Small Membrane Protein Research Articles

The small membrane protein CcoS is involved in cofactor insertion into the cbb3-type cytochrome c oxidase.

Respiratory complexes, such as cytochrome oxidases, are cofactor-containing multi-subunit protein complexes that are critically important for energy metabolism in all domains of life. Their intricate assembly strictly depends on accessory proteins, which coordinate subunit associations and cofactor deliveries. The small membrane protein CcoS was previously identified as an essential assembly factor to produce an active cbb3-type cytochrome oxidase (cbb3-Cox) in Rhodobacter capsulatus, but its function remained unknown. Here we show that the ΔccoS strain assembles a heme b deficient cbb3-Cox, in which the CcoN-CcoO subunit association is impaired. Chemical crosslinking demonstrates that CcoS interacts with the CcoN and CcoP subunits of cbb3-Cox, and that it stabilizes the interaction of the Cu-chaperone SenC with cbb3-Cox. CcoS lacks heme- or Cu-binding motifs, and we did not find evidence for direct heme or Cu binding; rather our data indicate that CcoS, together with SenC, coordinates heme and Cu insertion into cbb3-Cox.

Analysis of the structure and interactions of the SARS-CoV-2 ORF7b accessory protein

SARS-CoV-2 carries a sizeable number of proteins that are accessory to replication but may be essential for virus-host interactions and modulation of the host immune response. Here, we investigated the structure and interactions of the largely unknown ORF7b, a small membranous accessory membrane protein of SARS-CoV-2. We show that structural predictions indicate a transmembrane (TM) leucine zipper for ORF7b, and experimentally confirm the predominantly α-helical secondary structure within a phospholipid membrane mimetic by solid-state NMR. We also show that ORF7b forms heterogeneous higher-order multimers. We determined ORF7b interactions with cellular TM leucine zipper proteins using both biochemical and NMR approaches, providing evidence for ORF7b interaction with the TM domains of E-cadherin, as well as phospholamban. Our results place ORF7b as a hypothetical interferer in cellular processes that utilize leucine zipper motifs in transmembrane multimerization domains.

Translation profiling of stress-induced small proteins reveals a novel link among signaling systems.

Signaling networks allow adaptation to stressful environments by activating genes that counteract stressors. Small proteins (≤ 50 amino acids long) are a rising class of stress response regulators. Escherichia coli encodes over 150 small proteins, most of which lack phenotypes and their biological roles remain elusive. Using magnesium limitation as a stressor, we identify stress-induced small proteins using ribosome profiling, RNA sequencing, and transcriptional reporter assays. We uncover 17 small proteins with increased translation initiation, several of them transcriptionally upregulated by the PhoQ-PhoP two-component signaling system, crucial for magnesium homeostasis. Next, we describe small protein-specific deletion and overexpression phenotypes, underscoring their physiological significance in low magnesium stress. Most remarkably, we elucidate an unusual connection via a small membrane protein YoaI, between major signaling networks - PhoR-PhoB and EnvZ-OmpR in E. coli, advancing our understanding of small protein regulators in cellular signaling.

Cholesterol Accelerates Aggregation of α-Synuclein Simultaneously Increasing the Toxicity of Amyloid Fibrils.

A hallmark of Parkinson disease (PD) is a progressive degeneration of neurons in the substantia nigra pars compacta, hypothalamus, and thalamus. Although the exact etiology of irreversible neuronal degeneration is unclear, a growing body of experimental evidence indicates that PD could be triggered by the abrupt aggregation of α-synuclein (α-Syn), a small membrane protein that is responsible for cell vesicle trafficking. Phospholipids uniquely alter the rate of α-Syn aggregation and, consequently, change the cytotoxicity of α-Syn oligomers and fibrils. However, the role of cholesterol in the aggregation of α-Syn remains unclear. In this study, we used Caenorhabditis elegans that overexpressed α-Syn to investigate the effect of low (15%), normal (30%), and high (60%) concentrations of cholesterol on α-Syn aggregation. We found that an increase in the concentration of cholesterol in diets substantially shortened the lifespan of C. elegans. Using biophysical methods, we also investigated the extent to which large unilamellar vesicles (LUVs) with low, normal, and high concentrations of cholesterol altered the rate of α-Syn aggregation. We found that only lipid membranes with a 60% concentration of cholesterol substantially accelerated the rate of protein aggregation. Cell assays revealed that α-Syn fibrils formed in the presence of LUVs with different concentrations of cholesterol exerted very similar levels of cytotoxicity to rat dopaminergic neurons. These results suggest that changes in the concentration of cholesterol in the plasma membrane, which in turn could be caused by nutritional preferences, could accelerate the onset and progression of PD.

