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1.
Cell ; 152(1-2): 210-23, 2013 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-23332756

RESUMO

The primary cilium is required for Sonic hedgehog (Shh) signaling in vertebrates. In contrast to mutants affecting ciliary assembly, mutations in the intraflagellar transport complex A (IFT-A) paradoxically cause increased Shh signaling. We previously showed that the IFT-A complex, in addition to its canonical role in retrograde IFT, binds to the tubby-like protein, Tulp3, and recruits it to cilia. Here, we describe a conserved vertebrate G-protein-coupled receptor, Gpr161, which localizes to primary cilia in a Tulp3/IFT-A-dependent manner. Complete loss of Gpr161 in mouse causes midgestation lethality and increased Shh signaling in the neural tube, phenocopying Tulp3/IFT-A mutants. Constitutive Gpr161 activity increases cAMP levels and represses Shh signaling by determining the processing of Gli3 to its repressor form. Conversely, Shh signaling directs Gpr161 to be internalized from cilia, preventing its activity. Thus, Gpr161 defines a morphogenetic pathway coupling protein kinase A activation to Shh signaling during neural tube development.


Assuntos
Cílios/metabolismo , Embrião de Mamíferos/metabolismo , Tubo Neural/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Dados de Sequência Molecular , Filogenia , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Alinhamento de Sequência
2.
Cell ; 145(4): 513-28, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21565611

RESUMO

Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.


Assuntos
Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Transdução de Sinais , Animais , Ataxina-10 , Centrossomo/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Proteínas Hedgehog/metabolismo , Humanos , Doenças Renais Císticas/metabolismo , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Doenças Renais Policísticas/genética , Retinose Pigmentar , Peixe-Zebra
3.
PLoS Genet ; 19(8): e1010609, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37585454

RESUMO

Diabetic retinopathy (DR) is a common complication of diabetes. Approximately 20% of DR patients have diabetic macular edema (DME) characterized by fluid leakage into the retina. There is a genetic component to DR and DME risk, but few replicable loci. Because not all DR cases have DME, we focused on DME to increase power, and conducted a multi-ancestry GWAS to assess DME risk in a total of 1,502 DME patients and 5,603 non-DME controls in discovery and replication datasets. Two loci reached GWAS significance (p<5x10-8). The strongest association was rs2239785, (K150E) in APOL1. The second finding was rs10402468, which co-localized to PLVAP and ANKLE1 in vascular / endothelium tissues. We conducted multiple sensitivity analyses to establish that the associations were specific to DME status and did not reflect diabetes status or other diabetic complications. Here we report two novel loci for risk of DME which replicated in multiple clinical trial and biobank derived datasets. One of these loci, containing the gene APOL1, is a risk factor in African American DME and DKD patients, indicating that this locus plays a broader role in diabetic complications for multiple ancestries. Trial Registration: NCT00473330, NCT00473382, NCT03622580, NCT03622593, NCT04108156.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Edema Macular , Humanos , Edema Macular/genética , Edema Macular/complicações , Retinopatia Diabética/genética , Retinopatia Diabética/complicações , Estudo de Associação Genômica Ampla , Apolipoproteína L1/genética , Fatores de Risco
4.
Nat Chem Biol ; 19(1): 55-63, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36577875

RESUMO

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.


Assuntos
Proteínas Nucleares , Fatores de Transcrição , Proteínas Nucleares/metabolismo , Microscopia Crioeletrônica , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ligases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
5.
J Am Soc Nephrol ; 31(9): 2065-2082, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32764138

RESUMO

BACKGROUND: Circulating APOL1 lyses trypanosomes, protecting against human sleeping sickness. Two common African gene variants of APOL1, G1 and G2, protect against infection by species of trypanosomes that resist wild-type APOL1. At the same time, the protection predisposes humans to CKD, an elegant example of balanced polymorphism. However, the exact mechanism of APOL1-mediated podocyte damage is not clear, including APOL1's subcellular localization, topology, and whether the damage is related to trypanolysis. METHODS: APOL1 topology in serum (HDL particles) and in kidney podocytes was mapped with flow cytometry, immunoprecipitation, and trypanolysis assays that tracked 170 APOL1 domain-specific monoclonal antibodies. APOL1 knockout podocytes confirmed antibody specificity. RESULTS: APOL1 localizes to the surface of podocytes, with most of the pore-forming domain (PFD) and C terminus of the Serum Resistance Associated-interacting domain (SRA-ID), but not the membrane-addressing domain (MAD), being exposed. In contrast, differential trypanolytic blocking activity reveals that the MAD is exposed in serum APOL1, with less of the PFD accessible. Low pH did not detectably alter the gross topology of APOL1, as determined by antibody accessibility, in serum or on podocytes. CONCLUSIONS: Our antibodies highlighted different conformations of native APOL1 topology in serum (HDL particles) and at the podocyte surface. Our findings support the surface ion channel model for APOL1 risk variant-mediated podocyte injury, as well as providing domain accessibility information for designing APOL1-targeted therapeutics.


