pathway (PPP) and glycolysis are directly involved in the dynamics and regulation of glioma cell proliferation and migration. The PPP is implicated in cellular redox homeostasis and the modulation of signaling pathways, which play a fundamental role in the progression of tumors to malignant grades, metastasis, and drug resistance. Several studies have shown that in glioblastoma cells, the activity, expression, and metabolic flux of some PPP enzymes increase, leading to heightened activity of the pathway. This generates higher levels of DNA, lipids, cholesterol, and amino acids, favoring rapid cell proliferation. Due to the crucial role played by the PPP in the development of glioma cells, enzymes from this pathway have been proposed as potential therapeutic targets. This review summarizes and highlights the role that the PPP plays in glioma cells and focuses on the key functions of the enzymes and metabolites generated by this pathway, as well as the regulation of the PPP. The studies described in this article enrich the understanding of the PPP as a therapeutic tool in the search for pharmacological targets for the development of a new generation of drugs to treat glioma.

Fatty acid desaturases (FADs) belong to of the oxygenase superfamily. They play important roles in metabolic pathways and adaption mechanisms in a wide range of organisms, including bacteria and humans. These enzymes dehydrogenate a single bond in the acyl chain of fatty acids (FAs), forming a double bond. Multiple parameters influence the precise position of double bond formation and acyl chain docking in the catalytic pocket of various FADs, such as the length of an acyl chain, the position of previously generated double bonds, the location of the enzyme's metal catalytic site, and so on. The "counting" mode differs from one FAD to another. The cyanobacterium Synechocystis sp. strain PCC 6803 has four FADs (Δ9, Δ12, Δ6, and Δ15 or ω3) that synthesize mono-, di-, tri-, and tetraenoic FAs. The substrate preferences and "counting" modes for the first three FADs have been identified, but the substrate specificity for the terminal ω3-FAD remains uncertain. We used molecular cloning, heterologous expression with exogenous FAs, and molecular docking to explore the substrate selectivity and counting mode of ω3-FAD. Our results show that ω3-FAD "counts" from the carboxyl (Δ) end, introduces a double bond between 15 and 16 atoms, and requires a specific acyl substrate configuration with two pre-existing double bonds at Δ⁹ and Δ¹² positions.

Anencephaly, the most severe type of neural tube defects (NTDs) in humans, occurs between the third and fourth gestational weeks (GW), involves the cranial part of the NT and results in the absence of the forebrain and skull. Exposed to amniotic fluid toxicity, neural tissue is degraded and prevented from development. Currently, little is known about the molecular bases of the disease and the possible involvement of glycans. In this context, considering the role played by gangliosides (GGs) in fetal brain development and the previous achievements of ion mobility separation (IMS) mass spectrometry (MS) in biomarker discovery, we report here on the introduction of this advanced analytical technique in NTD research, and its optimization for a comprehensive determination of anencephaly gangliosidome. Three native GG extracts from residual brains of anencephalic fetuses in 28, 35 and 37 GW were comparatively profiled by IMS MS, structurally analyzed by IMS MS/MS, and finally assessed against a native GG mixture from normal fetal brain. IMS MS provided data on 343 anencephaly gangliosides vs. only 157 known before and revealed for the first time the incidence of the entire penta-to octasialylated series. The comparative assay disclosed variations in GG expression with fetal age and a correlation of the pattern with the developmental stage. In contrast to the normal fetal brain, the neural tissue in anencephaly was found to contain an elevated number of polysialogangliosides and a lower expression of O-Ac- and GalNAc-modified glycoforms. These species worth further detailed investigation as new potential anencephaly markers.

Oxidative stress arises from an imbalance between reactive species (RS) production and the antioxidant defense, increasing the brain susceptibility to neurodegenerative and psychiatric diseases. Besides, changes in the expression or activity of neurotransmitter metabolism enzymes, such as monoamine oxidases (MAO), are also associated with mental disorders, including depression. Considering this, antioxidant and MAO-A activity inhibitory potential of six 2,3-chalcogenodihydrobenzofurans (2,3-DHBF) was investigated through in vitro and in silico tests. Compounds 1 to 5 incorporate sulfur (S) as chalcogen, whereas compound 6 integrates tellurium (Te). A screening (compounds 1-6) of cerebral MAO-A activity showed inhibitory activity for the compounds 2, 4, 5, and 6. Among sulfur compounds, compound 2 demonstrated superior scores in docking studies, yielding a value of - 9.9 kcal/mol. Selected for concentration-response curves, compounds 2 (with S) and 6 (with Te) inhibited MAO-A at concentrations equal to or higher than 25 μM. In a redox screening test, only compound 6 showed antioxidant effects. Concentration-response curves indicated that compound 6 reduced lipid peroxidation and protein carbonylation levels in mouse brain tissue (≥0.5 μM), as well as reduced RS levels (≥1 μM). Furthermore, the compound 6 (≥5 μM) was effective in reducing the ferric ion (FRAP). In radical scavenging tests such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), compound 6 showed significant results in concentrations from 50 μM and mimicked the enzyme glutathione S-transferase (GST) at 100 μM. In summary, this study demonstrated the cerebral antioxidant and/or MAO-A inhibition properties of 2,3-DHBF, presenting potential as neuroprotective candidates.