Membrane Protein Components Research Articles

EIF5A controls mitoprotein import by relieving ribosome stalling at TIM50 translocase mRNA.

Efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A binds ribosomes, alleviating stalling at polyproline-encoding sequences. eIF5A impacts mitochondrial function across species, though the precise molecular mechanism is unclear. We found that eIF5A depletion in yeast reduces the translation and levels of the TCA cycle and oxidative phosphorylation proteins. Loss of eIF5A causes mitoprotein precursors to accumulate in the cytosol and triggers a mitochondrial import stress response. We identify an essential polyproline protein as a direct target of eIF5A: the mitochondrial inner membrane protein and translocase component Tim50. Thus, eIF5A controls mitochondrial protein import by alleviating ribosome stalling along Tim50 mRNA at the mitochondrial surface. Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response and translation of oxidative phosphorylation genes. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by promoting efficient translation and reducing ribosome stalling of co-translationally imported proteins, thereby positively impacting the mitochondrial import process.

Expression of stress responsive genes enables Limosilactobacillus reuteri to cross-protection against acid, bile salt, and freeze-drying.

Limosilactobacillus reuteri effectively colonizing the gut, secretes antimicrobial compounds and strengthens immune system function. Considering these health benefits, increasing its stress assessments efficiency could improve its commercial viability. In this work, the resistance of L. reuteri FP41 to acid, bile salts, and freeze-drying was examined. The findings showed that strain FP41 demonstrated a strong resistance to acid/bile salt stresses. The transcriptome revealed a significant up-regulation of various stress response genes, including those related to membrane integrity, glutamine metabolism, OsmC family protein, ABC transporters, and chaperonin. Subsequent research demonstrated that overexpression of three stress response-specific proteins, including glutamate decarboxylase GatD, osmotically induced bacterial protein OsmC, and membrane protein component CsbD, significantly increased the survival rate of L. reuteri Z204 under acid/bile salts stress. Notably, overexpression of the OsmC, CsbD, and GatD proteins also enhanced the survival of L. reuteri after freeze-drying. The development of a unique cross-protection method is highlighted in this study, that might significantly increase cellular resistance to acid, bile salts, and cold stresses. This finding could significantly impact the way that L. reuteri is employed in industrial manufacturing processes.

TGF - β/SMAD signaling pathway and protein molecules in the treatment of liver fibrosis: A natural lipid membrane protein of exosomes

In recent years, exosomes, as an important medium of intercellular information transmission, have received extensive attention for their potential in the treatment of liver fibrosis. The purpose of this study was to investigate the role of exosome natural lipid membrane proteins in the treatment of liver fibrosis, with emphasis on the regulatory mechanism through the TGF-β/SMAD signaling pathway. Exosomes were extracted from healthy human hepatocytes and their membrane protein components were identified by mass spectrometry. Subsequently, the effects of these exosomes and their membrane proteins on the TGF-β/SMAD signaling pathway were examined using in vitro cell models and mouse liver fibrosis models. Western blot, qPCR and immunofluorescence were used to analyze the expression of fibrosis markers and the activity of signaling pathways. In vitro cell experiments, fibrotic cells showed an obvious reversal trend after treating exosome membrane proteins. In a mouse model of liver fibrosis, the injection of exosome membrane proteins significantly improved the degree of fibrosis in liver tissue.

Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

Cutting-Edge HEK293T Protein-Integrated Lipid Nanostructures: Boosting Biocompatibility and Efficacy.

