RESUMO
Nuclear receptor-binding SET domain-containing 2 (NSD2) plays important roles in gene regulation, largely through its ability to dimethylate lysine 36 of histone 3 (H3K36me2). Despite aberrant activity of NSD2 reported in numerous cancers, efforts to selectively inhibit the catalytic activity of this protein with small molecules have been unsuccessful to date. Here, we report the development of UNC8153, a novel NSD2-targeted degrader that potently and selectively reduces the cellular levels of both NSD2 protein and the H3K36me2 chromatin mark. UNC8153 contains a simple warhead that confers proteasome-dependent degradation of NSD2 through a novel mechanism. Importantly, UNC8153-mediated reduction of H3K36me2 through the degradation of NSD2 results in the downregulation of pathological phenotypes in multiple myeloma cells including mild antiproliferative effects in MM1.S cells containing an activating point mutation and antiadhesive effects in KMS11 cells harboring the t(4;14) translocation that upregulates NSD2 expression.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Regulação da Expressão Gênica , Linhagem Celular Tumoral , Regulação para BaixoRESUMO
Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.
Assuntos
Leucemia Mieloide Aguda , Proteína-Arginina N-Metiltransferases , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Oxirredução , Estresse Oxidativo , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Nuclear receptor-binding SET domain-containing 2 (NSD2) is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two proline-tryptophan-tryptophan-proline (PWWP) domains and five plant homeodomains (PHDs) believed to serve as chromatin reading modules. Here, we report a chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1, antagonizes PWWP1 interaction with nucleosomal H3K36me2 and selectively engages endogenous NSD2 in cells. UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 that result from translocations prevalent in multiple myeloma (MM). Mutations of other NSD2 chromatin reader domains also increase NSD2 nucleolar localization and enhance the effect of UNC6934. This chemical probe and the accompanying negative control UNC7145 will be useful tools in defining NSD2 biology.
Assuntos
Nucléolo Celular/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Sondas Moleculares/química , Domínios Proteicos , Proteínas Repressoras/metabolismo , Metilação , Mieloma Múltiplo/metabolismo , Nucleossomos/metabolismoRESUMO
Increased activity of the lysine methyltransferase NSD2 driven by translocation and activating mutations is associated with multiple myeloma and acute lymphoblastic leukemia, but no NSD2-targeting chemical probe has been reported to date. Here, we present the first antagonists that block the protein-protein interaction between the N-terminal PWWP domain of NSD2 and H3K36me2. Using virtual screening and experimental validation, we identified the small-molecule antagonist 3f, which binds to the NSD2-PWWP1 domain with a Kd of 3.4 µM and abrogates histone H3K36me2 binding to the PWWP1 domain in cells. This study establishes an alternative approach to targeting NSD2 and provides a small-molecule antagonist that can be further optimized into a chemical probe to better understand the cellular function of this protein.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Simulação por Computador , Cristalografia por Raios X , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Histona-Lisina N-Metiltransferase/efeitos dos fármacos , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Domínios Proteicos , Proteínas Repressoras/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25's catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a Tacrolimo/metabolismo , Proliferação de Células , Imunofluorescência , Humanos , Células Tumorais CultivadasRESUMO
PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.
Assuntos
Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Sondas Moleculares/farmacologia , Cristalografia por Raios X , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Células HEK293 , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/ultraestrutura , Histonas/metabolismo , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Domínios Proteicos , S-Adenosilmetionina/metabolismoRESUMO
Gene duplications increase organismal robustness by providing freedom for gene divergence or by increasing gene dosage. The yeast histone chaperones Fpr3 and Fpr4 are paralogs that can assemble nucleosomes in vitro; however, the genomic locations they target and their functional relationship is poorly understood. We refined the yeast synthetic genetic array approach to enable the functional dissection of gene paralogs. Applying this method to Fpr3 and Fpr4 uncovered redundant, cooperative, and divergent functions. While Fpr3 is uniquely involved in chromosome segregation, Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. We find that the TRAMP5 RNA exosome is critical for fitness in Δfpr3Δfpr4 yeast and leverage this information to identify an important role for Fpr4 at the 5' ends of protein coding genes. Additionally, Fpr4 and TRAMP5 negatively regulate RNAs from the nontranscribed spacers of ribosomal DNA. Yeast lacking Fpr3 and Fpr4 exhibit a genome instability phenotype at the ribosomal DNA, which implies that these histone chaperones regulate chromatin structure and DNA access at this location. Taken together. we provide genetic and transcriptomic evidence that Fpr3 and Fpr4 operate separately, cooperatively, and redundantly to regulate a variety of chromatin environments.