Epithelial membrane protein 1 in human cancer: a potential diagnostic biomarker and therapeutic target.

Epithelial membrane protein 1 (EMP1) is a member of the small hydrophobic membrane protein subfamily. EMP1 is aberrantly expressed in various tumor tissues and governs multiple cellular behaviors (e.g., proliferation, differentiation, and migration). The resultant regulation of the cancer pathway is responsible for the metastasis of cancer cells and determines the risk of malignant tumor progression. This review provides an updated overview of EMP1 as either an oncogene or a tumor suppressor contingent on the cancer type and summarizes its upstream regulators and downstream target genes. This systematic review summarizes our current understanding of the role of EMP1 in malignant tumor development, including critical functional mechanisms and implications for its potential use as the biomarker and therapeutic target.

A cell-free system for functional studies of small membrane proteins

Numerous small proteins have been discovered across all domains of life, among which many are hydrophobic and predicted to localize to the cell membrane. Based on a few that are well-studied, small membrane proteins are regulators involved in various biological processes, such as cell signaling, nutrient transport, drug resistance, and stress response. However, the function of most identified small membrane proteins remains elusive. Their small size and hydrophobicity make protein production challenging, hindering function discovery. Here, we combined a cell-free system with lipid sponge droplets and synthesized small membrane proteins in vitro. Lipid sponge droplets contain a dense network of lipid bilayers, which accommodates and extracts newly synthesized small membrane proteins from the aqueous surroundings. Using small bacterial membrane proteins MgrB, SafA, and AcrZ as proof of principle, we showed that the in vitro produced membrane proteins were functionally active, for example, modulating the activity of their target kinase as expected. The cell-free system produced small membrane proteins, including one from human, up to micromolar concentrations, indicating its high level of versatility and productivity. Furthermore, AcrZ produced in this system was used successfully for in vitro co-immunoprecipitations to identify interaction partners. This work presents a robust alternative approach for producing small membrane proteins, which opens a door to their function discovery in different domains of life.

Inhibitors of the small membrane (M) protein viroporin prevent Zika virus infection.

Flaviviruses, including Zika virus (ZIKV), are a significant global health concern, yet no licensed antivirals exist to treat disease. The small Membrane (M) protein plays well-defined roles during viral egress and remains within virion membranes following release and maturation. However, it is unclear whether M plays a functional role in this setting. Here, we show that M forms oligomeric membrane-permeabilising channels in vitro, with increased activity at acidic pH and sensitivity to the prototypic channel-blocker, rimantadine. Accordingly, rimantadine blocked an early stage of ZIKV cell culture infection. Structure-based channel models, comprising hexameric arrangements of two trans-membrane domain protomers were shown to comprise more stable assemblages than other oligomers using molecular dynamics (MD) simulations. Models contained a predicted lumenal rimantadine binding site, as well as a second druggable target region on the membrane-exposed periphery. In silico screening enriched for repurposed drugs/compounds predicted to bind to either one site or the other. Hits displayed superior potency in vitro and in cell culture compared with rimantadine, with efficacy demonstrably linked to virion-resident channels. Finally, rimantadine effectively blocked ZIKV viraemia in preclinical models, supporting that M constitutes a physiologically relevant target. This could be explored by repurposing rimantadine, or development of new M-targeted-therapies.

Lipoteichoic acid biosynthesis by Staphylococcus aureus is controlled by the MspA protein.

Staphylococcus aureus produces a plethora of virulence factors critical to its ability to establish an infection and cause disease. We have previously characterized a small membrane protein, MspA, which has pleiotropic effects on virulence and contributes to S. aureus pathogenicity in vivo. Here we report that mspA inactivation triggers overaccumulation of the essential cell wall component, lipoteichoic acid (LTA), which, in turn, decreases autolytic activity and leads to increased cell size due to a delay in cell separation. We show that MspA directly interacts with the enzymes involved in LTA biosynthesis (LtaA, LtaS, UgtP, and SpsB), interfering with their normal activities. MspA, in particular, interacts with the type I signal peptidase SpsB, limiting its cleavage of LtaS into its active form. These findings suggest that MspA contributes to maintaining a physiological level of LTA in the cell wall by interacting with and inhibiting the activity of SpsB, thereby uncovering a critical role for the MspA protein in regulating cell envelope biosynthesis and pathogenicity.IMPORTANCEThe S. aureus cell envelope, comprising the cytoplasmic membrane, a thick peptidoglycan layer, and the anionic polymers lipoteichoic acid and wall teichoic acids, is fundamental for bacterial growth and division, as well as being the main interface between the pathogen and the host. It has become increasingly apparent that the synthesis and turnover of cell envelope components also affect the virulence of S. aureus. In this study, we show that MspA, an effector of S. aureus virulence, contributes to the maintenance of normal levels of lipoteichoic acid in the cell wall, with implications on cell cycle and size. These findings further our understanding of the connections between envelope synthesis and pathogenicity and suggest that MspA represents a promising target for the development of future therapeutic strategies.

