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Objective: Micronutrient status, specifically vitamin D and iron, represent modifiable factors for optimizing military readiness. The primary purpose of this investigation was to determine associations between micronutrient deficiency (i.e., iron status and 25-hydroxy-vitamin D [25(OH)D]) and operationally relevant outcomes (i.e., skeletal health, musculoskeletal injury) at baseline and post-10 weeks of arduous military training. Methods: A total of 227 (177 men, 50 women) Marine Officer Candidates School (OCS) candidates who completed OCS training with complete data sets were included in this analysis. Vitamin D and iron status indicators were collected at two timepoints, pre (baseline) and post OCS. Musculoskeletal outcomes at the mid- and proximal tibial diaphysis were assessed via peripheral quantitative computed tomography. Results: Micronutrient status declined following OCS training in men and women and was associated with musculoskeletal outcomes including greater bone strength (strength strain index) at the mid-diaphysis site in those with optimal status (M = 38.26 mm3, SE = 15.59) versus those without (M = −8.03 mm3, SE = 17.27). In women (p =.037), endosteal circumference was greater in the deficient group (M = 53.26 mm, SE = 1.19) compared with the optimal group (M = 49.47 mm, SE = 1.31) at the proximal diaphysis. In men, greater baseline hepcidin concentrations were associated with an increased likelihood of suffering musculoskeletal injury during training. Conclusions: Vitamin D and iron status declined over the course of training, suggesting impaired micronutrient status. Differences in musculoskeletal outcomes by micronutrient group suggests optimal vitamin D and ferritin concentrations may exert beneficial effects on bone fatigability and fracture reduction during military training. [ABSTRACT FROM AUTHOR]

Nanodiscs, consisting of a lipid bilayer surrounded by membrane scaffold proteins (MSPs), are extensively used to study membrane proteins (MPs) because they provide a stable lipid environment. However, the precise mechanism governing MP behavior within the nanodisc remains elusive. Here, we examined the cryo‐EM structures of various MPs reconstituted in nanodiscs from EMPIAR. By analyzing the heterogeneity and interactions in the nanodiscs, we discovered that MPs display a distinct spatial preference toward the edges of the nanodisc shells. Furthermore, MPs can establish direct, amphipathic interactions with the MSPs, causing a reduction in local protein dynamics. These interactions may rearrange MSP‐MSP interactions into MP‐MSP interactions. Collectively, we provide structural insights into how nanodiscs contribute to MP structural behavior and dynamics. Impact statement Nanodiscs are used to study membrane proteins (MPs), but the mechanisms governing the behavior of MPs within nanodiscs remain elusive. Here, we provide structural insights into how nanodiscs contribute to the behavior of MPs, which will aid the interpretation of cryo‐EM studies performed using nanodiscs. Nanodiscs are used to study membrane proteins (MPs), but the mechanisms governing the behavior of MPs within nanodiscs remain elusive. Here, we provide structural insights into how nanodiscs contribute to the behavior of MPs, which will aid the interpretation of cryo‐EM studies performed using nanodiscs. [ABSTRACT FROM AUTHOR]

Mycoplasma synoviae is an important avian pathogen that causes respiratory infections and arthritis symptoms in chickens and turkeys, resulting in significant economic damage to the poultry farming industry worldwide. Cell adhesion is a vital stage of Mycoplasma infection, and the proteins associated with this process play an important role in its pathogenesis. Elongation factor thermo stable (EF-Ts) is an important factor in prokaryotic biosynthesis that serves as a guanosine exchange factor for elongation factor thermo unstable (EF-Tu). To date, little is known about the role of EF-Ts in Mycoplasma infection. In this study, we identified EF-Ts as an immunogenic protein in M. synoviae through liquid chromatography with tandem mass spectrometry (LC–MS/MS) screening. We constructed an E. coli recombinant expression vector and prepared a highly efficient rabbit antiserum. Immunoblot analysis and suspension immunofluorescence revealed that the EF-Ts is located in both the cell membrane and cytoplasm. The prepared rabbit EF-Ts antiserum exhibited complement-dependent Mycoplasma-killing activity and inhibited the adhesion of rEF-Ts and M. synoviae to DF-1 cells. An in-vitro binding assay showed that EF-Ts could bind to fibronectin (Fn) and chicken plasminogen (cPlg) in a dose-dependent manner. In addition, EF-Ts could internalize into cells through lipid rafts and clathrin-dependent endocytosis and induce DF-1 cell proliferation. In conclusion, our studies demonstrated that MS EF-Ts is a potentially immunogenic, novel adhesion protein that acts as a critical virulence factor in M. synoviae adhesion to host cells during infection. These studies further deepen our understanding of the pathogenic mechanism of M. synoviae. [ABSTRACT FROM AUTHOR]

ABSTRACT Pinto beans, an underutilized legume, are abundant in protein content and contain a variety of beneficial phytonutrients. However, the commonly used protein extraction method, alkaline extraction, is associated with several drawbacks. These drawbacks include low extraction yield and purity as well as the production of large amounts of wastewater that can lead to environmental hazards. In this regard, membrane technology has gained considerable recognition as a superior method for extracting proteins. A combined processing scheme was developed, which included alkaline extraction at pH 10.5, ultrafiltration with a concentration factor of 5.5, diafiltration with a diavolume of 4, and isoelectric precipitation at pH 4.5 followed by freeze drying. The specific functional characteristics (nitrogen solubility index, water and oil holding capacity, and emulsifying and foaming properties) of the protein concentrates were assessed and compared with those of a commercially available soybean protein isolate. Based on pinto bean flour containing 23.9% protein, 85.5% of the protein was recovered in the products of this process: precipitated protein concentrate (PPC) with 86.4% protein, acid‐soluble protein concentrate (ASP‐C) with 56.3% protein, and meal residue with 6.1% protein. The mass yields were 17.3% in PPC, 3.9% in ASP‐C, and 54% in the meal residue. The precipitated protein showed higher emulsifying activity, and the acid‐soluble protein showed a high nitrogen solubility index (NSI) and oil‐holding capacity. Both proteins had comparable foaming properties to commercial soy protein isolate. The project demonstrated the feasibility of protein production from pinto beans and highlighted the proteins' useful food functionality and good potential for commercialization. [ABSTRACT FROM AUTHOR]

Membrane fusion is an essential biological process that merges two separate lipid bilayers into a whole one. Membrane fusion proteins facilitate this process by bringing lipid bilayers in close proximity to reduce the repulsive energy between membranes. Along with their interactions with membranes, the structures and dynamics of membrane fusion proteins are key to elucidating the mechanisms of membrane fusion. Solid‐state NMR (SSNMR) spectroscopy has unique advantages in determining the structures and dynamics of membrane fusion proteins in their membrane‐bound states. It has been extensively applied to reveal conformational changes in intermediate states of viral membrane fusion proteins and to characterize the critical lipid–membrane interactions that drive the fusion process. In this review, we summarize recent advancements in SSNMR techniques for studying membrane fusion proteins and their applications in elucidating the mechanisms of membrane fusion. [ABSTRACT FROM AUTHOR]

The low‐density lipoprotein receptor (LDLr) is the first member of a closely related transmembrane protein family. It is known for its involvement in various physiological processes, mainly in the regulation of lipid metabolism, especially in the brains of mammals and zebrafish. In zebrafish, two ldlr genes (ldlra and b) have been identified and their distribution in the brain is not well documented. Recently, the roles of ldlr and its inhibitor pcsk9 in regenerative process after telencephalic brain injury have been discussed. In this study, we explored the expression patterns of these genes during zebrafish development. We found that ldlra expression was detected at the end of the pharyngula period (48 hpf) and increased during the larval stage. Conversely, ldlrb expression was observed from zygotic to larval stages. Using techniques like in situ hybridization and taking advantage of transgenic fish, we demonstrated the widespread distribution of ldlra, ldlrb and pcsk9 in the brain of adult zebrafish. Specifically, these genes were expressed in neurons and neural stem cells and also at lower levels in endothelial cells. As expected, intraperitoneal injection of fluorescent‐labelled LDLs resulted in their uptake by cerebral endothelial cells in a homeostatic context, whereas they diffused within the brain parenchyma after telencephalic injury. However, after intracerebroventricular injections into animals, LDL particles were not taken up by neural stem cells. In conclusion, our results provide additional evidence for LDLr expression in the brain of adult zebrafish. These results raise the question of the role of LDLr in the cholesterol/lipid imbalance in cerebral complications. [ABSTRACT FROM AUTHOR]

Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is a membrane protein that plays an important role in a variety of immune and non‐immune functions. Such functions are regulated by its activity as a homophilic ligand but also through its ability to interact as a heterophilic ligand with various host proteins. These include CEACAM5, T cell immunoglobulin‐mucin like protein‐3 (TIM‐3) and, potentially, protein death protein 1 (PD‐1). Furthermore, CEACAM1 is targeted by various pathogens to allow them to invade a host and bypass an effective immune response. Clinically, CEACAM1 plays an important role in infectious diseases, autoimmunity and cancer. In this review, we describe the structural basis for CEACAM1 interactions as a homophilic and heterophilic ligand. We discuss the regulation of its monomeric, dimeric and oligomeric states in cis and trans binding as well as the consequences for eliciting downstream signalling activities. Furthermore, we explore the potential role of avidity in determining CEACAM1's activities. [ABSTRACT FROM AUTHOR]

Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as capping protein, Arp2/3, myosin I linker (CARMIL), CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin-capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1/myotrophin binds to the F-actin-binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin, and actin monomers in solution, recreating key features of cell physiology. [Display omitted] • Active capping protein (CP) levels determine actin network growth and organization • V-1 inhibits CP and decreases Arp2/3-based actin network growth • Low levels of CARMIL activate CP by displacing V-1, allowing CP to cap barbed ends • Protein interactions may provide fine-tuning for feedback of actin assembly Mooren et al. show that membrane-bound CARMIL activates capping protein (CP) by dissociating CP from V-1, thereby promoting actin polymerization nucleated by Arp2/3. The activity of CP is a critical determinant for polarized and structured growth of the actin network. Interactions among the proteins provide several points of feedback control. [ABSTRACT FROM AUTHOR]

SUMMARY: Soybean is an important plant source of protein worldwide. Increasing demands for soybean can be met by improving the quality of its seed protein. In this study, GmCG‐1, which encodes the β‐conglycinin α′ subunit, was identified via combined genome‐wide association study and transcriptome analysis. We subsequently knocked down GmCG‐1 and its paralogues GmCG‐2 and GmCG‐3 with CRISPR‐Cas9 technology and generated two stable multigene knockdown mutants. As a result, the β‐conglycinin content decreased, whereas the 11S/7S ratio, total protein content and sulfur‐containing amino acid content significantly increased. Surprisingly, the globulin mutant exhibited salt tolerance in both the germination and seedling stages. Little is known about the relationship between seed protein composition and the salt stress response in soybean. Metabonomics and RNA‐seq analysis indicated that compared with the WT, the mutant was formed through a pathway that was more similar to that of active salicylic acid biosynthesis; however, the synthesis of cytokinin exhibited greater defects, which could lead to increased expression of plant dehydrin‐related salt tolerance proteins and cell membrane ion transporters. Population evolution analysis suggested that GmCG‐1, GmCG‐2, and GmCG‐3 were selected during soybean domestication. The soybean accessions harboring GmCG‐1Hap1 presented relatively high 11S/7S ratios and relatively high salt tolerance. In conclusion, knockdown of the β‐conglycinin α and α′ subunits can improve the nutritional quality of soybean seeds and increase the salt tolerance of soybean plants, providing a strategy for designing soybean varieties with high nutritional value and high salt tolerance. Significance Statement: CRISPR‐CAS9 generated soybean mutants of β‐conglycinin α and α′ subunits increases sulfur‐containing amino acids and protein contents, further enhances the salt tolerance, thus providing a new strategy for designing soybean varieties with both high nutritional value and high salt tolerance. [ABSTRACT FROM AUTHOR]

Transmembrane potassium ion channels are crucial for ion transport, metabolism, and signaling, and serve as promising targets for anti-cancer therapies. However, their hydrophobic transmembrane nature requires detergents, posing a major bottleneck for experimental handling. In this paper, we present a structural bioinformatics study of six experimentally determined and twelve modeled potassium channel structures, in which hydrophobic amino acids (L, I/V, and F) were systematically replaced with neutral hydrophilic ones (Q, T, and Y), making the proteins more water-soluble. QTY (computationally predicted) and native (experimental and repredicted) variants show remarkable structural similarity (RMSD: ~0.50 Å – ~2.14 Å) despite significant sequence differences. QTY variants, both rigid and refined with MD simulations, maintain comparable to native variants stability, solvent-accessible surface area (SASA), and ionic, aromatic, and van der Waals interactions but differ in the grand average of hydropathy (GRAVY), solubility, and hydrophobic contacts. Overall, our study presents a computational approach for designing hydrophilic potassium ion channels while maintaining the native global structure that could potentially simplify their practical use by eliminating the need for detergents. [ABSTRACT FROM AUTHOR]

Background: Cellular carcinogenesis is often marked by the accumulation of lipid droplets (LDs) due to reprogrammed lipid metabolism. LDs are dynamic organelles that primarily store intracellular triacylglycerol (TAG) and cholesteryl esters (CEs). Transmembrane protein 68 (TMEM68), a potential modifier of human breast cancer risk and outcomes, functions as a diacylglycerol acyltransferase, synthesizing TAG. However, the specific roles of TMEM68 in breast cancer cells remain unclear. Methods: Gene expression profiling interactive analysis and survival analysis were conducted. TMEM68 was overexpressed or knockdown in breast cancer cells to assess its impact on cell proliferation, migration and invasion. Targeted quantitative lipidomic analysis and quantitative polymerase chain reaction were used to profile lipid alterations and examine gene expression related to lipid metabolism following changes in TMEM68 levels. Results: TMEM68 gene was upregulated in breast cancer patients and higher TMEM68 levels were associated with poorer survival outcomes. Overexpression of TMEM68 increased breast cancer cell proliferation and invasion, whereas knockdown had minimal or no impact on reducing proliferation and invasion. Altering TMEM68 levels resulted in corresponding changes in TAG levels and cytoplasmic LDs, with overexpression increasing both and knockdown decreasing them. Lipidomic analysis revealed that TMEM68 regulated TAG levels and altered diacylglycerol content in breast cancer cells. Additionally, TMEM68 influenced the metabolism of glycerophospholipids, CEs and acylcarnitines. TMEM68 also modified the expression of key genes encoding enzymes related to neutral lipid metabolism, including TAG and CEs. Conclusions: TMEM68 is highly expressed in breast cancer and negatively correlated with survival. Its overexpression promotes breast cancer cell proliferation while knockdown has varied effects depending on TMEM68 levels. TMEM68 regulates intracellular TAG and LDs contents along with alterations in glycerophospholipids. These findings suggest that TMEM68 may drive breast cancer cells proliferation by modulating TAG and LD content. [ABSTRACT FROM AUTHOR]

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of all investigated TMD-effectors depended on a C-terminal secretion signal. A deeper analysis of the TMD-effector SidF showed that this signal needed to be presented towards the cytoplasmic side of the inner membrane and that a small periplasmic loop was required for efficient translocation. We propose that strongly hydrophobic TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell. Author summary: Legionella is a water-borne bacterial pathogen that can cause severe and often fatal pneumonia. It employs a so-called type VI b secretion system to inject hundreds of different effector proteins in order to establish an intracellular niche inside its natural host amoeba or inside alveolar macrophages of the lung. Many of these effector proteins are hydrophobic transmembrane proteins that find their final destination in one of the host cell's biological membranes. Here we present evidence that some of these transmembrane-effectors are secreted in a two-step secretion process with an intermediate in the inner membrane of Legionella. Extraction from this membrane and subsequent secretion likely occur at the cytoplasmic side of the membrane as these transmembrane effectors require C-terminal secretion signals facing inside. Our study highlights the amazing capability of type VI b secretion systems to secrete soluble and transmembrane-effectors from different subcellular locations of the bacterial cell. [ABSTRACT FROM AUTHOR]

