Protein Function Research Articles

Innovating non-small cell lung cancer treatment with novel TM-GL/NPs nanoparticles for Glycitin delivery

Sojae semen praeparatum is a traditional Chinese medicine, and its active component, Glycitin, has shown potential in the treatment of non-small cell lung cancer (NSCLC). The purpose of this investigation is to examine the mechanism of action of the effective components of sojae semen praeparatum in the treatment of NSCLC, with a special emphasis on Glycitin, and to explore the integration of nanotechnology in delivering pharmaceutical agents. Key effective components were selected through network pharmacology analysis and functional analysis, and protein–protein interaction analysis and functional enrichment were performed using transcriptomics and metabolomics data to identify the key NSCLC-related target genes and regulatory mechanisms of action of the active components of sojae semen praeparatum. Glycitin-loaded NPs encapsulated in tumor-associated fibroblast membranes were developed to verify their characterization and safety, and their therapeutic effects in inhibiting the malignant phenotype of NSCLC cells through targeting the DNA topoisomerase II alpha (TOP2A) protein were validated. The results indicate that Glycitin exhibits significant anti-tumor activity by affecting the function of the TOP2A protein, thereby inhibiting tumor proliferation and metastasis. This research presents proof of the crucial function of Glycitin in managing NSCLC using sojae semen praeparatum, and sheds light on the possibilities of nanotechnology in drug delivery mechanisms, offering a novel avenue for NSCLC therapy research.

Protein structure and interactions elucidated with in-cell NMR for different cell cycle phases and in 3D human tissue models

Most of our knowledge of protein structure and function originates from experiments performed with purified proteins resuspended in dilute, buffered solutions. However, most proteins function in crowded intracellular environments with complex compositions. Significant efforts have been made to develop tools to study proteins in their native cellular settings. Among these tools, in-cell NMR spectroscopy has been the sole technique for characterizing proteins in the intracellular space of living cells at atomic resolution and physiological temperature. Nevertheless, due to technological constraints, in-cell NMR studies have been limited to asynchronous single-cell suspensions, precluding obtaining information on protein behavior in different cellular states. In this study, we present a methodology that allows for obtaining an atomically resolved NMR readout of protein structure and interactions in living human cells synchronized in specific cell cycle phases and within 3D models of human tissue. The described approach opens avenues for investigating how protein structure or drug recognition responds to cell-cell communication or changes in intracellular space composition during transitions among cell cycle phases.

Perforin-2 is overexpressed in Kikuchi-Fujimoto disease.

Kikuchi-Fujimoto disease (KFD) is a self-limited lymphadenopathy characterized by subcortical necrosis and paracortical proliferation of lymphocytes, histiocytes and plasmacytoid dendritic cells with abundant apoptosis. The etiology of KFD remains unknown. Perforin-2 is a highly conserved transmembrane molecule capable of forming pores following intercellular interaction with bacteria and erythrocytes. Perforin-2 is also essential for the activation of type I interferon-induced JAK/STAT signaling and elimination of virus infection. Herein, we hypothesized that perforin-2 or macrophage-expressed gene 1 (MPEG1) protein function is dysregulated in KFD, resulting in an exaggerated immune response, possibly following exposure to an unknown infectious agent. Twelve cases of KFD were the study group. We isolated total RNA from fixed, paraffin-embedded whole tissue sections. We also assessed primary human B-cells and 4 cases of reactive follicular hyperplasia as controls. Perforin-2 mRNA levels were assessed by quantitative real-time PCR (qRT-PCR). We found that perforin-2 mRNA expression was significantly upregulated in 11 of 12 (92%) KFD cases as compared with control samples. These results suggest that expression of perforin-2 is dramatically upregulated in KFD cases. Increased levels of perforin-2 likely explain the abundant apoptosis in KFD. These data also support the hypothesis that upregulation of perforin-2 may be part of the host immune reaction to an unknown infectious agent that is the trigger for KFD.

