Heparin is essential for optimal cell signaling by FGF21 and for regulation of ßKlotho cellular stability.

While important insights were gained about how FGF21 and other endocrine fibroblast growth factors (FGFs) bind to Klotho proteins, the exact mechanism of Klotho/FGF receptor assembly that drives receptor dimerization and activation has not been elucidated. The prevailing dogma is that Klotho proteins substitute for the loss of heparan sulfate proteoglycan (HSPG) binding to endocrine FGFs by high-affinity binding of endocrine FGF molecules to Klotho receptors. To explore a potential role of HSPG in FGF21 signaling, we have analyzed the dynamic properties of FGF21-induced FGF21-ßKlotho-FGFR1c complexes on the surface of living wild-type (WT) or HSPG-deficient Chinese hamster ovary (CHO) cells by employing quantitative single-molecule fluorescence imaging analyses. Moreover, detailed analyses of FGF21 and FGF1 stimulation of cellular signaling pathways activated in WT or in HSPG-deficient CHO cells are also analyzed and compared. These experiments demonstrate that heparin is required for the formation of FGF21-ßKlotho-FGFR1c complexes on the cell membrane and that binding of heparin or HSPG to FGFR1c is essential for optimal FGF21 stimulation of FGFR1c activation, mitogen-activated protein kinase responses, and intracellular Ca2+ release. It is also shown that FGF1 binding stimulates assembly of ßKlotho and FGFR1c on cell membranes, resulting in endocytosis and degradation of ßKlotho. We conclude that heparin or HSPG is essential for FGF21 signaling and for regulation of ßKlotho cellular stability by acting as a coligand of FGFR1c.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Structures of ß-klotho reveal a 'zip code'-like mechanism for endocrine FGF signalling.

Canonical fibroblast growth factors (FGFs) activate FGF receptors (FGFRs) through paracrine or autocrine mechanisms in a process that requires cooperation with heparan sulfate proteoglycans, which function as co-receptors for FGFR activation. By contrast, endocrine FGFs (FGF19, FGF21 and FGF23) are circulating hormones that regulate critical metabolic processes in a variety of tissues. FGF19 regulates bile acid synthesis and lipogenesis, whereas FGF21 stimulates insulin sensitivity, energy expenditure and weight loss. Endocrine FGFs signal through FGFRs in a manner that requires klothos, which are cell-surface proteins that possess tandem glycosidase domains. Here we describe the crystal structures of free and ligand-bound ß-klotho extracellular regions that reveal the molecular mechanism that underlies the specificity of FGF21 towards ß-klotho and demonstrate how the FGFR is activated in a klotho-dependent manner. ß-Klotho serves as a primary 'zip code'-like receptor that acts as a targeting signal for FGF21, and FGFR functions as a catalytic subunit that mediates intracellular signalling. Our structures also show how the sugar-cutting enzyme glycosidase has evolved to become a specific receptor for hormones that regulate metabolic processes, including the lowering of blood sugar levels. Finally, we describe an agonistic variant of FGF21 with enhanced biological activity and present structural insights into the potential development of therapeutic agents for diseases linked to endocrine FGFs.

The strength and cooperativity of KIT ectodomain contacts determine normal ligand-dependent stimulation or oncogenic activation in cancer.

The receptor tyrosine kinase KIT plays an important role in development of germ cells, hematopoietic cells, and interstitial pacemaker cells. Oncogenic KIT mutations play an important "driver" role in gastrointestinal stromal tumors, acute myeloid leukemias, and melanoma, among other cancers. Here we describe the crystal structure of a recurring somatic oncogenic mutation located in the C-terminal Ig-like domain (D5) of the ectodomain, rendering KIT tyrosine kinase activity constitutively activated. The structural analysis, together with biochemical and biophysical experiments and detailed analyses of the activities of a variety of oncogenic KIT mutations, reveals that the strength of homotypic contacts and the cooperativity in the action of D4D5 regions determines whether KIT is normally regulated or constitutively activated in cancers. We propose that cooperative interactions mediated by multiple weak homotypic contacts between receptor molecules are responsible for regulating normal ligand-dependent or oncogenic RTK activation via a "zipper-like" mechanism for receptor activation.

FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho.

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

Structures of ligand-occupied ß-Klotho complexes reveal a molecular mechanism underlying endocrine FGF specificity and activity.