Assuntos
Apolipoproteína L1/análise , Membrana Celular/química , Podócitos/química , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Apolipoproteína L1/sangue , Apolipoproteína L1/química , Apolipoproteína L1/imunologia , Células CHO , Cricetulus , Humanos , Concentração de Íons de Hidrogênio , Podócitos/ultraestrutura , Domínios Proteicos
6.
J Am Soc Nephrol ; 31(9): 2044-2064, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32764142

RESUMO

BACKGROUND: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. METHODS: Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. RESULTS: Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. CONCLUSIONS: APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.


Assuntos
Apolipoproteína L1/análise , Membrana Celular/química , Retículo Endoplasmático/química , Podócitos/química , Animais , Anticorpos/imunologia , Apolipoproteína L1/imunologia , Apolipoproteínas L/análise , Células COS , Células Cultivadas , Chlorocebus aethiops , Reações Cruzadas , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Podócitos/ultraestrutura
7.
Bioconjug Chem ; 30(12): 3046-3056, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31726009

RESUMO

Disulfide-linked bioconjugates allow the delivery of pharmacologically active or other cargo to specific tissues in a redox-sensitive fashion. However, an understanding of the kinetics, subcellular distribution, and mechanism of disulfide cleavage in such bioconjugates is generally lacking. Here, we report a modular disulfide-linked TAMRA-BODIPY based FRET probe that can be readily synthesized, modified, and conjugated to a cysteine-containing biomolecule to enable real-time monitoring of disulfide cleavage during receptor-mediated endocytosis in cells. We demonstrate the utility of this probe to study disulfide reduction during HER2 receptor-mediated uptake of a Cys-engineered anti-HER2 THIOMAB antibody. We found that introduction of positive, but not negative, charges in the probe improved retention of the BODIPY catabolite. This permitted the observation of significant disulfide cleavage in endosomes or lysosomes on par with proteolytic cleavage of a similarly charged valine-citrulline peptide-based probe. In general, the FRET probe we describe should enable real-time cellular monitoring of disulfide cleavage in other targeted delivery systems for mechanistic or diagnostic applications. Furthermore, modifications to the released BODIPY moiety permit evaluation of physicochemical properties that govern lysosomal egress or retention, which may have implications for the development of next-generation antibody-drug conjugates.


Assuntos
Cisteína/química , Dissulfetos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Animais , Compostos de Boro , Monitoramento de Medicamentos/métodos , Endocitose , Endossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência/tendências , Humanos , Imunoconjugados , Lisossomos/metabolismo , Receptor ErbB-2/imunologia , Rodaminas
8.
Genes Dev ; 24(19): 2180-93, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20889716

RESUMO

Primary cilia function as a sensory signaling compartment in processes ranging from mammalian Hedgehog signaling to neuronal control of obesity. Intraflagellar transport (IFT) is an ancient, conserved mechanism required to assemble cilia and for trafficking within cilia. The link between IFT, sensory signaling, and obesity is not clearly defined, but some novel monogenic obesity disorders may be linked to ciliary defects. The tubby mouse, which presents with adult-onset obesity, arises from mutation in the Tub gene. The tubby-like proteins comprise a related family of poorly understood proteins with roles in neural development and function. We find that specific Tubby family proteins, notably Tubby-like protein 3 (TULP3), bind to the IFT-A complex. IFT-A is linked to retrograde ciliary transport, but, surprisingly, we find that the IFT-A complex has a second role directing ciliary entry of TULP3. TULP3 and IFT-A, in turn, promote trafficking of a subset of G protein-coupled receptors (GPCRs), but not Smoothened, to cilia. Both IFT-A and membrane phosphoinositide-binding properties of TULP3 are required for ciliary GPCR localization. TULP3 and IFT-A proteins both negatively regulate Hedgehog signaling in the mouse embryo, and the TULP3-IFT-A interaction suggests how these proteins cooperate during neural tube patterning.


Assuntos
Cílios/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Alinhamento de Sequência
9.
Nucleic Acids Res ; 43(2): 1189-203, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25550431

RESUMO

Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.