Regulation of N-Glycosylation of CDNF on Its Protein Stability and Function in Hypoxia/Reoxygenation Model of H9C2 Cells.

Myocardial ischemia-reperfusion (I/R) injury is a cause of high post-interventional mortality in patients with acute myocardial infarction (MI). Cerebral dopamine neurotrophic factor (CDNF) is an endoplasmic reticulum (ER) resident protein, and its expression and secretion are induced when tissues and cells are subjected to hypoxia, ischemia, or traumatic injury. As a novel cardiomyokine, CDNF plays a crucial role in the progression of myocardial I/R injury. In our previous study, we reported that the overexpression of CDNF inhibited tunicamycin-induced H9C2 cell apoptosis. Moreover, there is a unique N-glycosylation site at Asn57 in the CDNF protein, which likely affects its function in H9C2 cells. However, the detailed impact remains unexplored. In our current study, we observed elevated levels of CDNF in the serum of acute MI patients, myocardial tissue of I/R model mice, and H/R model H9C2 cells. To detect the effect of N-glycosylation on the CDNF protein, we constructed an Asn57 mutant (N57A) plasmid and found that the N57A protein presented similar intracellular localization to those of the wild-type CDNF protein. However, the N57A protein demonstrated reduced stability, and the mutant protein could not protect H/R-induced H9C2 cells from apoptosis. Moreover, this process may occur through the downregulation of the PI3K/Akt pathway. Therefore, N-glycosylation of CDNF may be essential for protein stability and its protective role in H/R injury in H9C2 cells.

Preparation and functional identification of various porcine cytokines.

The insufficiency of current Porcine Epidemic Diarrhea (PED) vaccines against highly pathogenic strains highlights the critical importance of enhancing mucosal immunity in the prevention and control of porcine enteric viral diseases. Due to limited research platforms, the understanding of the porcine mucosal immune system and its response mechanisms remains incomplete. This study employed prokaryotic expression and purification methods to obtain eight essential cytokines involved in mucosal immune responses (CD40L, IL-2, IL-6, TNF-α, IL-13, IL-17α, TGF-β, APRIL). By utilizing various cell models including porcine intestinal organoids, IPEC-J2, Vero-E6, porcine peripheral blood lymphocytes, and porcine Peyer's patch lymphocytes, the functions of these eight cytokines were identified through flow cytometry, immunoblotting, relative quantitative PCR, and CFSE proliferation assays. The results demonstrate that all eight purified proteins exhibit both protein activity and function. The purification of these molecules lays the groundwork for further exploration of the mucosal barrier of pigs and mucosal immune-related studies, as well as providing research tools for the prevention and control of enteric viral diseases in pigs.

Nanobody-thioesterase chimeras to specifically target protein palmitoylation

The complexity of the cellular proteome is massively expanded by a repertoire of chemically distinct reversible post-translational modifications (PTMs) that control protein localisation, interactions, and function. The temporal and spatial control of these PTMs is central to organism physiology, and mis-regulation of PTMs is a hallmark of many diseases. Here we present an approach to manipulate PTMs on target proteins using nanobodies fused to enzymes that control these PTMs. Anti-GFP nanobodies fused to thioesterases (which depalmitoylate protein cysteines) depalmitoylate GFP tagged substrates. A chemogenetic approach to enhance nanobody affinity for its target enables temporal control of target depalmitoylation. Using a thioesterase fused to a nanobody directed against the Ca(v)1.2 beta subunit we reduce palmitoylation of the Ca(v)1.2 alpha subunit, modifying the channel’s voltage dependence and arrhythmia susceptibility in stem cell derived cardiac myocytes. We conclude that nanobody enzyme chimeras represent an approach to specifically manipulate PTMs, with applications in both the laboratory and the clinic.