Recently, artificial exosomes have been developed to overcome the challenges of natural exosomes, such as production scalability and stability. In the production of artificial exosomes, the incorporation of membrane proteins into lipid nanostructures is emerging as a notable approach for enhancing biocompatibility and treatment efficacy. This study focuses on incorporating HEK293T cell-derived membrane proteins into liposomes to create membrane-protein-bound liposomes (MPLCs), with the goal of improving their effectiveness as anticancer therapeutics. MPLCs were generated by combining two key elements: lipid components that are identical to those in conventional liposomes (CLs) and membrane protein components uniquely derived from HEK293T cells. An extensive comparison of CLs and MPLCs was conducted across multiple in vitro and in vivo cancer models, employing advanced techniques such as cryo-TEM (tramsmission electron microscopy) imaging and FT-IR (fourier transform infrared spectroscopy). MPLCs displayed superior membrane fusion capabilities in cancer cell lines, with significantly higher cellular uptake. Additionally, MPLCs maintained their morphology and size better than CLs when exposed to FBS (fetal bovine serum), suggesting enhanced serum stability. In a xenograft mouse model using HeLa and ASPC cancer cells, intravenous administration of MPLCs MPLCs accumulated more in tumor tissues, highlighting their potential for targeted cancer therapy. Overall, these results indicate that MPLCs have superior tumor-targeting properties, possibly attributable to their membrane protein composition, offering promising prospects for enhancing drug delivery efficiency in cancer treatments. This research could offer new clinical application opportunities, as it uses MPLCs with membrane proteins from HEK293T cells, which are known for their efficient production and compatibility with GMP (good manufacturing practice) standards.

Iron-Based Nanoplatforms Achieve Hepatocellular Carcinoma Regression Through a Cascade of Effects.

Ferroptosis is a regulated form of cell death characterized by iron-dependent accumulation of associated lipid peroxides (LPO), which can induce cell death when a certain level is reached. However, the extremely complex tumor microenvironment (TME) has the characteristics of antioxidant, even if it induces ferroptosis of tumor cells, its killing effect on tumor cells is still very limited. To solve this problem, we constructed a novel nanomaterials (GOx/EC@Fe3O4@CCM). We evaluated the anticancer effect of this nanomaterial in inhibiting tumor growth through comprehensive in vitro and in vivo experiments. We successfully synthesized GOx/EC@Fe3O4 by one-pan synthesis method, then coated the Hepatocellular carcinoma cell membrane on its surface by co-extrusion technology, and finally synthesized the GOx/EC@Fe3O4@CCM nanoplatforms. We characterized the compounds in terms of morphology, particle size, and Zeta potential. In addition, we also studied the anti-tumor effect of GOx/EC@Fe3O4@CCM nanoplatforms from the following aspects, including the performance test of the nanoplatform, the intracellular effect of the nanoplatform, the anti-tumor effect in vitro, the intracellular ROS analysis, the intracellular effect of EC, and the anti-tumor effect in vivo. The iron-based carriers in GOx/EC@Fe3O4@CCM nanoplatforms are released and produce ferrous ions (Fe2+) in an acidic environment. Due to the limitation of the endogenous level of hydrogen peroxide (H2O2), we introduced GOx into the TME or tumor cells. Under the catalysis of GOx, glucose reacted rapidly to produce a large amount of H2O2, which then combined with Fe2+ to produce a large number of Hydroxyl radical (·OH). Its toxicity leads to dysfunction of cell membrane and organelles, and then causes cell damage. EC inhibits Nuclear factor erythroid 2-related factor 2 (Nrf2) in cancer cells, which effectively down-regulates downstream gene products, including NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HMOX1). A series of chain reactions reduce the escape effect of oxidative stress (OS) and effectively maintain a high level of intracellular oxidation. Furthermore, it induces sustained and intense ferroptosis in tumor cells. Finally, the use of cancer cell membrane modified nanoplatforms due to the homology of membrane protein components improves the tumor cell targeting of the nanoplatforms, showing significant tumor cell inhibition and killing effect in vivo. The results showed that the GOx/EC@Fe3O4@CCM nanoplatforms successfully induced significant ferroptosis of Hepatocellular carcinoma cells through a cascade effect, and finally effectively promoted cancer cell regression.

