Biochemistry and Biophysics Reports最新文献

Thirty years since the first report on the PilY1 protein in bacteria, only the C-terminal domain has been crystallized; there is no study in which the N-terminal domain, let alone the complete protein, has been crystallized. In our laboratory, we are interested in characterizing the Type IV Pili (T4P) of Acidithiobacillus thiooxidans. We performed an in silico characterization of PilY1 and other pilins of the T4P of this acidophilic bacterium. In silico characterization is crucial for understanding how proteins adapt and function under extreme conditions. By analyzing the primary and secondary structures of proteins through computational methods, researchers can gain valuable insights into protein stability, key structural features, and unique amino acid compositions that contribute to resilience in harsh environments. Here, it is presented a description of the particularities of At. thiooxidans PilY1 through predictor software and homology data. Our results suggest that PilY1 from At. thiooxidans may have the same role as has been described for other PilY1 associated with T4P in neutrophilic bacteria; also, its C-terminal interacts (interface interaction) with the minor pilins PilX, PilW and PilV. The N-terminal region comprises domains such as the vWA and the MIDAS, involved in signaling, ligand-binding, and protein-protein interaction. In fact, the vWA domain has intrinsically disordered regions that enable it to maintain its structure over a wide pH range, not only at extreme acidity to which At. thiooxidans is adapted. The results obtained helped us design the correct methodology for its heterologous expression. This allowed us partially experimentally characterize it by obtaining the N-terminal domain recombinantly and evaluating its acid stability through fluorescence spectroscopy. The data suggest that it remains stable across pH changes. This work thus provides guidance for the characterization of extracellular proteins from extremophilic organisms.

Neurodegenerative diseases including glaucoma affect insulin signaling, and insulin treatment has been shown to reverse the neurodegenerative loss of dendritic complexity in retinal ganglion cells. Therefore, strategies for enhancing or maintaining insulin signaling are worth pursuing to establish new therapies for these diseases. In the present study, we generated constitutively active insulin receptor (F-iIR) and insulin-like growth factor-1 receptor (F-iIGF1R) using a system that forces membrane localization of the intracellular domains of these receptors by farnesylation. Immunohistochemistry and Western blot analysis revealed that F-iIR and F-iIGF1R caused the activation of ERK and AKT in the absence of ligands in vitro. Our results suggest that in vivo effects of F-iIR and F-iIGF1R on the progression of neurodegenerative diseases should be investigated in the future.

Plasminogen activator inhibitor-1 (PAI-1/Serpin E1) is classically known for its antifibrinolytic activity via inhibiting uPA and tPA of the fibrinolytic pathway. PAI-1 has a paradoxical role in tumor progression, and its molecular functions are poorly understood. PAI-1 is a widely accepted secretory protease inhibitor, however, a study suggested the localization of PAI-1 in the cytoplasm and the nucleus. Besides the plethora of its biological functions as a secretory protein, intracellular localization, and functions of PAI-1 remain unexplored at the molecular level. In this study, using various in silico approaches, we showed that PAI-1 possesses a nuclear export signal. Using the CRM1-specific inhibitor leptomycin B, we demonstrated that PAI-1 has a functional CRM1-dependent NES, indicating the possibility of its nuclear localization. Further, we confirm that PAI-1 is localized in the nucleus of endothelial cells using fluorescence microscopy and immunoprecipitation. Notably, we identified an unconventional distribution of PAI-1 in the PML bodies of the nucleus of normal endothelial cells, while the protein was restricted in the cytoplasm of slow-growing cells. The data showed that the localization of PAI-1 in PML bodies is highly correlated with the growth potential of endothelial cells. This conditional nucleocytoplasmic shuttling of PAI-1 during the aging of cells could impart a strong link to its age-related functions and tumor progression. Together, this study identifies the novel behavior of PAI-1 that might be linked with cell aging and may be able to unveil the elusive role of PAI-1 in tumor progression.