Targeting MDM2 affects spastin protein levels and functions: implications for HSP treatment

Spastin is a microtubule (MT) severing enzyme that regulates several cell functions associated with MT dynamics. A reduction in spastin protein levels is responsible for approximately 40% of cases of Hereditary Spastic Paraplegia (HSP), a neurodegenerative disease. Currently, there is no cure for HSP but strategies to induce a recovery of spastin levels are emerging as potential therapeutic approaches. Here, we show that MDM2 interacts with spastin MT-interacting and trafficking (MIT) domain. By biochemical and functional experiments, we demonstrate that MDM2 binds spastin and regulates its levels in a post-transcriptional manner independently of the E3 ubiquitin ligase activity. Of relevance, treatment of spastin-deficient cells with the MDM2 inhibitor Nutlin-3a can restore spastin levels and functions, such as cytokinetic abscission and sorting of transferrin receptor. These findings identify MDM2 as a novel interactor of spastin and a potential druggable regulator of its protein levels.

CyanoTag: Discovery of protein function facilitated by high-throughput endogenous tagging in a photosynthetic prokaryote.

Despite their importance to aquatic ecosystems, global carbon cycling, and sustainable bioindustries, the genomes of photosynthetic bacteria contain large numbers of uncharacterized genes. Here, we develop high-throughput endogenous fluorescent protein tagging in the cyanobacterium Synechococcus elongatus PCC 7942. From 400 targets, we successfully tag over 330 proteins corresponding to >10% of the proteome. We use this collection to determine subcellular localization, relative protein abundances, and protein-protein interaction networks, providing biological insights into diverse processes-from photosynthesis to cell division. We build a high-confidence protein-protein interaction map for the major components of photosynthesis, associating previously uncharacterized proteins with different complexes and processes. In response to light changes, we visualize, on second timescales, the reversible formation, growth, and fusion of puncta by two Calvin cycle proteins, suggesting that biomolecular condensation provides spatiotemporal control of the Calvin cycle in cyanobacteria. We envision that these insights, cell lines, and optimized methods will facilitate rapid advances in cyanobacteria biology and, more broadly, all photosynthetic life.

Revealing the Potential of a Chimaera: a Peptide-Peptide Nucleic Acid Molecule Designed To Interact with the SARS-CoV-2 Nucleocapsid Protein.

Numerous RNA-binding proteins have modular structures with folded domains and intrinsically disordered regions, making their atomic characterization difficult. This severely limits the investigation of their modalities of interaction as well as the evaluation of possible ways to interfere with this process. We report herein a rational strategy for the design and synthesis of a ligand able to interfere with the protein function, monitoring the interaction through solution nuclear magnetic resonance spectroscopy. Our approach employs a chimaera composed of two different fragments, a peptide and a peptide-nucleic acid, allowing to incorporate in the resulting molecule key features to address RNA-protein interactions. Focusing on two constructs of the N protein from SARS-CoV-2, the globular N-terminal domain and a more extended one comprising also two flanking intrinsically disordered regions, we demonstrate the enhanced affinity of the designed peptide-peptide nucleic acid chimaera for the protein compared to a related peptide lacking π-π stacking contributions within the chain. Furthermore, we emphasize the increasingly recognized relevant and synergistic role of the intrinsically disordered regions in protein-ligand interaction.

Phosphodegrons in Health and Disease: From Cellular Homeostasis to Therapeutic Potential

Phosphodegrons are critical motifs that play a pivotal role in the regulation of protein stability and function via phosphorylation-dependent signaling pathways. These motifs serve as recognition elements for ubiquitin ligases, facilitating the targeted degradation of proteins. By modulating key cellular processes such as cell cycle progression, DNA repair, and apoptosis, phosphodegrons are essential for maintaining cellular homeostasis. Dysregulation of phosphodegrons has been implicated in a wide range of diseases, including cancer and neurodegenerative disorders, highlighting their potential as therapeutic targets. This review provides an overview of phosphodegron functions along with their biological significance in health and disease. Additionally, we discuss current methodologies for studying phosphodegrons and explore emerging trends in their identification and therapeutic targeting. By synthesizing recent advances in the field, this article aims to offer insights into the future directions and challenges in phosphodegron research, ultimately underscoring their importance in cellular regulation and disease pathology.