Availability of iron ions impacts physicochemical properties and proteome of outer membrane vesicles released by Neisseria gonorrhoeae

Outer membrane vesicles (OMVs) are bilayer structures released by bacteria for various purposes, e.g., response to environmental factors, bacterial communication, and interactions with host cells. One of the environmental variables bacteria need to react is the amount and availability of iron, a crucial element for bacteria biology. We have investigated the impact of the iron amount and availability on OMV secretion by pathogenic Neisseria gonorrhoeae, which, depending on the infection site, challenges different iron availability. N. gonorrhoeae releases OMVs in iron starvation and repletion growth environments. However, OMVs differed in physicochemical features and proteome according to iron amount and availability during the bacteria growth, as was analyzed by Liquid Chromatography-Tandem Mass Spectrometry, Infrared spectroscopy with a Fourier transform infrared spectrometer, and Atomic Force Microscopy. OMVs from iron starvation and repletion conditions had a higher variation in size, different flexibility, and different membrane protein and lipid components than OMVs isolated from control growth conditions. These OMVs also varied qualitatively and quantitatively in their total proteome composition and contained proteins unique for iron starvation and repletion conditions. Thus, the modulation of OMVs' properties seems to be a part of N. gonorrhoeae adaptation to surroundings and indicates a new direction of antigonococcal proceeding.

Combined Structure- and Ligand-Based Approach for the Identification of Inhibitors of AcrAB-TolC in Escherichia coli.

The inhibition of efflux pumps is a promising approach to combating multidrug-resistant bacteria. We have developed a combined structure- and ligand-based model, using OpenEye software, for the identification of inhibitors of AcrB, the inner membrane protein component of the AcrAB-TolC efflux pump in Escherichia coli. From a database of 1391 FDA-approved drugs, 23 compounds were selected to test for efflux inhibition in E. coli. Seven compounds, including ivacaftor (25), butenafine (19), naftifine (27), pimozide (30), thioridazine (35), trifluoperazine (37), and meloxicam (26), enhanced the activity of at least one antimicrobial substrate and inhibited the efflux pump-mediated removal of the substrate Nile Red from cells. Ivacaftor (25) inhibited efflux dose dependently, had no effect on an E. coli strain with genomic deletion of the gene encoding AcrB, and did not damage the bacterial outer membrane. In the presence of a sub-minimum inhibitory concentration (MIC) of the outer membrane permeabilizer colistin, ivacaftor at 1 μg/mL reduced the MICs of erythromycin and minocycline by 4- to 8-fold. The identification of seven potential AcrB inhibitors shows the merits of a combined structure- and ligand-based approach to virtual screening.

Protecting the Eye Lens from Oxidative Stress through Oxygen Regulation.

Molecular oxygen is a primary oxidant that is involved in the formation of active oxygen species and in the oxidation of lipids and proteins. Thus, controlling oxygen partial pressure (concentration) in the human organism, tissues, and organs can be the first step in protecting them against oxidative stress. However, it is not an easy task because oxygen is necessary for ATP synthesis by mitochondria and in many biochemical reactions taking place in all cells in the human body. Moreover, the blood circulatory system delivers oxygen to all parts of the body. The eye lens seems to be the only organ that is protected from the oxidative stress through the regulation of oxygen partial pressure. The basic mechanism that developed during evolution to protect the eye lens against oxidative damage is based on the maintenance of a very low concentration of oxygen within the lens. This antioxidant mechanism is supported by the resistance of both the lipid components of the lens membrane and cytosolic proteins to oxidation. Any disturbance, continuous or acute, in the working of this mechanism increases the oxygen concentration, in effect causing cataract development. Here, we describe the biophysical basis of the mechanism and its correlation with lens transparency.

Architecture of a complete Bce-type antimicrobial peptide resistance module

Gram-positive bacteria synthesize and secrete antimicrobial peptides that target the essential process of peptidoglycan synthesis. These antimicrobial peptides not only regulate the dynamics of microbial communities but are also of clinical importance as exemplified by peptides such as bacitracin, vancomycin, and daptomycin. Many gram-positive species have evolved specialized antimicrobial peptide sensing and resistance machinery known as Bce modules. These modules are membrane protein complexes formed by an unusual Bce-type ABC transporter interacting with a two-component system sensor histidine kinase. In this work, we provide the first structural insight into how the membrane protein components of these modules assemble into a functional complex. A cryo-EM structure of an entire Bce module revealed an unexpected mechanism of complex assembly, and extensive structural flexibility in the sensor histidine kinase. Structures of the complex in the presence of a non-hydrolysable ATP analog reveal how nucleotide binding primes the complex for subsequent activation. Accompanying biochemical data demonstrate how the individual membrane protein components of the complex exert functional control over one another to create a tightly regulated enzymatic system.