Bacterial toxin‐antitoxin (TA) systems consist of a toxin that inhibits essential cellular processes, such as DNA replication, transcription, translation, or ATP synthesis, and an antitoxin neutralizing their cognate toxin. These systems have roles in programmed cell death, defense against phage, and the formation of persister cells. Here, we characterized the previously identified Staphylococcus aureus TA system, tsaAT, which consists of two putative membrane proteins: TsaT and TsaA. Expression of the TsaT toxin caused cell death and disrupted membrane integrity, whereas TsaA did not show any toxicity and neutralized the toxicity of TsaT. Furthermore, subcellular fractionation analysis demonstrated that both TsaA and TsaT localized to the cytoplasmic membrane of S. aureus expressing either or both 3xFLAG‐tagged TsaA and 3xFLAG‐tagged TsaT. Taken together, these results demonstrate that the TsaAT TA system consists of two membrane proteins, TsaA and TsaT, where TsaT disrupts membrane integrity, ultimately leading to cell death. Although sequence analyses showed that the tsaA and tsaT genes were conserved among Staphylococcus species, amino acid substitutions between TsaT orthologs highlighted the critical role of the 6th residue for its toxicity. Further amino acid substitutions indicated that the glutamic acid residue at position 63 in the TsaA antitoxin and the cluster of five lysine residues in the TsaT toxin are involved in TsaA's neutralization reaction. This study is the first to describe a bacterial TA system wherein both toxin and antitoxin are membrane proteins. These findings contribute to our understanding of S. aureus TA systems and, more generally, give new insight into highly diverse bacterial TA systems. [ABSTRACT FROM AUTHOR]

Alcohol is the most consumed and abused psychoactive drug globally, but the molecular mechanisms driving alcohol action and its associated behaviors in the brain remain enigmatic. Here, we have discovered a transmembrane protein TMEM132B that is a GABA A receptor (GABA A R) auxiliary subunit. Functionally, TMEM132B promotes GABA A R expression at the cell surface, slows receptor deactivation, and enhances the allosteric effects of alcohol on the receptor. In TMEM132B knockout (KO) mice or TMEM132B I499A knockin (KI) mice in which the TMEM132B-GABA A R interaction is specifically abolished, GABAergic transmission is decreased and alcohol-induced potentiation of GABA A R-mediated currents is diminished in hippocampal neurons. Behaviorally, the anxiolytic and sedative/hypnotic effects of alcohol are markedly reduced, and compulsive, binge-like alcohol consumption is significantly increased. Taken together, these data reveal a GABA A R auxiliary subunit, identify the TMEM132B-GABA A R complex as a major alcohol target in the brain, and provide mechanistic insights into alcohol-related behaviors. [Display omitted] • Single-pass transmembrane protein TMEM132B is a GABA A R auxiliary subunit • TMEM132B promotes GABA A R expression at the cell surface and slows receptor deactivation • TMEM132B enhances the allosteric effects of alcohol on various GABA A Rs • Genetic inactivation of TMEM132B in mice alters alcohol-related behaviors Identification of TMEM132B as a GABA A R auxiliary subunit that modulates alcohol action in the brain at both the molecular and behavioral levels. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: TAG‐assisted peptide synthesis technology enables optimal conservation of Fmoc amino acid raw materials and chemical solvents while eliminating the need for intricate chromatographic purification processes. This work presents a 4,4'‐diphenylphosphonoxy diphenylcarbinol tag‐mediated liquid‐phase synthesis approach for preparing side‐to‐tail cyclopeptides macolacin which has strong activity against gram‐negative bacteria, and its 15 analogues containing four N‐methylation modified cyclopeptides, as well as an investigation of their structure‐activity relationship (SAR). The synthesis of macolacin analogues primarily focuses on the modifications of the N‐methylation group of Ile‐7 and the tail fatty acyl chain of macolacin. The incorporation of N‐methylation for Ile‐7, along with the dihalogenated or monohalogenated benzoic acids for tail modification, exhibited remarkable antibacterial efficacy and minimal hepatocellular toxicity in vitro. The present study identified an N‐methylation‐modified antimicrobial cyclopeptide Ma14 that exhibits rapid bactericidal efficacy against A. baumanii, etc., while showing reduced hepatocellular toxicity. Molecular docking simulations were conducted to investigate the binding of cyclopeptides to the outer membrane protein BamA of A. baumannii. The findings demonstrated the stable binding interactions of the cyclopeptides with the BamA protein and then presented a novel approach to explain the bacteriostatic mechanism of macolacin‐based cyclopeptide antibiotics. [ABSTRACT FROM AUTHOR]

Viroporins possess important potential as antiviral targets due to their critical roles during virus life cycles, spanning from virus entry to egress. Although the antiviral amantadine targets the M2 viroporin of influenza A virus, successful progression of other viroporin inhibitors into clinical use remains challenging. These challenges relate in varying proportions to a lack of reliable full‐length 3D‐structures, difficulties in functionally characterising individual viroporins, and absence of verifiable direct binding between inhibitor and viroporin. This review offers perspectives to help overcome these challenges. We provide a comprehensive overview of the viroporin family, including their structural and functional features, highlighting the moldability of their energy landscapes and actions. To advance the field, we suggest a list of best practices to aspire towards unambiguous viroporin identification and characterisation, along with considerations of potential pitfalls. Finally, we present current and future scenarios of, and prospects for, viroporin targeting drugs. [ABSTRACT FROM AUTHOR]

[Display omitted] Hypothesis: We hypothesize that simultaneous incorporation of ion channel peptides (in this case, potassium channel as a model) and hydrophobic magnetite Fe 3 O 4 nanoparticles (hFe 3 O 4 NPs) within lipidic hexagonal mesophases, and aligning them using an external magnetic field can significantly enhance ion transport through lipid membranes. Experiments: In this study, we successfully characterized the incorporation of gramicidin membrane ion channels and hFe 3 O 4 NPs in the lipidic hexagonal structure using SAXS and cryo-TEM methods. Additionally, we thoroughly investigated the conductive characteristics of freestanding films of lipidic hexagonal mesophases, both with and without gramicidin potassium channels, utilizing a range of electrochemical techniques, including impedance spectroscopy, normal pulse voltammetry, and chronoamperometry. Findings: Our research reveals a state-of-the-art breakthrough in enhancing ion transport in lyotropic liquid crystals as matrices for integral proteins and peptides. We demonstrate the remarkable efficacy of membranes composed of hexagonal lipid mesophases embedded with K+ transporting peptides. This enhancement is achieved through doping with hFe 3 O 4 NPs and exposure to a magnetic field. We investigate the intricate interplay between the conductive properties of the lipidic hexagonal structure, hFe 3 O 4 NPs, gramicidin incorporation, and the influence of Ca2+ on K+ channels. Furthermore, our study unveils a new direction in ion channel studies and biomimetic membrane investigations, presenting a versatile model for biomimetic membranes with unprecedented ion transport capabilities under an appropriately oriented magnetic field. These findings hold promise for advancing membrane technology and various biotechnological and biomedical applications of membrane proteins. [ABSTRACT FROM AUTHOR]

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1' position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii, a common nosocomial pathogen, is known for its rapid acquisition of antimicrobial resistance, underscoring the urgent need to develop an effective vaccine against this pathogen. Outer membrane protein 22 (Omp22) regulates the biogenesis of outer membrane vesicles to transport virulence-promoting factors into the host cells and facilitates the progression of A. baumannii infection. In this study, we used a mouse model to assess a vaccine's immunogenicity and protective efficacy using recombinant Omp22 protein within the hypervariable region of flagellin (FliC-Omp22). FliC-Omp22 demonstrated superior protection following challenge with a lethal dose of multidrug-resistant (MDR) A. baumannii strain 58ST compared to Omp22 alone. In addition, it elicited increased IgG1/IgG2a and IL-4/IFN-γ ratios, indicating a predominant Th2 immune response. Furthermore, the FliC-Omp22 vaccination elicited strong specific antibodies that inhibited the adhesion and invasion of A. baumannii 58ST and enhanced the opsonic killing activity against the pathogen. FliC-Omp22 immunization significantly reduced bacterial loads in infected mice's spleen, lungs, and liver, thereby improving their survival against the lethal infection caused by MDR A. baumannii 58ST. This study suggests that integrating Omp22 into the hypervariable domain of flagellin holds promise for developing an effective vaccine against A. baumannii infections. [ABSTRACT FROM AUTHOR]