An inducible mouse model of OI type V reveals aberrant osteogenesis caused by Ifitm5 c.-14C > T mutation.

Osteogenesis imperfecta (OI) Type V is typically characterized by radial head dislocation, calcification of interosseous membrane and hyperplastic callus. It is caused by the c.-14C > T mutation in the 5' UTR of IFITM5 gene, adding five amino acids (MALEP) to the N-terminal of IFITM5 protein. Previous studies have suggested a neomorphic function of the MALEP-IFITM5 protein. However, the underlying mechanisms remain unclear due to embryonic lethality in previous mouse models. Therefore, we developed an inducible mouse model (Ifitm5flox c.-14C > T) that could be induced by Cre expressed at different developmental stages to explore the pathogenic effects of the neomorphic MALEP-IFITM5. The mutant Ifitm5 allele could be regulated by the endogenous regulatory elements after Cre recombination, maintaining its spatiotemporal expression pattern and physiological level. Specifically, Prx1-Cre; Ifitm5flox c.-14C > T mutant mice were born with fractures in all limbs, showing impaired ossification and enhanced chondrogenesis associated with increased SOX9 abundance. Analyses of single-cell RNA sequencing data revealed arrested osteogenesis in Prx1-Cre; Ifitm5flox c.-14C > T mouse. A major population of cells expressing both osteogenic and chondrogenic signature genes was identified in the mutant mouse. Reduced expression of SP7 and SOST in the cortical regions of mutant mice confirmed delayed osteocyte maturation and compromised osteogenesis. Elevated bone marrow adipocytes were found in the adult mutant mice. Ectopic chondrogenesis and SOX9 expression were also observed in the perichondrium regions of Col1a1-Cre; Ifitm5flox c.-14C > T and Ocn-Cre; Ifitm5flox c.-14C > T mutant mice. The inducible Ifitm5flox c.-14C > T mouse model and integrated single-cell transcriptomic analyses elucidated that ectopic expression of SOX9 and disrupted homeostatic balance among osteogenesis, chondrogenesis and adipogenesis may contribute to the pathogenesis caused by MALEP-IFITM5, helping to gain deeper insights into the molecular mechanisms of type V OI.

Liver macrophage-derived exosomal miRNA-342-3p promotes liver fibrosis by inhibiting HPCAL1 in stellate cells

BackgroundThe progression of liver fibrosis involves complex interactions between hepatic stellate cells (HSCs) and multiple immune cells in the liver, including macrophages. However, the mechanism of exosomes in the crosstalk between liver macrophages and HSCs remains unclear.MethodExosomes were extracted from primary mouse macrophages and cultured with HSCs, and the differential expression of microRNAs was evaluated using high-throughput sequencing technology. The functions of miR-342-3p in exosomes were verified by qPCR and luciferase reporter gene experiments with HSCs. The function of the target gene Hippocalcin-like protein 1 (HPCAL1) in HSCs was verified by Western blotting, qPCR, cellular immunofluorescence and co-IP in vivo and in vitro.ResultsWe demonstrated that exosomal microRNA-342-3p derived from primary liver macrophages could activate HSCs by inhibiting the expression of HPCAL1 in HSCs. HPCAL1, which is a fibrogenesis suppressor, could inhibit TGF-β signaling in HSCs by regulating the ubiquitination of Smad2 through direct interactions with its EF-hand 4 domain.ConclusionThis study reveals a previously unidentified profibrotic mechanism of crosstalk between macrophages and HSCs in the liver and suggests an attractive novel therapeutic strategy for treating fibroproliferative liver diseases.Graphical

Biophysical and structural insights into AAV genome ejection.