Abstract 3841: SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro

Abstract Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can occur in laminin-111-containing Matrigel and requires the PI3K pathway. However, the membrane protein component and signaling pathways involved in this process are unknown. Methods: In an established in vitro model of VM of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized gene silencing and blocking antibodies to elucidate the signaling pathways involved in this process. RNASeq was used to identify novel transcripts and siRNA was utilized to verify the requirement of candidate RNA/protein in tubular formation. Results: siRNA and antibody blocking of integrin β1, but not β3, prevented VM formation in vitro. Individual silencing of cortactin, TKS5, MMP-2, MMP-9 and MMP-14 affected tubular formation. RNAseq analysis suggested that VM has minimal dependence on de novo transcriptional activity yet reported a strong upregulation of small Integral Membrane Protein 11 (SMIM11A). This result was verified by qPCR and siRNA silencing of SMIM11A prevented VM formation. Discussion: Laminin 111 may interact with integrin β1, and the consequent activation of the PI3K pathway could potentially remodel cytoskeletal proteins and promote the activity of MMPs. We report for the first time a biological role for SMIM11a. This gene is regulated at the RNA level and is required for the formation of tubular structure in vitro. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Javiera Pradenas, Nicole Babbitt, Pamela González, Cristina Bertocchi, Gareth Owen. SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3841.

Analysis of epidemiology and novel mutation of Helicobacter pylori antibiotic resistance in South Korea

BackgroundHelicobacter pylori is a Gram‐negative bacterium associated with upper gastrointestinal disease, peptic ulcer, mucosa‐associated lymphoid tissue lymphoma and eradication treatment is recommended because it is one of the causes of gastric cancer. However, resistance to some antibiotics used for treatment has developed, the eradication rate is declining worldwide. This study focused on antibiotic susceptibility test (AST) of H. pylori isolated from patient clinical specimens. The aim of this study is to identify the resistance epidemiology of H. pylori and to elucidate the resistance mechanism through gene analysis.methodsH. pylori isolated from patient clinical specimens is identified and AST for amoxicillin (AMX) and clarithromycin (CLR) are performed, and the results are analyzed through Whole Genome Sequencing (WGS) and MultiLocus Sequence Typing (MLST), respectively. The strain type of the MLST result is compared with the CLR resistance minimum inhibitory concentration (MIC) value to analyze the significant points.H. pylori is cultured on EYE Agar plate and incubated in Anaerobic Jar for 2~5 days with microaerophilic air condition (5% O2, 10% CO2, 85% N2). Cultured isolates are identified by MALDI‐TOF system and performed AST using Epsilometer test to determining MIC.Results366 H. pylori were isolated from 1952 clinical patients, and 36 AMX‐resistant H. pylori and 101 CLR‐resistant H. pylori were identified. Among the identified H. pylori, 9 AMX high MIC strains were selected and WGS was performed, and 12 CLR high MIC strains were selected and MLST was performed.As the WGS result of 9 clinical strains resistant to AMX, single nucleotide polymorphism (SNP) of the potential target gene was analyzed with reference to ATCC 26695. Six genes, Penicillin binding protein1A (PBP1), PBP2, NhaC, hofH, hofC, and hefC, were selected as potential target genes. As a result of the analysis, 8, 7, 4, 9, 16, and 2 SNPs were found, respectively, and it was specifically confirmed that there were many SNPs in the outer membrane component Gene. However, No correlation was found between AMX MIC value and target SNP.12 For the CLR resistance strain, MLST was performed using 7 housekeeping genes (atpA, efp, trpC, ppa, mutY, yphC, Ure1). In 12 strain, 10 CLR‐resistance H. pylori are not identified strain type(ST) to The National Center for Biotechnology Information(NCBI) database because they have a lots of mutation in 7 housekeeping gene. 2 CLR‐resistance H. pylori are identified the new type strains.ConclusionIn this study, we conclude that hofC, hofH related to outer membrane protein component may be associated to the AMX resistance. And additional research is continued to need about other resistance potential gene, especially hopC.