Assuntos
Anticorpos , RNA Interferente Pequeno/administração & dosagem , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Linhagem Celular , Endossomos/metabolismo , Camundongos , Neoplasias/genética , Engenharia de Proteínas , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo
10.
Traffic ; 15(3): 255-72, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24329939

RESUMO

CD22 is a cell surface glycoprotein restricted to normal and malignant B-cells and is the target of several anti-CD22 antibody-based cancer therapies. For therapeutic antibody-payload conjugates, it is important to understand the subcellular trafficking of anti-CD22 antibodies to optimize antibody and/or linker-drug properties to maximize antitumor efficacy. It is agreed that anti-CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell-type dependent. No studies examined anti-CD22 trafficking to either pathway in B-cells over time by dual immunofluorescence microscopy, likely partly because multiple samples of suspension cells are tedious to stain. We overcame this by using DropArray™, a novel wall-less 96-well plate technology allowing rapid simultaneous staining of suspension or adherent cells in small (10-20 µL) volumes. We examined the time-course of trafficking of five different anti-CD22 antibodies in eight B-cell lines representing four B-cell cancer types and show that in all cases antibodies internalize within 5 min and recycle, with only small amounts eventually trafficking to lysosomes. CD22 also localizes to recycling endosomes at steady state in the absence of antibody. Our data may help explain the differential efficacies of anti-CD22 antibodies conjugated to different therapeutic payloads.


Assuntos
Endocitose , Microscopia de Fluorescência/métodos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Análise Serial de Tecidos/métodos , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Humanos
11.
J Biol Chem ; 290(49): 29663-75, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26451044

RESUMO

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway.


Assuntos
AMP Cíclico/metabolismo , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Cílios/metabolismo , DNA Complementar/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C3H , Microscopia de Fluorescência , Dados de Sequência Molecular , RNA Interferente Pequeno/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptor Smoothened , Peixe-Zebra , Proteína Gli3 com Dedos de Zinco
12.
Nature ; 455(7211): 406-10, 2008 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-18754008

RESUMO

Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer.


Assuntos
Proteínas Hedgehog/metabolismo , Neoplasias/metabolismo , Comunicação Parácrina/fisiologia , Células Estromais/metabolismo , Animais , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ligantes , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/genética , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened
13.
Sci Rep ; 14(1): 3765, 2024 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-38355600

RESUMO

Homozygous Apolipoprotein L1 (APOL1) variants G1 and G2 cause APOL1-mediated kidney disease, purportedly acting as surface cation channels in podocytes. APOL1-G0 exhibits various single nucleotide polymorphisms, most commonly haplotype E150K, M228I and R255K ("KIK"; the Reference Sequence is "EMR"), whereas variants G1 and G2 are mostly found in a single "African" haplotype background ("EIK"). Several labs reported cytotoxicity with risk variants G1 and G2 in KIK or EIK background haplotypes, but used HEK-293 cells and did not verify equal surface expression. To see if haplotype matters in a more relevant cell type, we induced APOL1-G0, G1 and G2 EIK, KIK and EMR at comparable surface levels in immortalized podocytes. G1 and G2 risk variants (but not G0) caused dose-dependent podocyte death within 48h only in their native African EIK haplotype and correlated with K+ conductance (thallium FLIPR). We ruled out differences in localization and trafficking, except for possibly greater surface clustering of cytotoxic haplotypes. APOL1 surface expression was required, since Brefeldin A rescued cytotoxicity; and cytoplasmic isoforms vB3 and vC were not cytotoxic. Thus, APOL1-EIK risk variants kill podocytes in a dose and haplotype-dependent manner (as in HEK-293 cells), whereas unlike in HEK-293 cells the KIK risk variants did not.


Assuntos
Podócitos , Humanos , Podócitos/metabolismo , Haplótipos , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Células HEK293 , Variação Genética
14.
Cell Rep ; 43(6): 114310, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38838223

RESUMO

Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19, HIV, and apolipoprotein-L1 (APOL1) nephropathy, but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids, primary endothelial cells, and patient samples, we demonstrate that IFN-γ induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing, immunoblotting, and quantitative fluorescence-based assays reveal that IFN-γ-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-γ signaling inhibits APOL1 expression, prevents upregulation of pyroptosis-associated genes, and rescues vascular networks. Multiomic analyses in patients with COVID-19, proteinuric kidney disease, and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-γ signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis, suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy, which can be targeted therapeutically.