Recombinant adeno-associated virus (rAAV) is comprised of non-enveloped capsids that can package a therapeutic transgene and are currently being developed and utilized as gene therapy vectors. The therapeutic efficiency of rAAV is dependent on successful cytoplasmic trafficking and transgene delivery to the nucleus. It is hypothesized that an increased understanding of the effects of the cellular environment and biophysical properties of the capsid as it traffics to the nucleus could provide insight to improve vector efficiency. The AAV capsid is exposed to increasing [H+] during endo-lysosomal trafficking. Exposure to low pH facilitates the externalization of the viral protein 1 unique region (VP1u). This VP1u contains a phospholipase A2 domain required for endosomal escape and nuclear localization signals that facilitate nuclear targeting and entry. The viral genome is released either after total capsid disassembly or via a concerted DNA ejection mechanism in the nucleus. This study presents the characterization of genome ejection (GE) for two diverse serotypes, AAV2 and AAV5, using temperature. The temperature required to disassemble the virus capsid (TM) is significantly higher than the temperature required to expose the transgene (TE) for both serotypes. This was verified by quantitative PCR (qPCR) and transmission electron microscopy. Additionally, the absence of VP1/VP2 in the capsids and a decrease in pH increase the temperature of GE. Furthermore, cryo-electron microscopy structures of the AAV5 capsid pre- and post-GE reveal dynamics at the twofold, threefold, and fivefold regions of the capsid interior consistent with a concerted egress of the viral genome.IMPORTANCEThe development of recombinant adeno-associated virus (rAAV) capsids has grown rapidly in recent years, with five of the eight established therapeutics gaining approval in the past 2 years alone. Clinical progression with AAV2 and AAV5 represents a growing need to further characterize the molecular biology of these viruses. The goal of AAV-based gene therapy is to treat monogenic disorders with a vector-delivered transgene to provide wild-type protein function. A better understanding of the dynamics and conditions enabling transgene release may improve therapeutic efficiency. In addition to their clinical importance, AAV2 and 5 were chosen in this study for their diverse antigenic and biophysical properties compared to more closely related serotypes. Characterization of a shared genome ejection process may imply a conserved mechanism for all rAAV therapies.

Exploiting the Molecular Properties of Fibrinogen to Control Bleeding Following Vascular Injury

The plasma protein fibrinogen is critical for haemostasis and wound healing, serving as the structural foundation of the blood clot. Through a complex interaction between coagulation factors, the soluble plasma fibrinogen is converted to insoluble fibrin networks, which form the skeleton of the blood clot, an essential step to limit blood loss after vascular trauma. This review examines the molecular mechanisms by which fibrinogen modulates bleeding, focusing on its interactions with other proteins that maintain fibrin network stability and prevent premature breakdown. Moreover, we also cover the role of fibrinogen in ensuring clot stability through the physiological interaction with platelets. We address the therapeutic applications of fibrinogen across various clinical contexts, including trauma-induced coagulopathy, postpartum haemorrhage, and cardiac surgery. Importantly, a full understanding of protein function will allow the development of new therapeutics to limit blood loss following vascular trauma, which remains a key cause of mortality worldwide. While current management strategies help with blood loss following vascular injury, they are far from perfect and future research should prioritise refining fibrinogen replacement strategies and developing novel agents to stabilise the fibrin network. Exploiting fibrinogen’s molecular properties holds significant potential for improving outcomes in trauma care, surgical interventions and obstetric haemorrhage.