SMIM1 absence is associated with reduced energy expenditure and excess weight

Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments. We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1-/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan. We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure. This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them. This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant.

Mechanism of an animal toxin-antidote system.

Toxin-antidote systems are selfish genetic elements composed of a linked toxin and antidote. The peel-1 zeel-1 toxin-antidote system in C. elegans consists of a transmembrane toxin protein PEEL-1 which acts cell autonomously to kill cells. Here we investigate the molecular mechanism of PEEL-1 toxicity. We find that PEEL-1 requires a small membrane protein, PMPL-1, for toxicity. Together, PEEL-1 and PMPL-1 are sufficient for toxicity in a heterologous system, HEK293T cells, and cause cell swelling and increased cell permeability to monovalent cations. Using purified proteins, we show that PEEL-1 and PMPL-1 allow ion flux through lipid bilayers and generate currents which resemble ion channel gating. Our work suggests that PEEL-1 kills cells by co-opting PMPL-1 and creating a cation channel.

SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-β-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Aquaporins: Multifunctional Players in Plant Growth, Development and Stress Responses

Aquaporins are small membrane proteins belonging to the group of Major Intrinsic proteins (MIPs) that facilitate the transport of water and small molecules across biological membranes. They were initially identified as water channels and later it has become clear that they also play important roles in several other physiological processes in plants. Plants have different types of aquaporins in terms of their location, gating properties, and solute specificity and can mainly divide into four major subfamilies. They are tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), plasma membrane intrinsic proteins (PIPs), and the small basic intrinsic proteins (SIPs). All these aquaporins play a variety of physiological activities at all stages of plant growth and development. Also, they are believed to play a significant part in the plant’s defense mechanisms against biotic and abiotic stresses. This review comprehensively addresses the roles of diverse aquaporin homologs in growth and development and their response to different environmental factors.

Type I toxin-antitoxin systems in bacteria: from regulation to biological functions.

Toxin-antitoxin systems are ubiquitous in the prokaryotic world and widely distributed among chromosomes and mobile genetic elements. Several different toxin-antitoxin system types exist, but what they all have in common is that toxin activity is prevented by the cognate antitoxin. In type I toxin-antitoxin systems, toxin production is controlled by an RNA antitoxin and by structural features inherent to the toxin messenger RNA. Most type I toxins are small membrane proteins that display a variety of cellular effects. While originally discovered as modules that stabilize plasmids, chromosomal type I toxin-antitoxin systems may also stabilize prophages, or serve important functions upon certain stress conditions and contribute to population-wide survival strategies. Here, we will describe the intricate RNA-based regulation of type I toxin-antitoxin systems and discuss their potential biological functions.

Phoenixin knockout mice show no impairment in fertility or differences in metabolic response to a high-fat diet, but exhibit behavioral differences in an open field test.

Phoenixin (PNX) is a conserved secreted peptide that was identified 10 years ago with numerous studies published on its pleiotropic functions. PNX is associated with estrous cycle length, protection from a high-fat diet, and reduction of anxiety behavior. However, no study had yet evaluated the impact of deleting PNX in the whole animal. We sought to evaluate a mouse model lacking the PNX parent gene, small integral membrane protein 20 (Smim20), and the resulting effect on reproduction, energy homeostasis, and anxiety. We found that the Smim20 knockout mice had normal fertility and estrous cycle lengths. Consistent with normal fertility, the hypothalamii of the knockout mice showed no changes in the levels of reproduction-related genes, but the male mice had some changes in energy homeostasis-related genes, such as melanocortin receptor 4 (Mc4r). When placed on a high-fat diet, the wildtype and knockout mice responded similarly, but the male heterozygous mice gained slightly less weight. When placed in an open field test box, the female knockout mice traveled less distance in the outer zone, indicating alterations in anxiety or locomotor behavior. In summary, the homozygous knockout of PNX did not alter fertility and modestly alters a few neuroendocrine genes in response to a high-fat diet, especially in the female mice. However, it altered the behavior of mice in an open field test. PNX therefore may not be crucial for reproductive function or weight, however, we cannot rule out possible compensatory mechanisms in the knockout model. Understanding the role of PNX in physiology may ultimately lead to an enhanced understanding of neuroendocrine mechanisms involving this enigmatic peptide.

