Journal of Membrane Biology最新文献

Progesterone (P4) acts as a key conserved signalling molecule in vertebrate reproduction. P4 is especially important for mature sperm physiology and subsequent reproductive success. "CatSpermasome", a multi-unit molecular complex, has been suggested to be the main if not the only P4-responsive atypical Ca²⁺-ion channel present in mature sperm. Altogether, here we analyse the protein sequences of CatSper1-4 from more than 500 vertebrates ranging from early fishes to humans. CatSper1 becomes longer in mammals due to sequence gain mainly at the N-terminus. Overall the conservation of full-length CatSper1-4 as well as the individual TM regions remain low. The lipid-water-interface residues (i.e. a 5 amino acid stretch sequence present on both sides of each TM region) also remain highly diverged. No specific patterns of amino acid distributions were observed. The total frequency of positively charged, negatively charged or their ratios do not follow in any specific pattern. Similarly, the frequency of total hydrophobic, total hydrophilic residues or even their ratios remain random and do not follow any specific pattern. We noted that the CatSper1-4 genes are missing in amphibians and the CatSper1 gene is missing in birds. The high variability of CatSper1-4 and gene-loss in certain clades indicate that the "CatSpermasome" is not the only P4-responsive ion channel. Data indicate that the molecular evolution of CatSper is mostly guided by diverse hydrophobic ligands rather than only P4. The comparative data also suggest possibilities of other Ca²⁺-channel/s in vertebrate sperm that can also respond to P4.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

Concerted robust opening of cardiac ryanodine receptors' (RyR2) Ca²⁺ release 1oplasmic reticulum (SR) is fundamental for normal systolic cardiac function. During diastole, infrequent spontaneous RyR2 openings mediate the SR Ca²⁺ leak that normally constrains SR Ca²⁺ load. Abnormal large diastolic RyR2-mediated Ca²⁺ leak events can cause delayed after depolarizations (DADs) and arrhythmias. The RyR2-associated mechanisms underlying these processes are being extensively studied at multiple levels utilizing various model animals. Since there are well-described species-specific differences in cardiac intracellular Ca²⁺ handing in situ, we tested whether or not single RyR2 function in vitro retains this species specificity. We isolated RyR2-rich heavy SR microsomes from mouse, rat, rabbit, and human ventricular muscle and quantified RyR2 function using identical solutions and methods. The single RyR2 cytosolic Ca²⁺ sensitivity was similar across these species. However, there were significant species differences in single RyR2 mean open times in both systole and diastole-like solutions. In diastole-like solutions, single rat/mouse RyR2 open probability and frequency of long openings (> 6 ms) were similar, but these values were significantly greater than those of either single rabbit or human RyR2s. We propose these in vitro single RyR2 functional differences across species stem from the species-specific RyR2 regulatory environment present in the source tissue. Our results show the single rabbit RyR2 functional attributes, particularly in diastole-like conditions, replicate those of single human RyR2 best among the species tested.

Osteoarthritis is a common joint disease characterized by damage to the joint cartilage that occurs throughout the entire joint tissue. This damage primarily manifests as pain in the affected area. In clinical practice, medication is commonly used to relieve pain, but the treatment's effectiveness is poor and recurrent attacks are likely. Schisandrin B is the most abundant biphenylcyclohexene lignan found in the traditional Chinese medicine Schisandra chinensis, and it possesses various pharmacological effects. This study aims to investigate the protective effect of Schisandrin B on mitochondrial damage in osteoarthritis (C28I2 cells) under an inflammatory environment induced by LPS. Cell proliferation and activity, scratch tests, and LDH release tests are utilized to assess cell growth and migration ability. The immunofluorescence assay was used to detect the expression levels of proliferation and apoptosis proteins. The Western Blot assay was used to detect the expression levels of mitochondrial fusion and division proteins. The JC-1 assay was used to detect changes in mitochondrial membrane potential. The mitochondrial fluorescence probe assay was used to detect mitochondrial activity. Through research, it was found that Schisandrin B promotes the proliferation, growth, and migration of C28I2 cells, reduces apoptosis of C28I2 cells, balances mitochondrial fusion and division, stabilizes mitochondrial membrane potential, and promotes mitochondrial activity in an LPS induced inflammatory environment.

Cancer is one of the main causes of death among humans, second only to cardiovascular diseases. In recent years, numerous studies have been conducted on the pathophysiology of cancer, and it has been established that this disease is developed by a group of stem cells known as cancer stem cells (CSCs). Thus, cancer is considered a stem cell disease; however, there is no comprehensive consensus about the characteristics of these cells. Several different signaling pathways including Notch, Hedgehog, transforming growth factor-β (TGF-β), and WNT/β-catenin pathways cause the self-renewal of CSCs. CSCs change their metabolic pathways in order to access easy energy. Therefore, one of the key objectives of researchers in cancer treatment is to destroy CSCs. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the protection of CSCs from reactive oxygen species (ROS) and chemotherapeutic agents by regulating antioxidants and detoxification enzymes. Human epidermal growth factor receptor 2 (HER2) is a member of the tyrosine kinase receptor family, which contributes to the protection of cancer cells against treatment and implicated in the invasion, epithelial-mesenchymal transition (EMT), and tumorigenesis. Aldehyde dehydrogenases (ALDHs) are highly active in CSCs and protect the cells against damage caused by active aldehydes through the regulation of aldehyde metabolism. On the other hand, ALDHs promote the formation and maintenance of tumor cells and lead to drug resistance in tumors through the activation of various signaling pathways, such as the ALDH1A1/HIF-1α/VEGF axis and Wnt/β-catenin, as well as changing the intracellular pH value. Given the growing body of information in this field, in the present narrative review, we attempted to shed light on the function of Nrf2, HER2, and ALDH in CSCs.