The microenvironment of the endometrial immune system is crucial to the success of placental implantation and healthy pregnancy. However, the functionalities of immune cells across various stages of the reproductive cycle have yet to be fully comprehended. To address this, we conducted advanced bioinformatic analysis on 230,049 high-quality single-cell transcriptomes from healthy endometrial samples obtained during the proliferative, secretory, early pregnancy, and late pregnancy stages. Our investigation has unveiled that proliferative natural killer (NK) cells, a potential source of endometrial NK cells, exhibit the most robust proliferative and differentiation potential during non-pregnant stages. We have also identified similar differentiation trajectories of NK cells originating from proliferative NK cells across four stages. Notably, during early pregnancy, NK cells demonstrate the highest oxidative phosphorylation metabolism activity, and, in conjunction with macrophages and T cells, exhibit the strongest type II interferon response. With spatial transcriptome data, we have discerned that the most robust immune-non-immune interactions are associated with the promotion and inhibition of cell proliferation, differentiation and migration during four stages. Furthermore, we have compiled lists of stage-specific risk genes implicated in reproductive diseases, which hold promise as potential disease biomarkers. Our study provides insights into the dynamics of the endometrial immune microenvironment during different reproductive cycle stages, thus serving as a reference for detecting pathological changes during pregnancy.

Fluorescence microscopy is an important tool for cell biology and cancer research. Present-day approach of implementing advanced optical microscopy methods combined with immunofluorescence labelling of specific proteins in cells is now able to deliver optical super-resolution up to ∼25 nm. Here we perform super-resolved imaging using standard immunostaining protocol combined with easy stochastic optical reconstruction microscopy (easySTORM) to observe structural differences of two cytoskeleton elements, actin and tubulin in three different cell types namely human bone marrow-derived mesenchymal stem cells (MSCs), human glioblastoma (U87MG) and breast cancer (MDAMB-231) cells. The average width of the actin bundle obtained from STORM images of stem cells is observed to be larger than the same for U87MG and MDAMB-231 cells. No significant difference is however noticed in the width of the tubulin within the same cells. We also study the functional effect on the 2D migration potential of MDAMB-231 cells silenced for NICD1 and β-catenin. Although similar migration speed is observed for cells with the above two conditions compared to their control cells, easySTORM images show that widths of the actin in MDAMB-231 cells in β-catenin silenced is significantly lower than the same in control cells. Such minute differences however are not observable in widefield images. The outcome of our easySTORM investigation should benefit the researchers carrying out detailed investigations of the cellular structure and potential therapeutic applications.

In recent years, it has become clear that the cytotoxicity of γ-irradiation of cells is increased under microgravity conditions. However, there has been no study of the effect of the gravity vector direction, rather than the magnitude, on γ-ray-induced cytotoxicity. Therefore, in this study, we inverted cultures of human bronchial epithelium BEAS-2B cells and human lung cancer A549 cells in order to change the gravity vector direction by 180° with respect to the cells and observed the cellular response to radiation in this state. We found that cells in inverted culture showed increased irradiation-induced production of reactive oxygen species and decreased expression of the antioxidant protein thioredoxin-1 compared to cells in normal culture. Furthermore, the DNA damage response was delayed in γ-irradiated cells in inverted culture, and the number of unrepaired DNA sites was increased, compared to irradiated cells in normal culture. γ-Ray-induced cell death and the number of G₂-M arrested cells were increased in inverted culture, in accordance with the decreased capacity for DNA repair. Our findings suggest that the gravity vector direction, as well as its magnitude, alters the cellular response to radiation.