Implication of protein post translational modifications in gastric cancer

Gastric cancer (GC) is one of the most common and highly lethal malignant tumors worldwide, and its occurrence and development are regulated by multiple molecular mechanisms. Post-translational modifications (PTM) common forms include ubiquitylation, phosphorylation, acetylation and methylation. Emerging research has highlighted lactylation and glycosylation. The diverse realm of PTM and PTM crosstalk is linked to many critical signaling events involved in neoplastic transformation, carcinogenesis and metastasis. This review provides a comprehensive overview of the impact of PTM on the occurrence and progression of GC. Specifically, aberrant PTM have been shown to alter the proliferation, migration, and invasion capabilities of GC cells. Moreover, PTM are closely associated with resistance to chemotherapeutic agents in GC. Notably, this review also discusses the phenomenon of PTM crosstalk, highlighting the interactions among PTM and their roles in regulating signaling pathways and protein functions. Therefore, in-depth investigation into the mechanisms of PTM and the development of targeted therapeutic strategies hold promise for advancing early diagnosis, treatment, and prognostic evaluation of GC, offering novel insights and future research directions.

OsHARBI1-1 enhances cadmium tolerance in yeast through YAP1 mediated modulation of cell wall integrity genes and catalase genes.

Harbinger transposase-derived 1 proteins (HARBI1s) play important roles in plant growth, development, and response to abiotic stress. OsHARBI1-1 has been identified as a gene encoding HARBI1-1 protein in rice and has been shown to be responsive to Cadmium (Cd) stress. However, the function of OsHARBI1-1 protein under heavy metal stress remains unclear. In this study, the function of a novel rice Cd-responsive gene, OsHARBI1-1, under Cd stress was characterized by heterologous expression in yeast. The heterologous expression OsHARBI1-1 conferred yeast with increased tolerance to Cd. In addition, the yeast cells expressing OsHARBI1-1 exhibited enhanced tolerance to Congo red and exhibited an increase in cell wall thickness under Cd stress, suggesting a potential correlation between increased Cd tolerance and cell wall thickness in the transgenic yeast. When OsHARBI1-1 was expressed in ∆yap1 or ∆yap1∆ycf1 yeast mutants, there was no significant difference in the tolerance of transgenic yeast to Cd and Congo red, as well as in cell wall thickness compared to the control. Meanwhile, the expression of cell wall integrity (CWI) genes and catalase genes in transgenic yeast was up-regulated in a YAP1-dependent manner under Cd or Congo red stress. The above facts supported the inference that OsHARBI1-1 may counteract Cd toxicity by enhancing the expression of YAP1, thereby increasing the thickness of the cell wall and activating the expression of catalase genes.

A structural genomics approach to investigate Dystrophin mutations and their impact on the molecular pathways of Duchenne muscular dystrophy

BackgroundDystrophin is a key protein encoded by the DMD gene, serves as a scaffold linking the cytoskeleton to the extracellular matrix that plays a critical role in muscle contraction, relaxation, and structural integrity. Mutations, particularly single-point amino acid substitutions, can lead to dysfunctional Dystrophin, causing muscular dystrophies, with Duchenne muscular dystrophy (DMD) being the most severe form.ObjectiveThis study aimed to evaluate the effects of 184 single-point amino acid substitutions on the structure and function of Dystrophin using computational approaches.MethodsMany computational tools were used to predict the impact of amino acid substitutions on protein stability, solubility, and function. Pathogenic potential was assessed using disease phenotype predictors and CADD scores, while allele frequency data from gnomAD contextualized mutation prevalence. Additionally, aggregation propensity, frustration analysis, and post-translational modification sites were analyzed for functional disruptions.ResultsOf the 184 substitutions analyzed, 50 were identified as deleterious, with 41 predicted to be pathogenic. Seventeen mutations were localized in the Calponin-homology (CH) 1 domain, a critical functional region of Dystrophin. Six substitutions (N26H, N26K, G47W, D98G, G109A, and G109R) were predicted to decrease protein solubility and were located in minimally frustrated regions, potentially compromising Dystrophin functionality and contributing to DMD pathogenesis.ConclusionThis study provides novel insights into the molecular mechanisms of DMD, highlighting specific mutations that disrupt Dystrophin’s solubility and function. These findings could inform future therapeutic strategies targeting Dystrophin mutations to address DMD pathogenesis.

Reaction Coordinates Are Optimal Channels of Energy Flow.

