RESUMEN
The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.
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Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Miocitos Cardíacos/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Secuencia de Aminoácidos , Animales , Ratones , Conformación MolecularRESUMEN
We report a 45 years old woman, bedridden due to severe bone pain, back pain, multiple spontaneous fractures over 10 years. She had low serum Phosphates. We detected a swelling in her right groin and suspected tumour induced osteomalacia. Resection of the tumour led to reversal of metabolic bone disease. Patient became ambulatory within 6 weeks of tumour resection.
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Neoplasias de Tejido Conjuntivo/diagnóstico , Osteomalacia/diagnóstico , Femenino , Humanos , Persona de Mediana Edad , Neoplasias de Tejido Conjuntivo/complicaciones , Osteomalacia/etiología , FosfatosRESUMEN
Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.
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Miosina Tipo V/química , Sitios de Unión , Transporte Biológico Activo/fisiología , Humanos , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Peroxisomas/química , Peroxisomas/genética , Peroxisomas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
ß-Glucocerebrosidase (GBA/GCase) mutations leading to misfolded protein cause Gaucher's disease and are a major genetic risk factor for Parkinson's disease and dementia with Lewy bodies. The identification of small molecule pharmacological chaperones that can stabilize the misfolded protein and increase delivery of degradation-prone mutant GCase to the lysosome is a strategy under active investigation. Here, we describe the first use of fragment-based drug discovery (FBDD) to identify pharmacological chaperones of GCase. The fragment hits were identified by using X-ray crystallography and biophysical techniques. This work led to the discovery of a series of compounds that bind GCase with nM potency and positively modulate GCase activity in cells.
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Sitio Alostérico , Descubrimiento de Drogas , Glucosilceramidasa , Glucosilceramidasa/metabolismo , Glucosilceramidasa/antagonistas & inhibidores , Glucosilceramidasa/química , Humanos , Cristalografía por Rayos X , Relación Estructura-Actividad , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Glomus tumor is a painful benign neoplasm which arises from the glomus body. The glomus body is neuromyoarterial receptor responsible for regulating the peripheral skin blood flow and local temperature. The classical clinical symptoms of the glomus tumor are acute local pain, pin point tenderness and hypersensitivity to cold. The commonest location of the glomus tumor is the subungual region of the distal phalanx of the fingers and toes. We hereby describe a very rare case of extradigital glomus tumor in the interspace between the scapula and the posterior chest wall causing paroxysmal severe pain on the movement of the right shoulder. Being deep in location, the tumor was not clinically palpable or identifiable. The lesion was eventually located on MRI. Surgical resection of the tumor led to complete resolution of patient's symptoms.
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Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).
Asunto(s)
Baculoviridae/genética , Clonación Molecular , Escherichia coli , Expresión Génica , Vectores Genéticos/genética , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.
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Cromatografía de Afinidad , Cromatografía en Gel , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.
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Baculoviridae/genética , Vectores Genéticos/genética , Proteínas de la Membrana , Animales , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Células Sf9 , SpodopteraRESUMEN
This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the BacMam system. This eukaryotic expression system was selected and a screening process established in 2016 to enable production of highly challenging human integral membrane proteins (IMPs), which are a significant component of our target list. Here, we discuss our recently developed platform for identifying expression and monodispersity of IMPs from 3 mL of HEK293 cells.
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Expresión Génica , Vectores Genéticos/genética , Proteínas de la Membrana , Células HEK293 , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of approximately 40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9-20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.
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Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Clonación Molecular , Cristalografía por Rayos X , Vectores Genéticos/genética , Genómica/métodos , Humanos , Datos de Secuencia Molecular , Proteínas/genética , Proteómica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.
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Acetatos/farmacología , Benzofuranos/química , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Acetatos/química , Sitios de Unión , Línea Celular , AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/agonistas , Factores de Intercambio de Guanina Nucleótido/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura MolecularRESUMEN
P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be approximately 30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was approximately 26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G(2)-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non-small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G(1). H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G(1) with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non-small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule.
