RESUMO
RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 µM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.
Assuntos
Neoplasias , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Linhagem Celular , Neoplasias/genética , Epigênese Genética , Proteínas Repressoras/genéticaRESUMO
Although aspartic intramembrane-cleaving proteases (I-CLIPs) are crucial switches of multiple signaling pathways and involved in several devastating diseases, little is known about their physiological regulation. We have recently identified Frey regulator of sperm-oocyte fusion 1 (Frey1) as an inhibitory protein of Signal Peptide Peptidase-like 2c (SPPL2c), a member of this protease family. Employing structure modeling along with cell-based inhibition and interaction studies, we identify a short motif within the Frey1 transmembrane domain essential for inhibition of SPPL2c. Intriguingly, this motif can be transplanted to the SPPL2c substrate PLN, thereby transforming it into an inhibitor of this enzyme. It can be adopted for the generation of Notch1-based γ-Secretase inhibitors demonstrating its versatile use among aspartic I-CLIPs. In summary, we describe a mechanism of aspartic I-CLIP inhibition which allows the targeted generation of specific inhibitors of these enzymes and might enable the identification of endogenous negative regulators of these enzymes.
Assuntos
Proteínas de Membrana , Sêmen , Masculino , Animais , Proteínas de Membrana/metabolismo , Proteólise , Sêmen/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Peptídeo Hidrolases/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismoRESUMO
Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.
Assuntos
Furões , Proteínas Hedgehog , Animais , Feminino , Humanos , Camundongos , Gravidez , Sistema Nervoso Central/metabolismo , Cílios/genética , Cílios/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Camundongos Knockout , Transdução de SinaisRESUMO
Interleukin-27 (IL-27) uniquely assembles p28 and EBI3 subunits to a heterodimeric cytokine that signals via IL-27Rα and gp130. To provide the structural framework for receptor activation by IL-27 and its emerging therapeutic targeting, we report here crystal structures of mouse IL-27 in complex with IL-27Rα and of human IL-27 in complex with SRF388, a monoclonal antibody undergoing clinical trials with oncology indications. One face of the helical p28 subunit interacts with EBI3, while the opposite face nestles into the interdomain elbow of IL-27Rα to juxtapose IL-27Rα to EBI3. This orients IL-27Rα for paired signaling with gp130, which only uses its immunoglobulin domain to bind to IL-27. Such a signaling complex is distinct from those mediated by IL-12 and IL-23. The SRF388 binding epitope on IL-27 overlaps with the IL-27Rα interaction site explaining its potent antagonistic properties. Collectively, our findings will facilitate the mechanistic interrogation, engineering, and therapeutic targeting of IL-27.
Assuntos
Interleucina-27 , Humanos , Camundongos , Animais , Receptor gp130 de Citocina/metabolismo , Receptores de Citocinas/metabolismo , Interleucina-12 , Citocinas , Anticorpos Monoclonais/farmacologia , Epitopos , Interleucina-23RESUMO
Kinesins are canonical molecular motors but can also function as modulators of intracellular signaling. KIF26A, an unconventional kinesin that lacks motor activity, inhibits growth-factor-receptor-bound protein 2 (GRB2)- and focal adhesion kinase (FAK)-dependent signal transduction, but its functions in the brain have not been characterized. We report a patient cohort with biallelic loss-of-function variants in KIF26A, exhibiting a spectrum of congenital brain malformations. In the developing brain, KIF26A is preferentially expressed during early- and mid-gestation in excitatory neurons. Combining mice and human iPSC-derived organoid models, we discovered that loss of KIF26A causes excitatory neuron-specific defects in radial migration, localization, dendritic and axonal growth, and apoptosis, offering a convincing explanation of the disease etiology in patients. Single-cell RNA sequencing in KIF26A knockout organoids revealed transcriptional changes in MAPK, MYC, and E2F pathways. Our findings illustrate the pathogenesis of KIF26A loss-of-function variants and identify the surprising versatility of this non-motor kinesin.
Assuntos
Cinesinas , Neurônios , Humanos , Animais , Camundongos , Cinesinas/genética , Neurônios/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Apoptose , Encéfalo/metabolismoRESUMO
Paracrine cell-cell communication is central to all developmental processes, ranging from cell diversification to patterning and morphogenesis. Precise calibration of signaling strength is essential for the fidelity of tissue formation during embryogenesis and tissue maintenance in adults. Membrane-tethered ubiquitin ligases can control the sensitivity of target cells to secreted ligands by regulating the abundance of signaling receptors at the cell surface. We discuss two examples of this emerging concept in signaling: (1) the transmembrane ubiquitin ligases ZNRF3 and RNF43 that regulate WNT and bone morphogenetic protein receptor abundance in response to R-spondin ligands and (2) the membrane-recruited ubiquitin ligase MGRN1 that controls Hedgehog and melanocortin receptor abundance. We focus on the mechanistic logic of these systems, illustrated by structural and protein interaction models enabled by AlphaFold. We suggest that membrane-tethered ubiquitin ligases play a widespread role in remodeling the cell surface proteome to control responses to extracellular ligands in diverse biological processes.