Contribution of FBLN5 to Unstable Plaques in Carotid Atherosclerosis via mir128 and mir532–3p Based on Bioinformatics Prediction and Validation

FBLN5, a member of the short fibulins in the fibulin family of extracellular matrix/matricellular proteins, is involved in interactions with components of the basement membrane and extracellular matrix proteins. It plays key roles in endothelial tissues in many vascular diseases. In this study, the relationship between FBLN5 and carotid atherosclerotic plaque stability as well as the regulatory roles of miRNAs were evaluated. Differential gene expression analyses and weighted gene co-expression network analysis (WGCNA) based on the GSE163154 dataset (including 16 samples without intraplaque hemorrhage and 27 samples with intraplaque hemorrhage) in GEO revealed that FBLN5 is related to plaque stability and is the most significantly differentially expressed gene. LASSO regression was used to evaluate genes obtained from the intersection of differentially expressed genes and clinically significant modules identified by WGCNA. A prediction model based on eight genes, including FBLN5, was constructed and showed an accuracy of 0.951 based on an ROC analysis. Low FBLN5 expression in plaque tissues was confirmed by immunohistochemistry and western blotting. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that FBLN5 acted mainly by the maintenance of the cellular matrix and reactive oxygen species production. miRNAs upstream of these eight predictive genes, including FBLN5, were identified and used to construct a network diagram. These results revealed that hsa-mir-128 and hsa-mir-532–3p were upstream regulatory factors of FBLN5, as verified by PCR assays of human plaque tissues demonstrating that both miRNAs were significantly up-regulated. Therefore, FBLN5 may play an important role in carotid atherosclerosis via hsa-mir-128 and hsa-mir-532–3p as well as become an essential target for treatment.

Modified Exosomes: a Good Transporter for miRNAs within Stem Cells to Treat Ischemic Heart Disease.

Stem cell-based therapy for ischemic heart disease (IHD) has become a promising but controversial strategy during the past two decades. The fate and effects of stem cells engrafted into ischemia myocardium are still not fully understood. Stem cell-derived exosomes, a subcategory of extracellular vesicles with nano size, have been considered as an efficient and safe transporter for microRNAs (miRNAs) and a central mediator of the cardioprotective potentials of the parental cells. Hypoxia, pharmacological intervention, and gene manipulation could alter the exosomal miRNAs cargos from stem cells and promote therapeutic potential. Furthermore, several bioengineering methods were also successfully applied to modify miRNAs content and components of exosomal membrane proteins recently. In this review, we outline relevant results about exosomal miRNAs from stem cells and focus on the current strategies to promote their therapeutic efficiency in IHD.

Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli

Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.

Bacteriophage isolated from non-target bacteria demonstrates broad host range infectivity against multidrug-resistant bacteria.

Antibiotic resistance represents a global health challenge. The emergence of multidrug-resistant (MDR) bacteria such as uropathogenic Escherichia coli (UPEC) has attracted significant attention due to increased MDR properties, even against the last line of antibiotics. Bacteriophage, or simply phage, represents an alternative treatment to antibiotics. However, phage applications still face some challenges, such as host range specificity and development of phage resistant mutants. In this study, using both UPEC and non-UPEC hosts, five different phages were isolated from wastewater. We found that the inclusion of commensal Escherichia coli as target hosts during screening improved the capacity to select phage with desirable characteristics for phage therapy. Whole-genome sequencing revealed that four out of five phages adopt strictly lytic lifestyles and are taxonomically related to different phage families belonging to the Myoviridae and Podoviridae. In comparison to single phage treatment, the application of phage cocktails targeting different cell surface receptors significantly enhanced the suppression of UPEC hosts. The emergence of phage-resistant mutants after single phage treatment was attributed to mutational changes in outer membrane protein components, suggesting the potential receptors recognized by these phages. The findings highlight the use of commensal E. coli as target hosts to isolate broad host range phage with infectivity against MDR bacteria.

Transcriptome and Literature Mining Highlight the Differential Expression of ERLIN1 in Immune Cells during Sepsis.