The outbreak and spread of African swine fever virus (ASFV) have caused considerable economic losses to the pig industry worldwide. Currently, to promote the development of effective ASF vaccines, especially subunit vaccines, more antigenic protein targets are urgently needed. In this work, six transmembrane proteins (I329L, E146L, C257L, EP153R, I177L, and F165R) were expressed in mammalian cell lines and screened with pig anti-ASFV serum. It was found that the E146L protein was an immunodominant protein antigen among the six selected proteins. Moreover, the E146L protein induced antibody responses in all immunized pigs. To gain insight into the antigenic characteristics of the E146L protein, three monoclonal antibodies (mAbs; 12H12, 15G1, and 15H10) were generated by immunizing BALB/c mice with the purified E146L protein. The epitopes of the mAbs were further finely mapped through a peptide fusion protein expression strategy. Finally, the epitopes of the mAbs were identified as 48PDESSIAYMRFRN61 of the mAb 12H12, 138TLTGLQRII146 of the mAb 15G1, and 30GWSPFKYSKGNT41 of the mAb 15H10. Furthermore, the epitope of mAb 15H10 was validated as the immunodominant epitope with ASFV-infected pig sera. The chemically synthesized mAb 15H10 epitope peptide (EP1) exhibited the most extensive immunoreactivity with artificially or naturally ASFV-infected pig sera. The epitope 15H10 is located on the surface of the E146L protein and is highly conserved. These findings provide insight into the structure and function of the E146L protein of ASFV. [ABSTRACT FROM AUTHOR]

Phospholamban (PLN) plays a crucial role in regulating sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Mutations within the PLN gene have been detected in patients with cardiomyopathy, with the heterozygous variant c.40_42delAGA (p.R14del) of PLN being the most prevalent. Investigations into the mechanisms underlying the pathology of PLN-R14del have revealed that cardiac cells from affected patients exhibit pathological aggregates containing PLN. Herein, we performed comprehensive molecular and cellular analyses to delineate the molecular aberrations associated with the formation of these aggregates. We determined that PLN aggregates contain autophagic proteins, indicating inefficient degradation via the autophagy pathway. Our findings demonstrate that the expression of PLN-R14del results in diminished autophagic flux due to impaired fusion between autophagosomes and lysosomes. Mechanistically, this defect is linked to aberrant recruitment of key membrane fusion proteins to autophagosomes, which is mediated in part by changes in Ca2+ homeostasis. Collectively, these results highlight a novel function of PLN-R14del in regulating autophagy, that may contribute to the formation of pathogenic aggregates in patients with cardiomyopathy. Prospective strategies tailored to ameliorate impaired autophagy may hold promise against PLN-R14del disease. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) play a crucial role in intercellular communication. Characterizing EV protein composition is essential to understand EV function(s). Isolating EVs from cell culture medium is a common approach to study EVs, but it remains unclear whether EVs isolated from in vitro conditions accurately reflect physiological conditions of the same source in vivo tissues. Here, we analyzed the protein composition of EVs isolated from freshly dissected mouse forebrain and primary dissociated mouse forebrain culture medium. In total, 3,204 and 3,583 proteins were identified in EVs isolated in vivo and in vitro, respectively. Among the proteins identified from both EV sources, there was substantial overlap (~86%). While the overall proteome compositions were very similar, in vitro EVs were relatively enriched with transmembrane/GPI-anchored membrane and cytosolic proteins (MISEV2023 category 1 and 2) typically associated with EVs. Conversely, while both in vivo and in vitro EVs express likely non-EV proteins (MISEV2023 category 3), the in vivo samples were significantly more enriched with these probable contaminants, specifically ribosomal proteins. Our findings highlight that in vitro EVs may be representative of in vivo EVs when isolated from the same source tissue using similar methodology; however, each population of EVs have differences in both total and, primarily, relative protein expression likely due to differing levels of co-eluting contaminants. Therefore, these points must be considered when interpreting results of EV studies further suggesting that improved methods of isolation to reduce non-EV contaminants should be further investigated. [ABSTRACT FROM AUTHOR]

Film-electrochemical electron paramagnetic resonance spectroscopy (FE-EPR) enables simultaneous electrochemical and spectroscopic characterisation of paramagnetic electron-transfer centres, including in soluble proteins. We now report a modified set-up FE-EPR with tuneable macroporous working electrodes and demonstrate the feasibility to investigate electron transfer in membrane proteins in their native membrane environment. [ABSTRACT FROM AUTHOR]

Exposure of inner mitochondrial membrane resident protein PHB2 (prohibitin 2) during autophagic removal of depolarized mitochondria (mitophagy) depends on the ubiquitin-proteasome system. This uncovering facilitates the PHB2 interaction with phagophore membrane-associated protein MAP1LC3/LC3. It is unclear whether PHB2 is exposed randomly at mitochondrial rupture sites. Prior knowledge and initial screening indicated that VDAC1 (voltage dependent anion channel 1) might play a role in this phenomenon. Through in vitro biochemical assays and imaging, we have found that VDAC1-PHB2 interaction increases during mitochondrial depolarization. Subsequently, this interaction enhances the efficiency of PHB2 exposure and mitophagy. To investigate the relevance in vivo, we utilized porin (equivalent to VDAC1) knockout Drosophila line. Our findings demonstrate that during mitochondrial stress, porin is essential for Phb2 exposure, Phb2-Atg8 interaction and mitophagy. This study highlights that VDAC1 predominantly synchronizes efficient PHB2 exposure through mitochondrial rupture sites during mitophagy. These findings may provide insights to understand progressive neurodegeneration. [ABSTRACT FROM AUTHOR]

Spf1 is an important P-type ATPase in Candida albicans, which functions as an endoplasmic reticulum calcium pump to maintain calcium homoeostasis. The deficiency of Spf1 attenuates the virulence of C. albicans. However, its impact on immune response remains to be investigated. This study discovered that deletion of SPF1 resulted in a reduction of endoplasmic reticulum-plasma membrane contacts, an important structure mediating material and information exchange. This effect was attributed to the reduced plasma membrane localisation of the crucial endoplasmic reticulum-plasma membrane tethering proteins Ist2 and Tcb1/3. The reduction of the contacts led to a decrease in secretion of the virulence factors phospholipase, secreted aspartyl protease (SAP), candidalysin, and the cell wall-anchored protein Hwp1 during infection. Immunofluorescence staining and quantitative PCR assays further showed that the SPF1 deletion led to a remarkable decrease in the levels of pro-inflammatory cytokines, suggesting the alleviation of the fungus-induced inflammatory response. Ultimately, the regulatory role of Spf1 in immune response significantly weakened the infectivity of C. albicans, and increased the survival rate of the hosts. This finding elucidated the role of fungal calcium pump-governed endoplasmic reticulum-plasma membrane contacts in regulation of immune response. It also makes it possible to regulate the host’s immune response via control of SPF1 expression and functions, providing a theoretical basis for treating fungal infections. [ABSTRACT FROM AUTHOR]

Biondi bodies are filamentous amyloid inclusions of unknown composition in ependymal cells of the choroid plexuses, ependymal cells lining cerebral ventricles and ependymal cells of the central canal of the spinal cord. Their formation is age-dependent and they are commonly associated with a variety of neurodegenerative conditions, including Alzheimer's disease and Lewy body disorders. Here, we show that Biondi bodies are strongly immunoreactive with TMEM239, an antibody specific for inclusions of transmembrane protein 106B (TMEM106B). Biondi bodies were labelled by both this antibody and the amyloid dye pFTAA. Many Biondi bodies were also labelled for TMEM106B and the lysosomal markers Hexosaminidase A and Cathepsin D. By transmission immuno-electron microscopy, Biondi bodies of choroid plexuses were decorated by TMEM239 and were associated with structures that resembled residual bodies or secondary lysosomes. By electron cryo-microscopy, TMEM106B filaments from Biondi bodies of choroid plexuses were similar (Biondi variant), but not identical, to the fold I that was previously identified in filaments from brain parenchyma. [ABSTRACT FROM AUTHOR]