Pro-SMP finder-A systematic approach for discovering small membrane proteins in prokaryotes.

Prokaryotic chromosomes contain numerous small open reading frames (ORFs) of less than 200 bases. Since high-throughput proteomics methods often miss proteins containing fewer than 60 amino acids, it is difficult to decern if they encode proteins. Recent studies have revealed that many small proteins are membrane proteins with a single membrane-anchoring α-helix. As membrane anchoring or transmembrane motifs are accurately identifiable with high confidence using computational algorithms like Phobius and TMHMM, small membrane proteins (SMPS) can be predicted with high accuracy. This study employed a systematic approach, utilizing well-verified algorithms such as Orfipy, Phobius, and Blast to identify SMPs in prokaryotic organisms. Our main search parameters targeted candidate SMPs with an open reading frame between 60-180 nucleotides, a membrane-anchoring or transmembrane region 15 and 30 amino acids long, and sequence conservation among other microorganisms. Our findings indicate that each prokaryote possesses many SMPs, with some identified in the intergenic regions of currently annotated chromosomes. More extensively studied microorganisms, such as Escherichia coli and Bacillus subtilis, have more SMPs identified in their genomes compared to less studied microorganisms, suggesting the possibility of undiscovered SMPs in less studied microorganisms. In this study, we describe the common SMPs identified across various microorganisms and explore their biological roles. We have also developed a software pipeline and an accompanying online interface for discovering SMPs (http://cs.indstate.edu/pro-smp-finder). This resource aims to assist researchers in identifying new SMPs encoded in microbial genomes of interest.

Mapping the architecture of the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in a liponanoparticle

Bacterial cell surface glycoconjugates are critical for cell survival and for interactions between bacteria and their hosts. Consequently, the pathways responsible for their biosynthesis have untapped potential as therapeutic targets. The localization of many glycoconjugate biosynthesis enzymes to the membrane represents a significant challenge for expressing, purifying, and characterizing these enzymes. Here, we leverage cutting-edge detergent-free methods to stabilize, purify, and structurally characterize WbaP, a phosphoglycosyl transferase (PGT) from the Salmonella enterica (LT2) O-antigen biosynthesis. From a functional perspective, these studies establish WbaP as a homodimer, reveal the structural elements responsible for dimerization, shed light on the regulatory role of a domain of unknown function embedded within WbaP, and identify conserved structural motifs between PGTs and functionally unrelated UDP-sugar dehydratases. From a technological perspective, the strategy developed here is generalizable and provides a toolkit for studying other classes of small membrane proteins embedded in liponanoparticles beyond PGTs.

In Silico Identification and Characterization of Rare Cold Inducible 2 (RCI2) Gene Family in Cotton.

RCI2/PMP3s are involved in biotic and abiotic stresses and have an influence on the regulation of many genes. RCI2/PMP3 genes, which particularly encode small membrane proteins of the PMP3 family, are involved in abiotic stress responses in plants. In this work, in silico studies were used to investigate RCI2's potential function in stress tolerance and organogenesis. We conducted an extensive study of the RCI2 gene family and revealed 36 RCI2 genes from cotton species that were distributed over 36 chromosomes of the cotton genome. Functional and phylogenetic examination of the RCI2/PMP3 gene family has been studied in Arabidopsis, but in cotton, the RCI2/PMP3 genes have not yet been studied. Phylogenetic and sequencing studies revealed that cotton RCI2s are conserved, with most of them categorized into six distinct clades. A chromosome distribution and localization study indicated that cotton RCI2 genes were distributed unevenly on 36 chromosomes with segmental duplications, suggesting that the cotton RCI2 family is evolutionarily conserved. Many cis-elements related to stress responsiveness, development, and hormone responsiveness were detected in the promoter regions of the cotton RCI2. Moreover, the 36 cotton RCI2s revealed tissue-specific expression patterns in the development of cotton performed by transcriptome analysis. Gene structure analysis indicated that nearly all RCI2 genes have two exons and one intron. All of the cotton RCI2 genes were highly sensitive to drought, abscisic acid, salt, and cold treatments, demonstrating that they may be employed as genetic objects to produce stress-resistant plants.