In skeletal muscle, the Ca²⁺ release flux elicited by a voltage clamp pulse rises to an early peak that inactivates rapidly to a much lower steady level. Using a double pulse protocol the fast inactivation follows an arithmetic rule: if the conditioning depolarization is less than or equal to the test depolarization, then decay (peak minus steady level) in the conditioning release is approximately equal to suppression (unconditioned minus conditioned peak) of the test release. This is due to quantal activation by voltage, analogous to the quantal activation of IP3 receptor channels. Two mechanisms are possible. One is the existence of subsets of channels with different sensitivities to voltage. The other is that the clusters of Ca²⁺-gated Ryanodine Receptor (RyR) β in the parajunctional terminal cisternae might constitute the quantal units. These Ca²⁺-gated channels are activated by the release of Ca²⁺ through the voltage-gated RyR α channels. If the RyR β were at the basis of quantal release, it should be modified by strong inhibition of the primary voltage-gated release. This was attained in two ways, by sarcoplasmic reticulum (SR) Ca²⁺ depletion and by voltage-dependent inactivation. Both procedures reduced global Ca²⁺ release flux, but SR Ca²⁺ depletion reduced the single RyR current as well. The effect of both interventions on the quantal properties of Ca²⁺ release in frog skeletal muscle fibers were studied under voltage clamp. The quantal properties of release were preserved regardless of the inhibitory maneuver applied. These findings put a limit on the role of the Ca²⁺-activated component of release in generating quantal activation.

The gastric H⁺,K⁺-ATPase is an integral membrane protein which derives energy from the hydrolysis of ATP to transport H⁺ ions from the parietal cells of the gastric mucosa into the stomach in exchange for K⁺ ions. It is responsible for the acidic environment of the stomach, which is essential for digestion. Acid secretion is regulated by the recruitment of the H⁺,K⁺-ATPase from intracellular stores into the plasma membrane on the ingestion of food. The similar amino acid sequences of the lysine-rich N-termini α-subunits of the H⁺,K⁺- and Na⁺,K⁺-ATPases, suggests similar acute regulation mechanisms, specifically, an electrostatic switch mechanism involving an interaction of the N-terminal tail with the surface of the surrounding membrane and a modulation of the interaction via regulatory phosphorylation by protein kinases. From a consideration of sequence alignment of the H⁺,K⁺-ATPase and an analysis of its coevolution with protein kinase C and kinases of the Src family, the evidence points towards a phosphorylation of tyrosine-7 of the N-terminus by either Lck or Yes in all vertebrates except cartilaginous fish. The results obtained will guide and focus future experimental research.

Plasma membrane proteins (PMPs) play pivotal roles in various cellular events and are crucial in disease pathogenesis, making their comprehensive characterization vital for biomedical research. However, the hydrophobic nature and low expression levels of PMPs pose challenges for conventional enrichment methods, hindering their identification and functional profiling. In this study, we presented a novel TurboID-based enrichment approach for PMPs that helped overcoming some of the existing limitations. We evaluated the efficacy of TurboID and its modified form, TurboID-START, in PMP enrichment, achieving efficient and targeted labelling of PMPs without the need for stable cell line generation. This approach resulted reduction in non-specific biotinylation events, leading to improved PMP enrichment and enabled assessment of the subcellular proteome associated with the plasma membrane. Our findings paved the way for studies targeting the dynamic nature of the plasma membrane proteome and aiming to capture transient associations of proteins with the plasma membrane. The novel TurboID-based enrichment approach presented here offers promising prospects for in-depth investigations into PMPs and their roles in cellular processes.

As one of the most prevalent malignancies among women, breast cancer (BC) is tightly linked to metabolic dysfunction. However, the correlation between mitochondrial metabolism-related genes (MMRGs) and BC remains unclear. The training and validation datasets for BC were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases, respectively. MMRG-related data were obtained from the Molecular Signatures Database. A risk score prognostic model incorporating MMRGs was established based on univariate, LASSO, and multivariate Cox regression analyses. Independent factors affecting BC prognosis were identified through regression analysis and presented in a nomogram. Single-sample gene set enrichment analysis was employed to assess the immune levels of high-risk (HR) and low-risk (LR) groups. The sensitivity of BC patients in the two groups to common anti-tumor drugs was evaluated by utilizing the Genomics of Drug Sensitivity in Cancer database. 12 MMRGs significantly associated with survival were selected from 1234 MMRGs. A 12-gene risk score prognostic model was built. In the multivariate regression analysis incorporating classical clinical factors, the MMRG-related risk score remained an independent prognostic factor. As revealed by tumor immune microenvironment analysis, the LR group with higher survival rates had elevated immune levels. The drug sensitivity results unmasked that the LR group demonstrated higher sensitivity to Irinotecan, Nilotinib, and Oxaliplatin, while the HR group demonstrated higher sensitivity to Lapatinib. The development of MMRG characteristics provides a comprehensive understanding of mitochondrial metabolism in BC, aiding in the prediction of prognosis and tumor microenvironment, and offering promising therapeutic choices for BC patients with different MMRG risk scores.