Coronavirus stands for a large family of viruses characterized by protruding spikes surrounding a lipidic membrane adorned with proteins. The present study explores the adhesion of transmissible gastroenteritis coronavirus (TGEV) particles on a variety of reference solid surfaces that emulate typical virus‐surface interactions. Atomic force microscopy informs about trapping effectivity and the shape of the virus envelope on each surface, revealing that the deformation of TGEV particles spans from 20% to 50% in diameter. Given this large deformation range, experimental Langmuir isotherms convey an unexpectedly moderate variation in the adsorption‐free energy, indicating a viral adhesion adaptability which goes beyond the membrane. The combination of an extended Helfrich theory and coarse‐grained simulations reveals that, in fact, the envelope and the spikes present complementary adsorption affinities. While strong membrane‐surface interaction lead to highly deformed TGEV particles, surfaces with strong spike attraction yield smaller deformations with similar or even larger adsorption‐free energies. [ABSTRACT FROM AUTHOR]

The dystrophin-glycoprotein-complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link the extracellular matrix and actin cytoskeleton. Breaking this connection is associated with diseases such as muscular dystrophy, yet cleavage of dystroglycan by matrix-metalloproteinases (MMPs) remains an understudied mechanism to disrupt the DGC. We determined the crystal structure of the membrane-adjacent domain (amino acids 491–722) of E. coli expressed human dystroglycan to understand MMP cleavage regulation. The structural model includes tandem immunoglobulin-like (IGL) and sperm/enterokinase/agrin-like (SEAL) domains, which support proteolysis in diverse receptors to facilitate mechanotransduction, membrane protection, and viral entry. The structure reveals a C-terminal extension that buries the MMP site by packing into a hydrophobic pocket, a unique mechanism of MMP cleavage regulation. We further demonstrate structure-guided and disease-associated mutations disrupt proteolytic regulation using a cell-surface proteolysis assay. Thus disrupted proteolysis is a potentially relevant mechanism for "breaking" the DGC link to contribute to disease pathogenesis. [Display omitted] • MMP cleavage in dystroglycan is regulated by a distinct structural mechanism • A C-terminal peptide extension buries the MMP site in a SEAL domain pocket • Structure-guided and disease-associated mutations lead to increased proteolysis • Proteolysis is a relevant mechanism to disrupt ECM to actin mechanical linkage Anderson et al. present the structure of the proteolysis-regulation domain of the ECM receptor dystroglycan, which anchors the dystrophin-glycoprotein-complex adhesive complex to the cytoskeleton. The matrix-metalloprotease (MMP) site is regulated by clamping a unique C-terminal extension to the SEA-like domain, and disruption of this regulation leads to enhanced MMP cleavage. [ABSTRACT FROM AUTHOR]

The outer mitochondrial membrane (OMM) creates a boundary that imports most of the mitochondrial proteome while removing extraneous or damaged proteins. How the OMM senses aberrant proteins and remodels to maintain OMM integrity remains unresolved. Previously, we identified a mitochondrial remodeling mechanism called the mitochondrial-derived compartment (MDC) that removes a subset of the mitochondrial proteome. Here, we show that MDCs specifically sequester proteins localized only at the OMM, providing an explanation for how select mitochondrial proteins are incorporated into MDCs. Remarkably, selective sorting into MDCs also occurs within the OMM, as subunits of the translocase of the outer membrane (TOM) complex are excluded from MDCs unless assembly of the TOM complex is impaired. Considering that overloading the OMM with mitochondrial membrane proteins or mistargeted tail-anchored membrane proteins induces MDCs to form and sequester these proteins, we propose that one functional role of MDCs is to create an OMM-enriched trap that segregates and sequesters excess proteins from the mitochondrial surface. [ABSTRACT FROM AUTHOR]

Background: Porcine epidemic diarrhea virus (PEDV) infection and transmission pose a serious threat to the global swine industry. The search for a new host factor with anti-PEDV effect may be an effective potential target for the development of novel antiviral drugs. Interferon-induced transmembrane proteins (IFITMs) play a crucial role in the innate immune response triggered by viral infection, and it has been suggested that IFITMs can block the early stages of viral replication, but the mechanism of action is currently unclear. The current study sheds light on the role of IFITM1 in PEDV infection. Specifically, overexpression of IFITM1 suppresses PEDV proliferation in IPEC-J2 cells, while knockdown of IFITM1 has the opposite effect. Collectively, these findings underscore IFITM1's inhibitory role in PEDV infection, with critical implications for the residues and structural motifs within its CTD. Results: The study demonstrates that IFITM1, an interferon-induced transmembrane protein, plays a critical role in the antiviral response against Porcine Epidemic Diarrhea Virus (PEDV). Notably: Overexpression of IFITM1 suppresses PEDV proliferation.IFITM1 co-localizes with PEDV virions in the cytoplasm surrounding the nucleus.Immunocolloidal gold electron microscopy reveals IFITM proteins embedded on the surface of PEDV virions.IFITM1 directly interacts with the N protein of PEDV.C-terminal domain mutations in IFITM1 compromise its inhibitory function against PEDV, with specific amino acid residues playing a pronounced role.These findings enhance our understanding of innate immunity and antiviral defense mechanisms, with potential implications for therapeutic strategies against PEDV infection. Conclusions: The study establishes IFITM1 as a key player in the antiviral response against PEDV. Its inhibitory function, co-localization with virions, and interaction with the N protein provide valuable insights. Notably, the CTD mutations of IFITM1 have a fundamental impact on its modulatory action. These findings contribute to our understanding of innate immunity and antiviral defense mechanisms, with potential implications for therapeutic strategies against PEDV infection. [ABSTRACT FROM AUTHOR]

A temporal neural code reliant on the pattern of spike times rather than spike rates offers a feasible mechanism for encoding information from weak periodic external stimuli, such as static or extremely low-frequency electromagnetic fields. Our model focuses on the influence of magnetic fields on neurotransmitter dynamics near the neuron membrane. Neurotransmitter binding to specific receptor sites on membrane proteins can regulate biochemical reactions. The duration a neurotransmitter spends in the bonded state serves as a metric for the magnetic field's capacity as a chemical regulator. By initiating a physical analysis of ligand-receptor binding, utilizing the alpha function for synaptic conductance, and employing a modified version of Bell's law, we quantified the impact of magnetic fields on the bond half-life time and, consequently, on postsynaptic spike timing. [ABSTRACT FROM AUTHOR]

The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases. Synopsis: Mitochondrial calcium uptake through the uniporter channel MCUC is critical to cell signaling, metabolism, physiology, and disease. This study provides an unbiased and quantitative map of the MCUC interactome and its remodelling, both under resting conditions and after genetic perturbations. Tandem affinity purification/mass-spectrometry-based approach identifies the MCUC protein interaction network. Mitochondrial inner membrane protein EFHD1 interacts with MCUC and inhibits mitochondrial calcium uptake. Loss of MCUB subunit results in an expansion and greater interconnection of the MCU protein network. A dynamic map of the MCUC interaction landscape reveals inhibition of mitochondrial calcium uptake by the mitochondrial intermembrane space protein EFHD1. [ABSTRACT FROM AUTHOR]