Sialidases catalyze the removal of terminal sialic acids from sialylated biomolecules, and their substrate preference is frequently indicated in terms of the glycosidic linkages cleaved (α2-3, α2-6, and α2-8) without mention of the remaining sub-terminal reducing-end saccharide moieties. Many human gut commensal and pathogenic bacteria secrete sialidases to forage for sialic acids, which are then utilized as an energy source or assimilated into membrane/capsular structural components. Infant gut commensals similarly utilize sialylated human milk oligosaccharides containing different glycosidic linkages. Here, we have studied the preference of the bacterial sialidases, BbSia2 from Bifidobacterium bifidum, CpNanI from Clostridium perfringens, and HpNanH from Glaesserella parasuis, for the glycosidic linkages, Siaα2-3Gal, Siaα2-6Gal, and Siaα2-6GlcNAc, by employing 2-Aminobenzamide-labeled human milk oligosaccharides, 3′-Sialyllactose (3′-SL), 6′-Sialyllactose (6′-SL), and Sialyllacto-N-tetraose-b (LSTb), respectively, as proxies for these glycosidic linkages. BbSia2, CpNanI, and HpNanH hydrolyzed these three oligosaccharides with the glycosidic linkage preferences, 3′-SL (Siaα2-3Gal) ≥ LSTb (Siaα2-6GlcNAc) ≥ 6′-SL (Siaα2-6Gal), 3′-SL (Siaα2-3Gal) ≥ 6′-SL (Siaα2-6Gal) > LSTb (Siaα2-6GlcNAc), and 3′-SL (Siaα2-3Gal) ≥ 6′-SL (Siaα2-6Gal) > LSTb (Siaα2-6GlcNAc), respectively. Our finding suggests that sub-terminal reducing-end saccharide moieties can profoundly influence the substrate preference of sialidases, and advocates for the characterization and indication of the substrate preference of sialidases in terms of both the glycosidic linkage and the sub-terminal reducing-end saccharide moiety.

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of type I collagen. This modification is critical for the formation of stable hydroxylysine-aldehyde derived collagen cross-links, thus, for the stability of collagen fibrils. Though dysfunction of LH2 causes Bruck syndrome, recessive osteogenesis imperfecta with joint contracture, the molecular mechanisms by which LH2 affects bone formation are still not well understood. Since the Plod2 knockout mice are embryonically lethal, we generated bone-specific LH2 conditional knockout mice (bsLH2-cKO) using the osteocalcin-Cre/loxP system, and evaluated phenotypes of femurs. LH2 mRNA and protein levels assessed by qPCR, immunohistochemistry and Data Independent Acquisition proteomics were all markedly low in bsLH2-cKO femurs when compared to controls. Lysine hydroxylation of both carboxy- and amino-terminal telopeptides of an α1(I) chain were significantly diminished resulting in reduction of the hydroxylysine-aldehyde derived cross-links. The collagen fibrils in bsLH2-cKO appeared to be thicker, often fused and irregular when compared to controls. In addition, bone mineral density and mechanical properties of bsLH2-cKO femurs were significantly impaired. Taken together, these data demonstrate that LH2-catalyzed modification and consequent cross-linking of collagen are critical for proper bone formation and mechanical strength.

Novel Geobacillus sp. DS3, isolated from the Sikidang Crater in Dieng, exhibits promising characteristics for industrial applications, particularly in thermostable α-amylase production. Recombinant technology was used to express thermostable α-amylase in E. coli BL21(DE3) to overcome high-temperature production challenges. The study aimed to express, purify, characterize, and explore potential applications of this novel enzyme. The enzyme was successfully expressed in E. coli BL21(DE3) at 18 °C for 20 h with 0.5 mM IPTG induction. Purification with Ni-NTA column yielded 69.23 % from the initial crude enzyme, with a 3.6-fold increase in specific activity. The enzyme has a molecular weight of ±70 kDa (±58 kDa enzyme+11 kDa SUMO protein). It exhibited activity over a wide temperature range (30–90 °C) and pH range (6–8), with optimal activity at 70 °C and pH 6 with great stability at 60 °C. Kinetic analysis revealed Km and Vmax values of 324.03 mg/ml and 36.5 U/mg, respectively, with dextrin as the preferred substrate without cofactor addition. As a metalloenzyme, it showed the best activity in the presence of Ca²⁺. The enzyme was used for porous starch production and successfully immobilized with chitosan, exhibiting improved thermal stability. After the fourth reuse, the immobilized enzyme maintained 62 % activity compared to the initial immobilization.