The three members of the endocrine fibroblast growth factor (FGF) family designated FGF19, FGF21, and FGF23 mediate their pleiotropic cellular effects by binding to and activating binary complexes composed of an FGF receptor (FGFR) bound to either α-Klotho or ß-Klotho receptors. Structural analyses of ligand-occupied Klotho extracellular domains have provided important insights concerning mechanisms underlying the binding specificities of FGF21 and FGF23 to ß-Klotho or α-Klotho, respectively. They have also demonstrated that Klotho proteins function as primary high-affinity receptors while FGFRs function as the catalytic subunits that mediate intracellular signaling. Here we describe the crystal structure the C-terminal tail of FGF19 (FGF19CT) bound to sKLB and demonstrate that FGF19CT and FGF21CT bind to the same binding site on sKLB, via a multiturn D-P motif to site 1 and via a S-P-S motif to the pseudoglycoside hydrolase region (site 2). Binding affinities to sKLB and cellular stimulatory activities of FGF19CT, FGF21CT, and a variety of chimeric mutants to cells expressing ß-Klotho together with FGFR1c or FGFR4 were also analyzed. These experiments as well as detailed comparison of the structures of free and ligand-occupied sKLB to the structure of ligand-occupied sKLA reveal a general mechanism for recognition of endocrine FGFs by Klotho proteins and regulatory interactions with FGFRs that control their pleiotropic cellular responses.

Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α).

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,ß) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions.

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants.

Augmentor α and ß (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand-receptor interactions.

Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-ß) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states.

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers.

Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT.

Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region.

Somatic oncogenic mutations in the receptor tyrosine kinase KIT function as major drivers of gastrointestinal stromal tumors and a subset of acute myeloid leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression of disease eventually occurs in virtually all patients. In this report, we describe inhibitory KIT antibodies that bind to the membrane-proximal Ig-like D4 of KIT with significant overlap with an epitope in D4 that mediates homotypic interactions essential for KIT activation. Crystal structures of the anti-KIT antibody in complex with KIT D4 and D5 allowed design of affinity-matured libraries that were used to isolate variants with increased affinity and efficacy. Isolated antibodies showed KIT inhibition together with suppression of cell proliferation driven by ligand-stimulated WT or constitutively activated oncogenic KIT mutant. These antibodies represent a unique therapeutic approach and a step toward the development of "naked" or toxin-conjugated KIT antibodies for the treatment of KIT-driven cancers.

Asymmetric receptor contact is required for tyrosine autophosphorylation of fibroblast growth factor receptor in living cells.

Tyrosine autophosphorylation of receptor tyrosine kinases plays a critical role in regulation of kinase activity and in recruitment and activation of intracellular signaling pathways. Autophosphorylation is mediated by a sequential and precisely ordered intermolecular (trans) reaction. In this report we present structural and biochemical experiments demonstrating that formation of an asymmetric dimer between activated FGFR1 kinase domains is required for transphosphorylation of FGFR1 in FGF-stimulated cells. Transphosphorylation is mediated by specific asymmetric contacts between the N-lobe of one kinase molecule, which serves as an active enzyme, and specific docking sites on the C-lobe of a second kinase molecule, which serves a substrate. Pathological loss-of-function mutations or oncogenic activating mutations in this interface may hinder or facilitate asymmetric dimer formation and transphosphorylation, respectively. The experiments presented in this report provide the molecular basis underlying the control of transphosphorylation of FGF receptors and other receptor tyrosine kinases.

Implantação de um serviço para pessoas com distúrbios do assoalho pélvico / Implementation of a service for people with pelvic floor disorders / Implementación de un servicio para personas con trastornos del suelo pélvico

Objetivo:objetivou-se descrever os procedimentos técnicos operacionais e dados clínicos relacionados à implantação de um programa de atenção à saúde das pessoas com distúrbios do assoalho pélvico em um serviço público de atenção secundária. Método: trata-se de um relato de experiência, baseado em vivências relacionadas à assistência acadêmico-profissional na implantação de serviço voltado aos distúrbios do assoalho pélvico na região do Cariri cearense, realizado de maio a julho de 2021. Resultados: para a implantação do serviço, adotaram-se as seguintes estratégias: rastreamento da rede de atenção à saúde da pessoa com distúrbios do assoalho pélvico; estruturação organizacional do serviço; captação de pessoas com disfunções pélvicas; início dos atendimentos; e seguimento terapêutico. Conclusão: face ao exposto, evidencia-se que o programa de atenção à saúde das pessoas com distúrbios do assoalho pélvico pôde ser implantado satisfatoriamente, tendo em vista a infraestrutura, ao expressivo quantitativo de atendimentos realizados e ao seguimento terapêutico alcançado. Assim, com este relato, espera-se contribuir para o desenvolvimento de novos serviços ambulatoriais voltados a essa área de atuação do enfermeiro estomaterapeuta e da equipe multidisciplinar.