Assuntos
Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas de Ligação a Tacrolimo/metabolismo , Cromatina/metabolismo , DNA Espaçador Ribossômico/genética , Epistasia Genética , Exossomos/metabolismo , Genes Supressores , Instabilidade Genômica , Imunofilinas/metabolismo , Transcrição Gênica , Transcriptoma/genéticaRESUMO
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.
Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
The methylation of proteins is integral to the execution of many important biological functions, including cell signalling and transcriptional regulation. Protein methyltransferases (PMTs) are a large class of enzymes that carry out the addition of methyl marks to a broad range of substrates. PMTs are critical for normal cellular physiology and their dysregulation is frequently observed in human disease. As such, PMTs have emerged as promising therapeutic targets with several inhibitors now in clinical trials for oncology indications. The discovery of chemical inhibitors and antagonists of protein methylation signalling has also profoundly impacted our general understanding of PMT biology and pharmacology. In this review, we present general principles for drugging protein methyltransferases or their downstream effectors containing methyl-binding modules, as well as best-in-class examples of the compounds discovered and their impact both at the bench and in the clinic.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Medicina de Precisão , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/classificação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/classificação , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.
Assuntos
Antígenos CD34/biossíntese , Linhagem Celular Tumoral , Deleção Cromossômica , Inversão Cromossômica , Cromossomos Humanos Par 3/genética , Cromossomos Humanos Par 7/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda , Proteínas de Neoplasias/biossíntese , Adulto , Antígenos CD34/genética , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/patologia , Cromossomos Humanos Par 3/metabolismo , Cromossomos Humanos Par 7/metabolismo , Técnicas de Cocultura , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Neoplasias/genética , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
In this issue of Structure, Haddad et al. (2018) solve the high-resolution trimeric crystal structure of human COMPASS-like components Dpy-30 and Ash2L (2:1) to unravel an uncharacterized interaction surface required for competent H3K4 methylation in cells and clarify Dpy-30's role in the allosteric regulation of KMT2 enzymes.
Assuntos
Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Ligação a DNA , Epigênese Genética , Humanos , Metilação , Proteína de Leucina Linfoide-Mieloide , Proteínas Nucleares , Processamento de Proteína Pós-Traducional , Fatores de TranscriçãoRESUMO
Quantitative analysis of morphological changes in a cell nucleus is important for the understanding of nuclear architecture and its relationship with pathological conditions such as cancer. However, dimensionality of imaging data, together with a great variability of nuclear shapes, presents challenges for 3D morphological analysis. Thus, there is a compelling need for robust 3D nuclear morphometric techniques to carry out population-wide analysis. We propose a new approach that combines modeling, analysis, and interpretation of morphometric characteristics of cell nuclei and nucleoli in 3D. We used robust surface reconstruction that allows accurate approximation of 3D object boundary. Then, we computed geometric morphological measures characterizing the form of cell nuclei and nucleoli. Using these features, we compared over 450 nuclei with about 1,000 nucleoli of epithelial and mesenchymal prostate cancer cells, as well as 1,000 nuclei with over 2,000 nucleoli from serum-starved and proliferating fibroblast cells. Classification of sets of 9 and 15 cells achieved accuracy of 95.4% and 98%, respectively, for prostate cancer cells, and 95% and 98% for fibroblast cells. To our knowledge, this is the first attempt to combine these methods for 3D nuclear shape modeling and morphometry into a highly parallel pipeline workflow for morphometric analysis of thousands of nuclei and nucleoli in 3D.