G-protein coupled receptors (GPCRs), the largest family of membrane proteins in human body, involve a great variety of biological processes and thus have become highly valuable drug targets. By binding with ligands (e.g., drugs), GPCRs switch between active and inactive conformational states, thereby performing functions such as signal transmission. The changes in binding pockets under different states are important for a better understanding of drug-target interactions. Therefore it is critical, as well as a practical need, to obtain binding sites in human GPCR structures. We report a database (called GPCR-BSD) that collects 127,990 predicted binding sites of 803 GPCRs under active and inactive states (thus 1,606 structures in total). The binding sites were identified from the predicted GPCR structures by executing three geometric-based pocket prediction methods, fpocket, CavityPlus and GHECOM. The server provides query, visualization, and comparison of the predicted binding sites for both GPCR predicted and experimentally determined structures recorded in PDB. We evaluated the identified pockets of 132 experimentally determined human GPCR structures in terms of pocket residue coverage, pocket center distance and redocking accuracy. The evaluation showed that fpocket and CavityPlus methods performed better and successfully predicted orthosteric binding sites in over 60% of the 132 experimentally determined structures. The GPCR Binding Site database is freely accessible at https://gpcrbs.bigdata.jcmsc.cn. This study not only provides a systematic evaluation of the commonly-used fpocket and CavityPlus methods for the first time but also meets the need for binding site information in GPCR studies. [ABSTRACT FROM AUTHOR]

Mutations in the interface of membrane protein (MP) complexes are key contributors to a broad spectrum of human diseases, primarily due to changes in their binding affinities. While various methods exist for predicting the mutation-induced changes in binding affinity (ΔΔG) in protein–protein complexes, none are specific to MP complexes. This study proposes a novel strategy for ΔΔG prediction in MP complexes, which combines linear and nonlinear models, to obtain a more robust model with improved prediction accuracy. We used multiple linear regression to extract informative features that influence the binding affinity in MP complexes, which included changes in the stability of the complex, conservation score, electrostatic interaction, relatively accessible surface area, and interface contacts. Further, using gradient boosting regressor on the selected features, we developed MPA-MutPred, a novel method specific for predicting the ΔΔG of membrane protein–protein complexes, and it is freely accessible at https://web.iitm.ac.in/bioinfo2/MPA-MutPred/. Our method achieved a correlation of 0.75 and a mean absolute error (MAE) of 0.73 kcal/mol in the jack-knife test conducted on a dataset of 770 mutants. We further validated the method using a blind test set of 86 mutations, obtaining a correlation of 0.85 and an MAE of 0.77 kcal/mol. We anticipate that this method can be used for large-scale studies to understand the influence of binding affinity change on disease-causing mutations in MP complexes, thereby aiding in the understanding of disease mechanisms and the identification of potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Summary: TMEM56, a gene coding a transmembrane protein, is abundantly expressed in erythroid cells. Despite this, its role in erythropoiesis has not been well characterized. In this study, we sought to clarify the function of TMEM56 in erythroid development, focusing specifically on its involvement in haem biosynthesis and cell cycle progression. To do this, we used CD34+ haematopoietic stem cells derived from umbilical cord blood and differentiated them into erythroid cells in an ex vivo model. Our results indicate that the loss of TMEM56 disrupts haem biosynthesis and impairs erythroid differentiation. Furthermore, deletion of Tmem56 in the erythroid lineage in murine models using erythropoietin receptor (EpoR)‐Cre revealed defects in erythroid progenitors within the bone marrow under both normal conditions and during haemolytic anaemia. These observations underscore the regulatory role of TMEM56 in maintaining erythroid lineage homeostasis. Taken together, our results unveil a previously unrecognized function of TMEM56 in erythroid differentiation and suggest its potential as an unfounded target for therapeutic strategies in the treatment of erythropoietic disorders. [ABSTRACT FROM AUTHOR]

We evaluated associations between muscle phenotype, positional role, and on‐ice performance in male U20 Danish national team ice hockey players. Sixteen players (10 forwards, six defensemen) participated in a game with activity tracking. Resting thigh muscle biopsies were analyzed for metabolic enzyme activity and protein expression linked to performance. On‐ice intermittent exercise capacity, repeated sprint ability, and maximal isometric knee‐extensor torque were also assessed. No significant position‐specific muscle phenotype characteristics were found, but forwards generally exhibited higher levels of several membrane proteins (p = 0.100–0.991). NAKα2, NAK∑, KATP, ClC‐1, and NHE1 showed significant correlations with total distance (r = 0.52–0.59, p = 0.016–0.046), however, within positions these only persisted for KATP (r = 0.70, p = 0.024) and NAKα2 (r = 0.57, p = 0.085) in forwards, where CS enzyme activity also displayed a strong association with distance covered (r = 0.75, p = 0.019). For high‐intensity skating, NAKα2 (r = 0.56, p = 0.025) and KATP (r = 0.50, p = 0.048) similarly exhibited the strongest associations, persisting within forwards (r = 0.63, p = 0.052 and r = 0.72; p = 0.018, respectively). In conclusion, although several muscle proteins involved in ion and metabolic regulation were associated with performance, only NAKα2 and KATP displayed consistent relationships within positions. Moreover, CS enzyme activity was strongly related to total distance within forwards, coherent with the proposed importance of oxidative capacity in intense intermittent exercise. [ABSTRACT FROM AUTHOR]

Fungal biosynthetic gene clusters often include genes encoding transmembrane proteins, which have been mostly thought to be transporters exporting the products. However, there is little knowledge about subcellular compartmentalization of transmembrane proteins essential for biosynthesis. Fungal mycotoxin cyclochlorotine is synthesized by non-ribosomal peptide synthetase, which is followed by modifications with three transmembrane UstYa-family proteins. Heterologous expression in Aspergillus oryzae revealed that total biosynthesis of cyclochlorotine requires additional two transporter proteins. Here, we investigated subcellular localizations of the five transmembrane proteins under heterologous expression in A. oryzae. Enhanced green fluorescent protein (EGFP) fusions to the transmembrane proteins, which were confirmed to normally function in cyclochlorotine production, were expressed together with organellar markers. All the transmembrane proteins exhibited localizations commonly in line of the trans -Golgi, endosomes, and vacuoles. This study suggests that subcellular compartmentalization of UstYa family proteins and transporters allows corporative functions of delivering intermediates and subsequent modifications, completing cyclochlorotine biosynthesis. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis (Mtb) cell envelope provides a protective barrier against the immune response and antibiotics. The mycobacterial membrane protein large (MmpL) family of proteins export cell envelope lipids and siderophores; therefore, these proteins are important for the basic biology and pathogenicity of Mtb. In particular, MmpL3 is essential and a known drug target. Despite interest in MmpL3, the structural data in the field are incomplete. Utilizing homology modeling, AlphaFold, and biophysical techniques, we characterized the cytoplasmic C‐terminal domain (CTD) of MmpL3 to better understand its structure and function. Our in silico models of the MmpL11TB and MmpL3TB CTD reveal notable features including a long unstructured linker that connects the globular domain to the last transmembrane (TM) in each transporter, charged lysine and arginine residues facing the membrane, and a C‐terminal alpha helix. Our predicted overall structure enables a better understanding of these transporters. [ABSTRACT FROM AUTHOR]

Infectious diseases have significantly shaped human history, leading to significant advancements in medical science. The discovery and development of antibiotics represented a critical breakthrough, but the rise of antibiotic-resistant pathogens now presents a serious global health threat. Due to the limitations of current synthetic antimicrobials, such as toxicity and environmental concerns, it is essential to explore alternative solutions. Algae, particularly microalgae and cyanobacteria, have emerged as promising sources of bioactive antimicrobial compounds. This review provides a comprehensive analysis of the antimicrobial properties of algal-derived compounds, including polysaccharides, fatty acids, and phenols, which have shown effectiveness against multi-drug-resistant bacteria. A co-occurrence bibliometric analysis using VOSviewer highlighted five key research clusters: antibiotic resistance, algal extracts, biosynthesis, water treatment, and novel pharmacological compounds. Furthermore, the primary mechanisms of action of these bioactive compounds, such as the inhibition of protein synthesis and cell membrane disruption, were identified, demonstrating their potential against both common and multi-resistant pathogens. Future research should prioritize optimizing algal biomass production, utilizing genetic and metabolic engineering, and creating innovative delivery systems to enhance the efficient production of bioactive compounds. [ABSTRACT FROM AUTHOR]