Objective:the objective was to describe the technical operational procedures and clinical data related to the implementation of a health care program for people with pelvic floor disorders in a public secondary care service. Method: this is an experience report, based on experiences related to academic and professional assistance in the implementation of a service aimed at pelvic floor disorders in the Cariri region of Ceará, carried out from May to July 2021. Results: for the implementation of the service, the following strategies were adopted: tracking the health care network for people with pelvic floor disorders; organizational structuring of the service; capturing people with pelvic dysfunctions; start of care; and therapeutic follow-up. Conclusion: in view of the above, it is evident that the health care program for people with pelvic floor disorders could be implemented satisfactorily, considering the infrastructure, the significant amount of care provided and the therapeutic follow-up achieved. Thus, with this report, it is expected to contribute to the development of new outpatient services aimed at this area of work of the stomatherapist nurse and the multidisciplinary team.

Objetivo:El objetivo es describir los procedimientos técnicos operativos y datos clínicos relacionados a la implementación de un programa de atención a la salud de las personas con trastornos del suelo pélvico en un servicio público de atención secundaria. Método: se trata de un reporte de experiencia, basado en vivencias relacionadas a la asistencia académico-profesional en la implementación de servicio destinado a los trastornos del suelo pélvico en la región del Cariri cearense, realizado de mayo a julio de 2021. Resultados: para la implementación del servicio, se adoptaron las siguientes estrategias: rastreo de la red de atención a la salud de personas con trastornos del suelo pélvico; estructuración organizacional del servicio; captación de personas con disfunciones pélvicas; inicio de la atención; y seguimiento terapéutico. Conclusión: frente a lo expuesto, queda evidente que el programa de atención a la salud de las personas con trastornos del suelo pélvico puede ser implementado satisfactoriamente, teniendo en cuenta la infraestructura, el importante número de atenciones realizadas y al seguimiento terapéutico alcanzado. Así, con este informe, se espera contribuir al desarrollo de nuevos servicios ambulatorios destinados a esta área de trabajo del enfermero estomaterapeuta y del equipo multidisciplinario.

Tuberculosis cavitaria en un lactante con meningitis: report de un caso / Cavitary tuberculosis in an infant with meningitis: case report

Se efectúa la descripción del caso de un lactante de 8 meses, quién presentó tuberculosis cavitária acompañada de Meningitis Tuberculosa. Posteriormente se hace una revisión de los aspectos clínicos y métodos de estudio diagnóstico actuales, enfatizando en la importancia que tienen los antecedentes epidemiológicos, para identificar la enfermedad

Mortalidad infantil en población huichol del estado de Jalisco / Infant mortality among the huichol population in the state of Jalisco

Introducción. La mortalidad infantil supone un importante problema de salud en los países desarrollados, especialmente entre los sectores marginados de los mismos, como son los grupos indígenas. Desgraciadamente, el subregistro y el manejo de cifras globales de mortalidad, impiden conocer la dimensión del problema en estos grupos específicos. El objetivo de este estudio fue conocer la magnitud de la mortalidad infantil entre la población huichola del estado de Jalisco. Material y métodos. Se realizó un estudio de tipo observacional descriptivo en población huichola del estado de Jalisco. Material y métodos. Se realizó un estudio de tipo observacional descriptivo en población huichola del estado de Jalisco. La información recabada se refirió al período comprendido entre agosto de 1990 y agosto de 1991 (un año). La información sobre mortalidad infantil se recogió a través de un cuestionario que se aplicó a 349 familias hicholas . Dicho cuestionario incluyó preguntas de identificación, número de nacidos durante el período estudiado, número de nacidos vivos que murieron antes del año de edad en el mismo período, sexo de los niños fallecidos y causa de muerte. Resultados. Los resultados informaron 208 niños nacidos vivos y 27 fallecimientos de niños menores de 1 año: 15 menores de 28 días y 12 de 28 hasta 364 días. La tasa de mortalidad infantil fue de 129.8 por 1000 nacidos vivos; las tasas de mortalidad neonatal y postneonatal fueron de 72.1 y 57.7 por 1000 nacidos vivos respectivamente. Dichas tasas resultaron más elevadas que las correspondientes a México y otros países del área panamericana. Las causas de las muertes fueron, en orden decreciente: infecciones, causa desconocida, síndrome de dificultad respiratoria, nacimiento pretérmino, bajo peso al nacer y deficiencias de la nutrición. Conclusiones. Existe una elevada mortalidad infantil entre la población huichol, lo cual prodría relacionarse con factores sociales, culturales y de servicios presentes en la zona. Asimismo, la elevada mortalidad infantil encontrada señala a la población huichola como una de las más desprotegidas de México y es reflejo de las considerables diferencias que, en materia de salud, persisten en el país