Assuntos
Trombospondinas , Via de Sinalização Wnt , Proteínas Hedgehog , Homeostase , Ligantes , Trombospondinas/química , Trombospondinas/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGß and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.
Assuntos
Quinase do Linfoma Anaplásico/química , Quinase do Linfoma Anaplásico/metabolismo , Citocinas/química , Citocinas/metabolismo , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico/classificação , Quinase do Linfoma Anaplásico/genética , Sítios de Ligação , Ativação Enzimática , Fator de Crescimento Epidérmico/química , Glicina , Células HEK293 , Humanos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Especificidade por SubstratoRESUMO
Birth defects result from interactions between genetic and environmental factors, but the mechanisms remain poorly understood. We find that mutations and teratogens interact in predictable ways to cause birth defects by changing target cell sensitivity to Hedgehog (Hh) ligands. These interactions converge on a membrane protein complex, the MMM complex, that promotes degradation of the Hh transducer Smoothened (SMO). Deficiency of the MMM component MOSMO results in elevated SMO and increased Hh signaling, causing multiple birth defects. In utero exposure to a teratogen that directly inhibits SMO reduces the penetrance and expressivity of birth defects in Mosmo-/- embryos. Additionally, tissues that develop normally in Mosmo-/- embryos are refractory to the teratogen. Thus, changes in the abundance of the protein target of a teratogen can change birth defect outcomes by quantitative shifts in Hh signaling. Consequently, small molecules that re-calibrate signaling strength could be harnessed to rescue structural birth defects.
Assuntos
Anormalidades Induzidas por Medicamentos/genética , Interação Gene-Ambiente , Proteínas Hedgehog/metabolismo , Penetrância , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Transdução de Sinais , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMO
Royal jelly is the queen-maker for the honey bee Apis mellifera, and has cross-species effects on longevity, fertility, and regeneration in mammals. Despite this knowledge, how royal jelly or its components exert their myriad effects has remained poorly understood. Using mouse embryonic stem cells as a platform, here we report that through its major protein component Royalactin, royal jelly can maintain pluripotency by activating a ground-state pluripotency-like gene network. We further identify Regina, a mammalian structural analog of Royalactin that also induces a naive-like state in mouse embryonic stem cells. This reveals an important innate program for stem cell self-renewal with broad implications in understanding the molecular regulation of stem cell fate across species.
Assuntos
Ácidos Graxos/farmacologia , Glicoproteínas/farmacologia , Proteínas de Insetos/farmacologia , Mamíferos/fisiologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Abelhas/metabolismo , Cromatina , Ácidos Graxos/química , Feminino , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glicoproteínas/química , Proteínas de Insetos/química , Lentivirus/genética , Lentivirus/metabolismo , Longevidade , Camundongos , Modelos Moleculares , Proteínas Recombinantes , Teratoma/patologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins.
Assuntos
Biologia Computacional/métodos , Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas/genética , Alinhamento de SequênciaRESUMO
Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.
Assuntos
Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Aminoácidos/metabolismo , Animais , Sítios de Ligação , Sequência Conservada , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Ligantes , Camundongos , Modelos Moleculares , Células NIH 3T3 , Receptores Patched , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptor Smoothened , Esteróis/química , Relação Estrutura-Atividade , Proteínas Wnt/metabolismoRESUMO
We report that the metazoan Wnt protease and signaling inhibitor TIKI shares sequence homology with bacterial TraB/PrgY proteins, inhibitors of pheromone signaling essential for propagation of antibiotic resistance. Our analysis suggests that these proteins represent an ancient metalloprotease clan regulating cellular communications across biological kingdoms.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Metaloproteases/metabolismo , Dobramento de Proteína , Proteínas Wnt/metabolismo , Animais , Domínio Catalítico , Comunicação Celular , Farmacorresistência Bacteriana/genética , Feromônios/antagonistas & inibidores , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Homologia de Sequência de AminoácidosRESUMO
Polo-like kinases (PLKs) are marked by C-terminal polo box modules with critical protein interaction and subcellular targeting roles. Slevin et al. in this issue of Structure reveal the architecture of a hidden set of polo boxes from the divergent PLK4, a critical player in centrosome duplication, shedding new light on the evolution of PLKs and their functionally related kinase ZYG-1.