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Antineoplásicos/uso terapéutico , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Flavonas/farmacología , Fase G2/efectos de los fármacos , Neoplasias Experimentales/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cisplatino/uso terapéutico , Quinasas Ciclina-Dependientes/metabolismo , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Neoplasias Experimentales/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
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Canales Catiónicos TRPP/química , Animales , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Mutación Missense , Riñón Poliquístico Autosómico Dominante/genética , Conformación Proteica en Hélice alfa , Dominios Proteicos , Células Sf9 , Spodoptera , Canales Catiónicos TRPP/genéticaRESUMEN
In vitro and ex vivo efficacies of four series of benzo[b]thiophene-2-carboxylic acid derivatives were studied against Mycobacterium tuberculosis H37Ra (MTB). Benzo[b]thiophenes were also tested in vitro against multidrug resistant Mycobacterium tuberculosis H37Ra (MDR-MTB), and 7b was found to be highly active against A- and D-MDR-MTB/MTB (MIC ranges 2.73-22.86 µg/mL). The activity of all benzo[b]thiophenes against M. bovis BCG (BCG) was also assessed grown under aerobic and under conditions of oxygen depletion. Compounds 8c and 8g showed significant activity with MICs of 0.60 and 0.61 µg/mL against dormant BCG. The low cytotoxicity and high selectivity index data against human cancer cell lines, HeLa, Panc-1, and THP-1 indicate the potential importance of the development of benzo[b]thiophene-based 1,3-diketones and flavones as lead candidates to treat mycobacterial infections. Molecular docking studies into the active site of DprE1 (Decaprenylphosphoryl-ß-d-ribose-2'-epimerase) enzyme revealed a similar binding mode to native ligand in the crystal structure thereby helping to understand the ligand-protein interactions and establish a structural basis for inhibition of MTB. In summary, its good activity in in vitro and ex vivo model, as well as its activity against multidrug-resistant M. tuberculosis H37Ra in a potentially latent state, makes 7b an attractive drug candidate for the therapy of tuberculosis.
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OBJECTIVES: This study aimed to investigate the anticancer potential of indigocarpan (1), a pterocarpan isolated from Indigofera aspalathoides, a plant found in India which has been used in Ayurveda for centuries for the treatment of oedematous tumours. METHODS: The antiproliferative activity in a panel of four human cancer cell lines was studied. The mechanism of its antiproliferative activity in human colorectal adenocarcinoma LS174T cells was investigated in detail. KEY FINDINGS: Indigocarpan (1) showed antiproliferative activity in a panel of four human cancer cell lines with IC50 s ranging from 180 to 250 µm. Indigocarpan induces p53-dependent p21 upregulation and apoptosis in LS174T cells, upregulates p53 and p21(WAF1) protein levels, enhances cleavage of caspase-3 and downregulates cyclin D1, cyclin B1 and PCNA protein levels, indicating its role in modulating cell cycle progression. Indigocarpan also exhibited a strong antioxidative effect in LS174T cells. CONCLUSIONS: Along with the antiproliferative capacity, the strong antioxidative property of the compound makes it a promising candidate for further development as an anticancer and chemopreventive compound.
Asunto(s)
Antioxidantes/farmacología , Proliferación Celular/efectos de los fármacos , Indigofera/química , Extractos Vegetales/farmacología , Pterocarpanos/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , India , Extractos Vegetales/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We describe a case of a middle-aged woman, who presented to us with fever, anorexia, abdominal distension from a massive hepatomegaly, low hemoglobin, and acute liver failure. A liver biopsy revealed B cell non-Hodgkin's lymphoma predominantly in the sinusoids with CD10, CD20, and Bcl-2 positive on immunohistochemistry. She initially responded well to chemotherapy but succumbed 6 months later to the recurrence of disease. Sinusoidal non-Hodgkin's lymphoma of the liver should be considered in the differential diagnosis of a patient with large hepatomegaly presenting with acute liver failure.
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Hematogones, which are normal precursors of B lymphocytes in the bone marrow, may be mistaken for blast cells on flow cytometry and histology when their numbers increase. We report such a case in a 16 months old male who was unsuccessfully treated for a pre-B cell ALL on the basis of flow cytometry of the bone marrow which showed a substantial population of CD19 and CD10 expressing 'blast' cells. A diagnosis of AML M7 was made on a subsequent bone marrow biopsy in which the blast cells expressed CD61 and Factor VIII, while they were negative for CD10 and CD20. Also present were a few CD10 and CD20 expressing small lymphoid cells, which were interpreted as hematogones. This report reiterates the problem of mistaking hematogones for 'blast' cells on flow cytometry, especially in the marrow of very young children where hematogones are often prominent.
RESUMEN
Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the SGC, we opted for the Ligation-Independent Cloning (LIC) method which provides the medium throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in a 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).
Asunto(s)
Clonación Molecular/métodos , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos/genética , Humanos , Proteínas Recombinantes/aislamiento & purificación , Transformación BacterianaRESUMEN
This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 ml of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures. Although not discussed in this chapter, the same process can also be applied to integral membrane proteins (IMPs) with slight adaptations to the purification procedure.
Asunto(s)
Expresión Génica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Animales , Baculoviridae/genética , Técnicas de Cultivo Celular por Lotes , Técnicas de Cultivo de Célula , Vectores Genéticos/genética , Humanos , Control de Calidad , Proteínas Recombinantes/aislamiento & purificación , Células Sf9 , TransfecciónRESUMEN
In Chapter 4 we described the SGC process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or crystallization experiments. Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.