Simple SummarySepsis is a disease affecting 19 million people worldwide, and accounts for 5 million deaths annually. Efforts in finding predictive markers of sepsis development have been difficult due to the complex clinical features of the disease. Public data repositories are valuable resources for mining gene expression changes across different studies. Using such resources, we observed a consistent increase in ERLIN1—a gene coding for an ER membrane prohibitin and regulator of cholesterol—in whole blood, and across a variety of immune cells, during sepsis or sepsis-like conditions. We verified this finding by exposing the whole blood of healthy volunteers to a combination of lipopolysaccharide and peptidoglycan in order to simulate sepsis. We observed an increase in ERLIN1 in whole-blood neutrophils and HL60 cell lines during sepsis; however, the protein was expressed differently in other immune blood cells. The current available studies on ERLIN1 and sepsis indicate a knowledge gap between the functions of ERLIN1, calcium balance, and cholesterol and fatty acid synthesis, and sepsis. Together with experimental data, we think that ERLIN1 is modulated differently in immune cells in response to infection, and has important implications for ER functions and/or ER membrane protein components during sepsis.Sepsis results from the dysregulation of the host immune system. This highly variable disease affects 19 million people globally, and accounts for 5 million deaths annually. In transcriptomic datasets curated from public repositories, we observed a consistent upregulation (3.26–5.29 fold) of ERLIN1—a gene coding for an ER membrane prohibitin and a regulator of inositol 1, 4, 5-trisphosphate receptors and sterol regulatory element-binding proteins—under septic conditions in healthy neutrophils, monocytes, and whole blood. In vitro expression of the ERLIN1 gene and proteins was measured by stimulating the whole blood of healthy volunteers to a combination of lipopolysaccharide and peptidoglycan. Septic stimulation induced a significant increase in ERLIN1 expression; however, ERLIN1 was differentially expressed among the immune blood cell subsets. ERLIN1 was uniquely increased in whole blood neutrophils, and confirmed in the differentiated HL60 cell line. The scarcity of ERLIN1 in sepsis literature indicates a knowledge gap between the functions of ERLIN1, calcium homeostasis, and cholesterol and fatty acid biosynthesis, and sepsis. In combination with experimental data, we bring forth the hypothesis that ERLIN1 is variably modulated among immune cells in response to cellular perturbations, and has implications for ER functions and/or ER membrane protein components during sepsis.

Direct homophilic interaction of LAMP2A with the two-domain architecture revealed by site-directed photo-crosslinks and steric hindrances in mammalian cells

ABSTRACT LAMP1 (lysosomal-associated membrane protein 1) and LAMP2 are the most abundant protein components of lysosome membranes. Both LAMPs have common structures consisting of a large lumenal domain composed of two domains (N-domain and C-domain, which are membrane-distal and -proximal, respectively), both with the β-prism fold, a transmembrane domain, and a short cytoplasmic tail. LAMP2 is involved in various aspects of autophagy, and reportedly forms high-molecular weight complexes at the lysosomal membrane. We previously showed that LAMP2 molecules coimmunoprecipitated with each other, but whether the homophilic interaction is direct or indirect has remained to be elucidated. In the present study, we demonstrated the direct homophilic interaction of mouse LAMP2A molecules, using expanded genetic code technologies that generate photo-crosslinking and/or steric hindrance at specified interfaces. Specifically, the results suggested that LAMP2A molecules assemble by facing each other with one side of the β-prism (defined as side A) of the C-domains. The N-domain truncation, which increased the coimmunoprecipitation of LAMP2A molecules in our previous study, permitted the nonspecific involvement of both sides of the β-prism (side A and side B). Thus, the presence of the N-domain restricts the LAMP2A interactions to side A-specific. The truncation of LAMP2A impaired the recruitment of GAPDH (a CMA-substrate) fused to the HaloTag protein to the surface of late endosomes/lysosomes (LE/Lys) and affected a process that generates LE/Lys. The present study revealed that the homophilic interaction of LAMP2A is direct, and the side A-specific, homophilic interaction of LAMP2A is required for the functional aspects of LAMP2A. Abbreviations: Aloc-Lys: N ε-allyloxycarbonyl-l-lysine; CMA: chaperone-mediated autophagy; FFE: free-flow electrophoresis; GAPDH-HT: glyceraldehyde-3-phosphate dehydrogenase fused to HaloTag protein; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LBPA: lysobisphosphatidic acid; LE/Lys: late endosome/lysosomes; MEFs: mouse embryonic fibroblasts; pBpa: p-benzoyl- l-phenylalanine