Assuntos
Centríolos , Proteínas Serina-Treonina Quinases/química , HumanosRESUMO
Mutations in the renal tumour suppressor protein, folliculin, lead to proliferative skin lesions, lung complications and renal cell carcinoma. Folliculin has been reported to interact with AMP-activated kinase, a key component of the mammalian target of rapamycin pathway. Most cancer-causing mutations lead to a carboxy-terminal truncation of folliculin, pointing to a functional importance of this domain in tumour suppression. We present here the crystal structure of folliculin carboxy-terminal domain and demonstrate that it is distantly related to differentially expressed in normal cells and neoplasia (DENN) domain proteins, a family of Rab guanine nucleotide exchange factors (GEFs). Using biochemical analysis, we show that folliculin has GEF activity, indicating that folliculin is probably a distantly related member of this class of Rab GEFs.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Fatores de Troca do Nucleotídeo Guanina/química , Sequência de Aminoácidos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Cristalografia por Raios X , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Eletroforese em Gel de Poliacrilamida , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismoRESUMO
The crystal structure of a Wnt morphogen bound to its Frizzled receptor ectodomain provides insights into the evolutionary provenance of this complex fold and offers an explanation for why Wnts utilize both lipid- and protein-mediated contacts to engage Frizzleds.
Assuntos
Proteínas Wnt/química , Animais , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Humanos , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Wnt/metabolismoRESUMO
TLRs are a family of pattern recognition receptors that recognize conserved molecular structures/products from a wide variety of microbes. Following recognition of ligands, TLRs recruit signaling adapters to initiate a pro-inflammatory signaling cascade culminating in the activation of several transcription factor families. Additionally, TLR signals lead to activation of PI3K, affecting many aspects of the cellular response, including cell survival, proliferation and regulation of the pro-inflammatory response. The recent discovery of BCAP as a TLR signaling adaptor, crucial for linking TLRs to PI3K activation, allows new questions of the importance of PI3K activation downstream of TLRs. Here, we summarize the current understanding of signaling pathways activated by TLRs and provide our perspective on TLR mediated activation of PI3K and its impact on regulating cellular processes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inflamação/enzimologia , Inflamação/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Humanos , Modelos BiológicosRESUMO
Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.
Assuntos
Anticorpos Neutralizantes/química , Interleucinas/química , Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Homologia Estrutural de Proteína , Baculoviridae , Sítios de Ligação , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucinas/antagonistas & inibidores , Interleucinas/genética , Cinética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Fator Estimulador de Colônias de Macrófagos/genética , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-kit/química , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transdução de Sinais/genética , Fator de Células-Tronco/química , TermodinâmicaRESUMO
Nectins (nectin1-4) and Necls [nectin-like (Necl1-5)] are Ig superfamily cell adhesion molecules that regulate cell differentiation and tissue morphogenesis. Adherens junction formation and subsequent cell-cell signaling is initiated by the assembly of higher-order receptor clusters of cognate molecules on juxtaposed cells. However, the structural and mechanistic details of signaling cluster formation remain unclear. Here, we report the crystal structure of poliovirus receptor (PVR)/Nectin-like-5/CD155) in complex with its cognate immunoreceptor ligand T-cell-Ig-and-ITIM-domain (TIGIT). The TIGIT/PVR interface reveals a conserved specific "lock-and-key" interaction. Notably, two TIGIT/PVR dimers assemble into a heterotetramer with a core TIGIT/TIGIT cis-homodimer, each TIGIT molecule binding one PVR molecule. Structure-guided mutations that disrupt the TIGIT/TIGIT interface limit both TIGIT/PVR-mediated cell adhesion and TIGIT-induced PVR phosphorylation in primary dendritic cells. Our data suggest a cis-trans receptor clustering mechanism for cell adhesion and signaling by the TIGIT/PVR complex and provide structural insights into how the PVR family of immunoregulators function.
Assuntos
Adesão Celular , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Transdução de Sinais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Receptores Imunológicos/químicaRESUMO
Interleukin-1 (IL-1)-family cytokines are mediators of innate and adaptive immunity. They exert proinflammatory effects by binding a primary receptor that recruits a receptor accessory protein to form a signaling-competent heterotrimeric complex. Here we present the crystal structure of IL-1ß bound to its primary receptor IL-1RI and its receptor accessory protein IL-1RAcP, providing insight into how IL-1-type cytokines initiate signaling and revealing an evolutionary relationship with the fibroblast growth factor receptor family.