Visual Analytics for Robust Investigations of Placental Aquaporin Gene Expression in Response to Maternal SARS-CoV-2 Infection

The human placenta is a multifunctional, disc-shaped temporary fetal organ that develops in the uterus during pregnancy, connecting the mother and the fetus. The availability of large-scale datasets on the gene expression of placental cell types and scholarly articles documenting adverse pregnancy outcomes from maternal infection warrants the use of computational resources to aid in knowledge generation from disparate data sources. Using maternal Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection as a case study in microbial infection, we constructed integrated datasets and implemented visual analytics resources to facilitate robust investigations of placental gene expression data in the dimensions of flow, curation, and analytics. The visual analytics resources and associated datasets can support a greater understanding of SARS-CoV-2-induced changes to the human placental expression levels of 18,882 protein-coding genes and at least 1233 human gene groups/families. We focus this report on the human aquaporin gene family that encodes small integral membrane proteins initially studied for their roles in water transport across cell membranes. Aquaporin-9 (AQP9) was the only aquaporin downregulated in term placental villi from SARS-CoV-2-positive mothers. Previous studies have found that (1) oxygen signaling modulates placental development; (2) oxygen tension could modulate AQP9 expression in the human placenta; and (3) SARS-CoV-2 can disrupt the formation of oxygen-carrying red blood cells in the placenta. Thus, future research could be performed on microbial infection-induced changes to (1) the placental hematopoietic stem and progenitor cells; and (2) placental expression of human aquaporin genes, especially AQP9.

Application of Intracerebral Aquaporin-4 Expression in Differential Diagnosis of Drowning: an Immunohistochemical Study Related to Timing of Autopsy.

The role of forensic pathologists is pivotal in definitively diagnosing drowning cases. Further differentiation becomes essential for distinguishing between freshwater drowning (FWD) and saltwater drowning (SWD). Aquaporins are small integral membrane proteins that serve as major water transport pathways in various cell types. AQP4 appears to be involved in mechanisms related to cerebral volume regulation. Our study aims to examine the expression of AQP4 in the brain as a potential marker for differentiating between FWD and SWD relating to autopsy-performing timing. A total of 23 cases were classified into three groups: FWD, SWD, and controls. All samples were classified upon autopsy-performing timing into two subgroups: within and after 72 hours of death. The samples were then processed for histological and immunohistochemical investigations. For autopsies performed within 72 hours of death, we found a significantly higher value of AQP4-positive astrocytes in cases of FWD compared to SWD and control groups. We also found a significantly lower AQP4 expression in SWD cases compared to the control group. For autopsies conducted after 72 hours, the immunohistochemical staining does not reveal the peripheral terminations of astrocytes, which appear blurred and only recognizable as halos. In conclusion, the data aligns with existing literature about autopsies performed within 72 hours. However, in autopsies conducted after 72 hours, uncertain and even opposed results are observed. The difference can be ascribed to the post-mortem transformative processes that take place upon the cessation of vital functions.

Molecular Regulation and Oncogenic Functions of TSPAN8.

Tetraspanins, a superfamily of small integral membrane proteins, are characterized by four transmembrane domains and conserved protein motifs that are configured into a unique molecular topology and structure in the plasma membrane. They act as key organizers of the plasma membrane, orchestrating the formation of specialized microdomains called "tetraspanin-enriched microdomains (TEMs)" or "tetraspanin nanodomains" that are essential for mediating diverse biological processes. TSPAN8 is one of the earliest identified tetraspanin members. It is known to interact with a wide range of molecular partners in different cellular contexts and regulate diverse molecular and cellular events at the plasma membrane, including cell adhesion, migration, invasion, signal transduction, and exosome biogenesis. The functions of cell-surface TSPAN8 are governed by ER targeting, modifications at the Golgi apparatus and dynamic trafficking. Intriguingly, limited evidence shows that TSPAN8 can translocate to the nucleus to act as a transcriptional regulator. The transcription of TSPAN8 is tightly regulated and restricted to defined cell lineages, where it can serve as a molecular marker of stem/progenitor cells in certain normal tissues as well as tumors. Importantly, the oncogenic roles of TSPAN8 in tumor development and cancer metastasis have gained prominence in recent decades. Here, we comprehensively review the current knowledge on the molecular characteristics and regulatory mechanisms defining TSPAN8 functions, and discuss the potential and significance of TSPAN8 as a biomarker and therapeutic target across various epithelial cancers.