Assuntos
Apolipoproteína L1 , Interferon gama , Nefropatias , Piroptose , Humanos , Apolipoproteína L1/metabolismo , Apolipoproteína L1/genética , COVID-19/metabolismo , COVID-19/patologia , COVID-19/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Interferon gama/metabolismo , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/genética , Piroptose/genética , SARS-CoV-2/metabolismo , Transdução de Sinais
15.
J Biol Chem ; 286(48): 41852-41861, 2011 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-21994939

RESUMO

Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer.


Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Capeamento Imunológico/efeitos dos fármacos , Receptor IGF Tipo 1/metabolismo , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/imunologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Humanos , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/imunologia , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/imunologia , Ubiquitinação
16.
Proc Natl Acad Sci U S A ; 106(11): 4254-9, 2009 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-19246386

RESUMO

The Hedgehog (Hh) pathway has been implicated in pancreatic cancer but its role remains controversial. To delineate the cell populations able to respond to Hh ligand stimulation, we expressed an oncogenic allele of Smoothened (SmoM2) to cell autonomously activate Hh signaling in the mouse pancreas. Surprisingly, we found that expression of SmoM2 in epithelial cells was not able to activate the pathway and had no impact on pancreatic development or neoplasia. In contrast, activation of Smo in the mesenchyme led to Hh pathway activation, indicating that only the tumor stroma is competent to transduce the Hh signal. Using a Ptc-LacZ reporter mouse, we show that Hh signaling is active in stromal cells surrounding Hh-expressing tumor epithelium in various mouse pancreatic cancer models. Activation of the Hh pathway in the tumor stroma of human pancreatic and metastatic cancer specimens was confirmed by quantitative RT-PCR of microdissected tissue samples. These data support a paracrine model of Hh-mediated tumorigenesis, in which tumor cells secrete Hh ligand to induce tumor-promoting Hh target genes in adjacent stroma.


Assuntos
Proteínas Hedgehog/fisiologia , Neoplasias Pancreáticas/patologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais , Células Estromais/patologia , Animais , Células Epiteliais , Regulação Neoplásica da Expressão Gênica , Mesoderma , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/patologia , Neoplasias Pancreáticas/etiologia , Comunicação Parácrina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor Smoothened
17.
J Biol Chem ; 285(34): 26570-80, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20504762

RESUMO

Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10-20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn(2+) cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.


Assuntos
Anticorpos Monoclonais/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Cálcio/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Epitopos , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Ligação Proteica , Engenharia de Proteínas
18.
Commun Biol ; 4(1): 916, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34316015

RESUMO

Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.


Assuntos
Apolipoproteína L1/química , Apolipoproteínas L/química , Domínios Proteicos , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Apolipoproteínas L/genética , Apolipoproteínas L/metabolismo , Humanos
19.
J Immunol Methods ; 480: 112767, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32119889

RESUMO

IgG antibodies have been used to treat many diseases including cancer. IgG antibody-drug conjugates (ADCs) deliver cytotoxic drugs to target cells for cell elimination, but they have dose limiting toxicity due to target-independent uptake, including pinocytotic uptake. Neonatal Fc receptor (FcRn) recycles pinocytosed IgG in a pH-dependent manner and is the receptor responsible for the long half-life of IgG. Use of IgG variants with stronger FcRn binding at pH 6.0 for ADCs might improve recycling efficiency and reduce toxicity. However, these variants have residual FcRn binding at pH 7.4, which could lead to FcRn-mediated uptake and higher toxicity. Thus, the uptake of such variants at pH 7.4 needs to be evaluated. Here we report a reproducible and quantitative assay using an inducible HM7 colorectal cancer cell line to measure IgG uptake at endogenous and overexpressed FcRn levels. Our assay had comparable reproducibility at pH 6.0, 6.8 and 7.4. The wild type (WT) IgG had similar uptake at endogenous and overexpressed FcRn levels, as expected for pinocytotic uptake. We found similar uptake of a WT IgG and a stronger FcRn binding T307Q/N434A variant (QA variant) at endogenous FcRn levels at pH 7.4, although the QA variant had higher uptake at overexpressed FcRn levels. The QA variant also had higher uptake than the WT IgG at overexpressed FcRn levels at pH 6.8. Our assay can be used to characterize the stronger FcRn binding variants to aid in selection of suitable variants with low uptake at pH 7.4 for use as ADCs.


Assuntos
Neoplasias Colorretais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulina G/metabolismo , Pinocitose , Receptores Fc/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligação Proteica , Receptores Fc/genética , Regulação para Cima
20.
Mol Cancer Ther ; 7(9): 2599-608, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18790743

RESUMO

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti-vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging.


Assuntos
Anticorpos Monoclonais/farmacologia , Glucose/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteassoma , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacos
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