This experimental study was performed to evaluate the alterations in the expression of a few subunits composing glutamate AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-D-aspartate) receptors in the hippocampal cells of Wistar rats in response to long-term fluoride (F-) exposure. The animals were given water with background 0.4 (control), 5, 20, and 50 ppm F- (as NaF) for 12 months. The cognitive capacities of rats were examined by novel object recognition (NOR), Y-maze test, and Morris water maze tests. RT-qPCR and Western blotting techniques were used to evaluate the expression of different AMPA and NMDA subunits at transcriptional and translational levels, respectively. Long-term F- poisoning disturbed the formation of hippocampus-dependent working spatial and long-term non-spatial memory. The expression of Gria1, Gria2, and Gria3 genes encoding different subunits of AMPA receptors were comparable in hippocampi of control and F--exposed animals, although the levels of both Grin2a and Grin2b mRNA increased. Long-term F- intake enhanced the ratio of phospho-GluA1/total-GluA1 proteins in subcellular fraction enriched with cytosolic proteins, while decreased content of GluA2 but elevated level of GluA3 were observed in subcellular fraction enriched with membrane proteins. Such changes were accompanied by increased phosphorylation of GluN2A and GluN2B subunits, higher ratios of GluN2A/GluN1 and GluN2B/GluN1 proteins in the cytosol, and GluN2A/GluN2B ratio in membranes. These changes indicate the predominance of Ca2+-permeable AMPARs in membranes and a shift between different NMDARs subunits in hippocampal cells of F--exposed rats, which is typical for neurodegeneration and can at least partially underly the observed disturbances in cognitive capacities of animals. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) presents limited therapeutic options and is associated with poor prognosis. Early detection and the development of novel therapeutic agents are therefore imperative. Fibroblast activation protein (FAP) is a membrane protein expressed on cancer-associated fibroblasts (CAFs) that plays an essential role in TNBC proliferation, migration, and invasion. Consequently, it is hypothesized that the Astatine (211At)-labeled FAP inhibitor (FAPI) selectively exerts anti-tumor effects through alpha-particle emission. In this study, we aimed to assess its theranostic capabilities by integrating [18F]FAPI-74 PET imaging with targeted alpha therapy using [211At]FAPI1 in TNBC models. Mice xenografts were established by transplanting MDA-MB-231 and HT1080 cells (control). As a parallel diagnostic method, [18F]FAPI-74 was administered for PET imaging to validate FAP expression. A single dose of [211At]FAPI1 (1.04 ± 0.10 MBq) was administered to evaluate the therapeutic efficacy. [18F]FAPI-74 exhibited high accumulation in MDA-MB-231 xenografts, and FAP expression was pathologically confirmed via immunostaining. The group that received [211At]FAPI1 (n = 11) demonstrated a significantly enhanced anti-tumor effect compared with the control group (n = 7) (p = 0.002). In conclusion, [18F]FAPI-74 PET imaging was successfully used to diagnose FAP expression, and as [211At]FAPI1 showed promising therapeutic efficacy in TNBC models, it is expected to be a viable therapeutic option. [ABSTRACT FROM AUTHOR]

The production of functionally active membrane proteins (MPs) in an adequate membrane environment is a key step in structural biology. Polymer–lipid particles based on styrene and maleic acid (SMA) represent a promising type of membrane mimic, as they can extract properly folded MPs directly from their native lipid environment. However, the original SMA polymer is sensitive to acidic pH levels, which has led to the development of several modifications: SMA-EA, SMA-QA, and others. Here, we introduce a novel SMA derivative with a negatively charged taurine moiety, SMA-tau, and investigate the formation and characteristics of lipid–SMA-EA and lipid–SMA-tau membrane-mimicking particles. Our findings demonstrate that both polymers can form nanodiscs with a patch of lipid bilayer that can undergo phase transitions at temperatures close to those of the lipid bilayer membranes. Finally, we discuss the potential applications of these SMAs for NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Simple Summary: Uveal melanoma (UM) is an aggressive cancer emerging from mutations in the GNAQ and GNA11 genes. These mutations are also linked to other conditions with distinct morphological features (skin capillary malformations, hemangiomas, Sturge–Weber syndrome, choroidal nevi, blue nevi, hepatic small vessel neoplasm, Blitz nevi, iris and ciliary body melanocytoma, primary central nervous system melanocytic neoplasms, and aldosterone-producing adenomas). By examining the molecular pathways influenced by these mutations, we provide insights into potential targeted therapies for UM and related disorders. Additionally, we address the involvement of SOX10-positive perivascular cells in the complex pathophysiology of GNAQ/GNA11-related conditions. Uveal melanoma (UM), recognized as the most prevalent primary intraocular malignancy in adults, is primarily driven by mutations in the GNAQ and GNA11 genes. These genetic alterations are also implicated in other conditions, which exhibit distinct morphological characteristics. In this article, we investigate the role of GNAQ and GNA11 mutations across varied disorders (e.g., UM, skin blue nevi, and hemangiomas), emphasizing the shared pathogenic mechanisms that connect them despite their differing clinical manifestations. By investigating the molecular pathways affected by these mutations, we provide insights into the potential for targeted therapies that could address not only UM but also other disorders associated with GNAQ/GNA11 mutations. Moreover, we discuss the role of SOX10-positive perivascular cells that may be implicated in the complex pathophysiology of GNAQ/GNA11-related entities. Understanding the common molecular foundation of these conditions opens new ways for research and treatment opportunities, potentially leading to more effective, personalized therapeutic strategies. [ABSTRACT FROM AUTHOR]

Simple Summary: STING agonists face challenges in breast cancer treatment due to their lower accumulation in tumors and rapid clearance from the body, resulting in a short duration of therapeutic effect. Novel phase-transitional liposomes loaded with STING agonists have been developed to overcome these limitations and are specifically designed for low-intensity focused ultrasound (LIFU)-assisted molecular imaging and precise treatment of breast cancer. With the assistance of LIFU, iRGD-modified liposomes (a targeting peptide) undergo a phase transition into microbubbles, leading to enhanced ultrasound molecular imaging of tumors and facilitating breast cancer immunotherapy by releasing STING agonists from the ultrasound-targeted liposomes. Background: Pharmacologically targeting the STING pathway offers a novel approach to cancer immunotherapy. However, small-molecule STING agonists face challenges such as poor tumor accumulation, rapid clearance, and short-lived effects within the tumor microenvironment, thus limiting their therapeutic potential. To address the challenges of poor specificity and inadequate targeting of STING in breast cancer treatment, herein, we report the design and development of a targeted liposomal delivery system modified with the tumor-targeting peptide iRGD (iRGD-STING-PFP@liposomes). With LIFU irradiation, the liposomal system exploits acoustic cavitation, where gas nuclei form and collapse within the hydrophobic region of the liposome lipid bilayer (transient pore formation), which leads to significantly enhanced drug release. Methods: Transmission electron microscopy (TEM) was used to investigate the physicochemical properties of the targeted liposomes. Encapsulation efficiency and in vitro release were assessed using the dialysis bag method, while the effects of iRGD on liposome targeting were evaluated through laser confocal microscopy. The CCK-8 assay was used to investigate the toxicity and cell growth effects of this system on 4T1 breast cancer cells and HUVEC vascular endothelial cells. A subcutaneous breast cancer tumor model was established to evaluate the tumor-killing effects and therapeutic mechanism of the newly developed liposomes. Results: The liposome carrier exhibited a regular morphology, with a particle size of 232.16 ± 19.82 nm, as indicated by dynamic light scattering (DLS), and demonstrated low toxicity to both HUVEC and 4T1 cells. With an encapsulation efficiency of 41.82 ± 5.67%, the carrier exhibited a slow release pattern in vitro after STING loading. Targeting results indicated that iRGD modification enhanced the system's ability to target 4T1 cells. The iRGD-STING-PFP@liposomes group demonstrated significant tumor growth inhibition in the subcutaneous breast cancer mouse model with effective activation of the immune system, resulting in the highest populations of matured dendritic cells (71.2 ± 5.4%), increased presentation of tumor-related antigens, promoted CD8+ T cell infiltration at the tumor site, and enhanced NK cell activity. Conclusions: The iRGD-STING-PFP@liposomes targeted drug delivery system effectively targets breast cancer cells, providing a new strategy for breast cancer immunotherapy. These findings indicate that iRGD-STING-PFP@liposomes could successfully deliver STING agonists to tumor tissue, trigger the innate immune response, and may serve as a potential platform for targeted immunotherapy. [ABSTRACT FROM AUTHOR]