Assuntos
Nucléolo Celular/fisiologia , Núcleo Celular/fisiologia , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Imageamento Tridimensional/métodos , Neoplasias da Próstata/patologia , Nucléolo Celular/patologia , Núcleo Celular/patologia , Humanos , Masculino , Células Tumorais CultivadasRESUMO
White-rot fungi, such as Dichomitus squalens, degrade all wood components and inhabit mixed-wood forests containing both soft- and hardwood species. In this study, we evaluated how D. squalens responded to the compositional differences in softwood [guaiacyl (G) lignin and higher mannan content] and hardwood [syringyl/guaiacyl (S/G) lignin and higher xylan content] using semi-natural solid cultures. Spruce (softwood) and birch (hardwood) sticks were degraded by D. squalens as measured by oxidation of the lignins using 2D-NMR. The fungal response as measured by transcriptomics, proteomics and enzyme activities showed a partial tailoring to wood composition. Mannanolytic transcripts and proteins were more abundant in spruce cultures, while a proportionally higher xylanolytic activity was detected in birch cultures. Both wood types induced manganese peroxidases to a much higher level than laccases, but higher transcript and protein levels of the manganese peroxidases were observed on the G-lignin rich spruce. Overall, the molecular responses demonstrated a stronger adaptation to the spruce rather than birch composition, possibly because D. squalens is mainly found degrading softwoods in nature, which supports the ability of the solid wood cultures to reflect the natural environment.
Assuntos
Basidiomycota/metabolismo , Polyporaceae/metabolismo , Madeira/química , Basidiomycota/enzimologia , Basidiomycota/genética , Betula/química , Betula/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lacase/genética , Lacase/metabolismo , Lignina/química , Lignina/metabolismo , Mananas/química , Mananas/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Picea/química , Picea/microbiologia , Madeira/microbiologiaRESUMO
FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.
Assuntos
Ciclo Celular , Microtúbulos/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Linhagem Celular , DNA/metabolismo , Instabilidade Genômica , Humanos , Mitose , Peptidilprolil Isomerase/fisiologia , Fosforilação , Polimerização , Proteína Quinase C/metabolismo , Proteínas de Ligação a Tacrolimo/fisiologiaRESUMO
Prolyl isomerases are defined by a catalytic domain that facilitates the cis-trans interconversion of proline residues. In most cases, additional domains in these enzymes add important biological function, including recruitment to a set of protein substrates. Here, we report that the N-terminal basic tilted helix bundle (BTHB) domain of the human prolyl isomerase FKBP25 confers specific binding to double-stranded RNA (dsRNA). This binding is selective over DNA as well as single-stranded oligonucleotides. We find that FKBP25 RNA-association is required for its nucleolar localization and for the vast majority of its protein interactions, including those with 60S pre-ribosome and early ribosome biogenesis factors. An independent mobility of the BTHB and FKBP catalytic domains supports a model by which the N-terminus of FKBP25 is anchored to regions of dsRNA, whereas the FKBP domain is free to interact with neighboring proteins. Apart from the identification of the BTHB as a new dsRNA-binding module, this domain adds to the growing list of auxiliary functions used by prolyl isomerases to define their primary cellular targets.
Assuntos
Conformação de Ácido Nucleico , Domínios Proteicos , Estrutura Secundária de Proteína , RNA de Cadeia Dupla/química , Proteínas de Ligação a Tacrolimo/química , Sequência de Bases , Western Blotting , Domínio Catalítico , Linhagem Celular Tumoral , Células HEK293 , Humanos , Microscopia Confocal , Modelos Moleculares , Ligação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Fluoruracila/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Interação Gene-Ambiente , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Muscle-eye-brain disease (MEB) and Walker Warburg Syndrome (WWS) belong to a spectrum of autosomal recessive diseases characterized by ocular dysgenesis, neuronal migration defects, and congenital muscular dystrophy. Until now, the pathophysiology of MEB/WWS has been attributed to alteration in dystroglycan post-translational modification. Here, we provide evidence that mutations in a gene coding for a major basement membrane protein, collagen IV alpha 1 (COL4A1), are a novel cause of MEB/WWS. Using a combination of histological, molecular, and biochemical approaches, we show that heterozygous Col4a1 mutant mice have ocular dysgenesis, neuronal localization defects, and myopathy characteristic of MEB/WWS. Importantly, we identified putative heterozygous mutations in COL4A1 in two MEB/WWS patients. Both mutations occur within conserved amino acids of the triple-helix-forming domain of the protein, and at least one mutation interferes with secretion of the mutant proteins, resulting instead in intracellular accumulation. Expression and posttranslational modification of dystroglycan is unaltered in Col4a1 mutant mice indicating that COL4A1 mutations represent a distinct pathogenic mechanism underlying MEB/WWS. These findings implicate a novel gene and a novel mechanism in the etiology of MEB/WWS and expand the clinical spectrum of COL4A1-associated disorders.