Changes in the structural components of the mucous membrane and plasma proteins of small intestine enterocytes in fetuses of cattle

The interrelationships of morphological changes of the mucosa of the large intestine with the dynamics of enzyme activity at different poles of enterocytes of cattle during the fetal period of ontogenesis are shown and analyzed. The work was performed on Holstein cattle aged two to nine months. The activity of hydrolytic and transport enzymatic systems in the basolateral membrane of cattle enterocytes depends less on the morphometric parameters of the large intestine than in the apical membrane. It is proved that during the fruitful period of ontogenesis there are dynamic transformations of enzyme systems of enterocytes of the large intestine of cattle which are connected with morphological changes of the mucous membrane of the large intestine. The activity of hydrolytic and transport enzymes in different domains of the plasma enterocytes of fetal cattle is interrelated with the morphometric parameters of the colon, in particular. the activity of alkaline phosphatase, γ-glutamyltransferase, Na+, K+-ATPase, Ca2 +, Mg2 +-ATPase and Mg-ATPase in the apical membrane of enterocytes is inversely related to the thickness of the intestinal wall with villi and the mucous membrane of the cavity (P ≤ 0.01–0.001). A significant correlation was found between the activity of alkaline phosphatase, γ-glutamyltransferase, Na+, K+-ATPase, Ca2 +, Mg2 +-ATPase and Mg-ATPase in the apical membrane of enterocytes with villi height (P ≤ 0.05). The activity of hydrolytic and transport enzymatic systems in the basolateral membrane of bovine fetal enterocytes depends to a lesser extent on the morphometric parameters of the large intestine than in the apical membrane. In particular, only the activity of alkaline phosphatase and γ-glutamyltransferase is inversely related to the thickness of the intestinal wall with villi and the mucous membrane of the jejunum with villi (P ≤ 0.01–0.001). The activity of transport enzymes is directly related to the morphometric parameters of the colon (P ≤ 0.01). The activity of these enzymes in the basolateral membrane does not significantly depend on the width of the villi, but is dependent on their height.

Simultaneous phycoremediation of petrochemical wastewater and lipid production by Chlorella vulgaris

A novel strategy of using microalgae Chlorella vulgaris for simultaneous bio-treatment of petrochemical wastewater and lipid production was developed in the present study. Phycoremediation was carried out in 30 days. The profile of fatty acids was identified, and the specifications of biodiesel including saponification value, iodine value, cetane number, long-chain saturated factor, cold filter plugging point, cloud point, allylic position equivalent and bis-allylic position equivalent were predicted by BiodieselAnalyzer® software. Besides, polycyclic aromatic hydrocarbons were determined in both wastewater samples and produced lipid. The observed data showed that biodiesel from C. vulgaris was superior to petrodiesel in terms of suitability in diesel engines. Moreover, contamination of petrochemical wastewater can influence the expression of a variety of genes in algae. To investigate the effectiveness of contamination on the expression of lipid synthesis as well as three photosynthesis genes, a real-time polymerase chain reaction assay was used to quantify transcript levels of PsaB (photosystem I reaction center protein subunit B), psbC (an integral membrane protein component of photosystem II), and rbcL (a large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase). Furthermore, the gene expression level of accD (acetyl-coenzyme A carboxylase carboxyl transferase subunit beta, chloroplastic) was studied to discover the effect of wastewater on lipid production. The results showed that when diluted petrochemical wastewater (50%) was used as a media for C. vulgaris cultivation, these genes expression significantly increased. For 50% diluted wastewater, the maximum removal of BOD, COD, total nitrogen, and total phosphor has been 30.36%, 10.89%, 69.89%, and 92.59%, respectively.