This study examined the effect of exposure of small and large intestinal epithelial cells to the bacterial lipopolysaccharide (LPS) on uptake of free form of vitamin B1, i.e., thiamin. The intestinal tract encounters two sources of thiamin: diet and the gut microbiota. Absorption of thiamin in both the small and large intestine occurs via a carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and -2). Complementary in vitro (human duodenal epithelial HuTu-80 cells and human colonic epithelial NCM460 cells), in vivo (mice), and ex vivo (human primary differentiated enteroid and colonoid monolayers) models were used. The results showed that exposure to LPS causes a significant inhibition in carrier-mediated [3H]-thiamin uptake by small and large intestinal epithelia, with no change in the levels of expression of THTR-1 and -2 mRNAs and their total cellular proteins. However, a significant decrease in the fractions of the THTR-1 and -2 proteins that are expressed at the cell membranes of these epithelial cells was observed. These effects of LPS appeared to involve a protein kinase A (PKA) signaling pathway as activating this pathway caused a reversal in the inhibition of thiamin uptake and level of expression of its transporters at the cell membrane. These findings demonstrate that exposure of gut epithelia to LPS (a situation that occurs under different pathological conditions) leads to inhibition in thiamin uptake due to a decrease in level of expression of its transporters at the cell membrane that is likely mediated via a PKA signaling pathway. NEW & NOTEWORTHY: This study shows that the exposure of gut epithelial cells to bacterial LPS negatively impact the uptake process of the free form of vitamin B1 (i.e., thiamin). This appears to be mediated via suppression in the level of thiamin transporters 1 and 2 (THTR-1 and -2) expression at the cell membrane and involves a protein kinase A (PKA) signaling pathway. [ABSTRACT FROM AUTHOR]

Introduction: Reactive oxygen species modulator 1 (Romo1) is a novel protein that is critically involved in the intracellular production of reactive oxygen species. Evidence has revealed that Romo1 is associated with treatment outcomes of various human malignancies, including lung cancer. However, the clinical implications of this protein in surgically resected lung cancers harboring epidermal growth factor receptor (EGFR) mutations have not been investigated. Methods: Data were collected from patients who underwent curative resection of EGFR-mutant lung adenocarcinoma. Romo1 protein expression levels were measured in the tumor tissue using immunohistochemical staining and evaluated semi-quantitatively using the histochemical score. Univariate and multivariate analyses were performed to identify the clinicopathological parameters that may be associated with clinical outcomes. Results: A total of 98 samples were analyzed. Using the cutoff H score of 200, the population was classified into low (n = 73) and high (n = 25) Romo1 groups. Romo1 expression was significantly higher in smokers, patients with stage III disease, and patients who experienced recurrence after surgery (all p < 0.05). Multivariate analyses showed that advanced-stage and poorly differentiated cancers were associated with shorter disease-free survival (DFS). In addition, high Romo1 expression was independently associated with poor DFS (hazard ratio = 2.18, 95% confidence interval: 1.10–4.32, p = 0.0261). Conclusions: Our data showed that Romo1 overexpression was significantly associated with early recurrence in patients with resected EGFR-mutant lung adenocarcinoma. Although large-scale studies are required, Romo1 may play a prognostic role in this patient population. [ABSTRACT FROM AUTHOR]

This study examines the efficacy of various artificial diets on the mortality rates of Bemisia tabaci (whiteflies) and evaluates the insecticidal properties of protein extracts from selected tropical plants. Colonies of B. tabaci were maintained in a controlled greenhouse environment and fed with different artificial diets. A bioassay method using vials with two hollow side arms was employed for administering the diets and for infestation with whiteflies. The biotype of B. tabaci was determined using a RAPD PCR test. Three distinct artificial diets were tested, alongside periodic renewals from natural hosts to maintain genetic vigor in the laboratory colony. Bioassays were conducted to assess the effectiveness of these diets and to determine the insecticidal activity of protein extracts from seven tropical plants. The results indicated that while the complex diet without TRIS led to 100% mortality within six days, other diets showed variable mortality rates with no significant difference in mean mortality. Our study found high mortality rates when CHAPS, a common solubilizing agent for membrane proteins, was used in the extraction process, suggesting its toxicity to B. tabaci. Among the tested plant protein extracts, four exhibited over 90% mortality at 72 hours, indicating potential as natural insecticides. The research highlights the need for careful selection of solvents in bioassays and points to the potential of young leaves from tropical plants as sources of anti-whitefly compounds. [ABSTRACT FROM AUTHOR]

Transmembrane protein 182 (TMEM182) is notably abundant in muscle and adipose tissue, but its role in the heart remains unknown. This study examined the contribution of TMEM182 in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. For this, we generated hiPSCs overexpressing TMEM182 in a doxycycline‐inducible manner and induced their differentiation into cardiomyocytes. On Day 12 of differentiation, expression of the cardiomyocyte markers, TNNT2 and MYH6, was significantly decreased in TMEM182‐overexpressing cells. Additionally, we found that phosphorylation of GSK‐3β (Ser9) and β‐catenin (Ser552) was increased during TMEM182 overexpression, suggesting activation of Wnt/β‐catenin signaling. We further focused on integrin‐linked kinase (ILK) as the mechanism by which TMEM182 activates Wnt/β‐catenin signaling. Evaluation showed that ILK expression was increased in cells overexpressing TMEM182. These results suggest that TMEM182 maintains Wnt/β‐catenin signaling in an activated state after mesoderm formation by increasing ILK expression, thereby suppressing hiPSCs differentiation into cardiomyocytes. [ABSTRACT FROM AUTHOR]

Symmetrical lesions in the temporal poles and external capsules on brain MRI are known as radiological markers of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); however, similar imaging findings have also been reported in neuromyelitis optica spectrum disorder (NMOSD), and this study investigated the frequency of such findings. The study included 55 NMOSD patients who met the 2015 international NMO diagnosis panel (IPND) criteria and were positive for aquaporin-4 antibodies (AQP4-Ab). Images were evaluated based on the consensus of two neuroradiologists, and brain lesions were detected in 33 patients, of whom 2 (6%) had symmetrical lesions in both the temporal poles and external capsules, and 1 (3%) had symmetrical lesions confined to the external capsules. Therefore, when symmetrical lesions in the temporal poles and external capsules are observed on MRI, NMOSD should be considered in the differential diagnosis. [ABSTRACT FROM AUTHOR]

About 30% of all bacterial proteins execute their function outside of the cytosol and must be inserted into or translocated across the cytoplasmic membrane. This requires efficient targeting systems that recognize N‐terminal signal sequences in client proteins and deliver them to protein transport complexes in the membrane. While the importance of these protein transport machineries for the spatial organization of the bacterial cell is well documented in multiple studies, the contribution of mRNA targeting and localized translation to protein transport is only beginning to emerge. mRNAs can exhibit diverse subcellular localizations in the bacterial cell and can accumulate at sites where new protein is required. This is frequently observed for mRNAs encoding membrane proteins, but the physiological importance of membrane enrichment of mRNAs and the consequences it has for the insertion of the encoded protein have not been explored in detail. Here, we briefly highlight some basic concepts of signal sequence‐based protein targeting and describe in more detail strategies that enable the monitoring of mRNA localization in bacterial cells and potential mechanisms that route mRNAs to particular positions within the cell. Finally, we summarize some recent developments that demonstrate that mRNA targeting and localized translation can sustain membrane protein insertion under stress conditions when the protein‐targeting machinery is compromised. Thus, mRNA targeting likely acts as a back‐up strategy and complements the canonical signal sequence‐based protein targeting. [ABSTRACT FROM AUTHOR]