Reaction coordinates (RCs) are the few essential coordinates of a protein that control its functional processes, such as allostery, enzymatic reaction, and conformational change. They are critical for understanding protein function and provide optimal enhanced sampling of protein conformational changes and states. Since the pioneering work in the late 1990s, identifying the correct and objectively provable RCs has been a central topic in molecular biophysics and chemical physics. This review summarizes the major advances in identifying RCs over the past 25 years, focusing on methods aimed at finding RCs that meet the rigorous committor criterion, widely accepted as the true RCs. Importantly, the newly developed physics-based energy flow theory and generalized work functional method provide a general and rigorous approach for identifying true RCs, revealing their physical nature as the optimal channels of energy flow in biomolecules.

XGBoost-enhanced ensemble model using discriminative hybrid features for the prediction of sumoylation sites

Posttranslational modifications (PTMs) are essential for regulating protein localization and stability, significantly affecting gene expression, biological functions, and genome replication. Among these, sumoylation a PTM that attaches a chemical group to protein sequences—plays a critical role in protein function. Identifying sumoylation sites is particularly important due to their links to Parkinson’s and Alzheimer’s. This study introduces XGBoost-Sumo, a robust model to predict sumoylation sites by integrating protein structure and sequence data. The model utilizes a transformer-based attention mechanism to encode peptides and extract evolutionary features through the PsePSSM-DWT approach. By fusing word embeddings with evolutionary descriptors, it applies the SHapley Additive exPlanations (SHAP) algorithm for optimal feature selection and uses eXtreme Gradient Boosting (XGBoost) for classification. XGBoost-Sumo achieved an impressive accuracy of 99.68% on benchmark datasets using 10-fold cross-validation and 96.08% on independent samples. This marks a significant improvement, outperforming existing models by 10.31% on training data and 2.74% on independent tests. The model’s reliability and high performance make it a valuable resource for researchers, with strong potential for applications in pharmaceutical development.

ZEB2 Gene Pathogenic Variants Across Protein-Coding Regions and Impact on Clinical Manifestations: A Review

Mowat–Wilson syndrome (MWS) is a rare multi-system genetic disorder caused by variants in the Zinc Finger E-Box-Binding Homeobox 2 (ZEB2) gene. ZEB2 is an autosomal dominant gene containing ten exons within the canonical version transcript (Isoform: O60315-1). The ZEB2 gene encodes six functional domains and seven non-domain regions. This review provides a comprehensive summary of pathogenic variants and their associated MWS clinical characteristics, focusing on ZEB2 pathogenic variants, functional protein domains and non-domain regions with clinical features. A systematic literature search from 2001 to 2023 and of unpublished datasets found 191 individuals with reported clinical features and genotypic data. Genetic defects and clinical manifestations were examined that presumably impact on the structure and function of the ZEB2 gene, thereby causing multiple developmental defects with corresponding clinical presentation. This study found more nonsense ZEB2 variants observed within exon 8, which encodes four of the six protein domains: the CtBP-interacting domain (CID), homeodomain (HD), SMAD-binding domain (SMD or SBD) and part of the N-terminal zinc finger cluster (N-ZF), suggesting exon 8 plays a crucial role in this protein structure and function with multi-organ involvement. Exon 8 defects were found to be statistically more represented for gastrointestinal findings when compared to other exons, while frameshift defects were more often seen for the typical MWS face in non-domain protein regions. In contrast, nonsense or other types of variants in exons 3, 4 and 5 which encode only flanking non-domain regions were observed more often, compared with other exons excluding exon 8, to be specifically involved in the MWS facial gestalt, brain malformations, developmental delay and intellectual disability. Deleterious ZEB2 frameshift (45%) and nonsense (38%) gene variants were most often observed with deletions at 6% and missense at 5%. The genotype and clinical relationships in MWS can provide insights into prognosis, morbidity, clinical surveillance strategies and counseling of family members.