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1.
Science ; 377(6602): 222-228, 2022 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-35857540

RESUMEN

G protein-coupled receptors (GPCRs) recruit ß-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage ß-arrestins but not G proteins, making them a model system for investigating the structural basis of ß-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that ß-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular ß-arrestin-coupling region. NMR studies of ACKR3 mutants defective in ß-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides ß-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.


Asunto(s)
Receptores CXCR , beta-Arrestinas , Regulación Alostérica , Ligandos , Espectroscopía de Resonancia Magnética , Mutación , Unión Proteica , Conformación Proteica , Receptores CXCR/química , Receptores CXCR/genética , beta-Arrestinas/química
2.
Nat Genet ; 54(5): 637-648, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35513723

RESUMEN

Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is characterized by clonal expansion of natural killer (NK) cells where the underlying genetic mechanisms are incompletely understood. In the present study, we report somatic mutations in the chemokine gene CCL22 as the hallmark of a distinct subset of CLPD-NK. CCL22 mutations were enriched at highly conserved residues, mutually exclusive of STAT3 mutations and associated with gene expression programs that resembled normal CD16dim/CD56bright NK cells. Mechanistically, the mutations resulted in ligand-biased chemokine receptor signaling, with decreased internalization of the G-protein-coupled receptor (GPCR) for CCL22, CCR4, via impaired ß-arrestin recruitment. This resulted in increased cell chemotaxis in vitro, bidirectional crosstalk with the hematopoietic microenvironment and enhanced NK cell proliferation in vivo in transgenic human IL-15 mice. Somatic CCL22 mutations illustrate a unique mechanism of tumor formation in which gain-of-function chemokine mutations promote tumorigenesis by biased GPCR signaling and dysregulation of microenvironmental crosstalk.


Asunto(s)
Quimiocina CCL22 , Células Asesinas Naturales , Trastornos Linfoproliferativos , Animales , Quimiocina CCL22/genética , Células Asesinas Naturales/patología , Activación de Linfocitos , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Ratones , Mutación
3.
Blood Cancer Discov ; 3(3): 194-207, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35176137

RESUMEN

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML. SIGNIFICANCE: We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.


Asunto(s)
Leucemia Mieloide Aguda , Adulto , Niño , Aberraciones Cromosómicas , Exones , Genómica , Humanos , Leucemia Mieloide Aguda/genética , Mutación , Recurrencia
4.
Science ; 371(6524): 86-90, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33384377

RESUMEN

Metamorphic proteins switch between different folds, defying the protein folding paradigm. It is unclear how fold switching arises during evolution. With ancestral reconstruction and nuclear magnetic resonance, we studied the evolution of the metamorphic human protein XCL1, which has two distinct folds with different functions, making it an unusual member of the chemokine family, whose members generally adopt one conserved fold. XCL1 evolved from an ancestor with the chemokine fold. Evolution of a dimer interface, changes in structural constraints and molecular strain, and alteration of intramolecular protein contacts drove the evolution of metamorphosis. Then, XCL1 likely evolved to preferentially populate the noncanonical fold before reaching its modern-day near-equal population of folds. These discoveries illuminate how one sequence has evolved to encode multiple structures, revealing principles for protein design and engineering.


Asunto(s)
Quimiocinas C/química , Evolución Molecular , Ingeniería de Proteínas , Pliegue de Proteína , Humanos , Multimerización de Proteína
5.
Methods Cell Biol ; 149: 259-288, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30616824

RESUMEN

The past decade has witnessed remarkable progress in the determination of G protein-coupled receptor (GPCR) structures, profoundly expanding our understanding of how GPCRs recognize ligands, become activated, and interact with intracellular signaling components. In recent years, numerous studies have used solution nuclear magnetic resonance (NMR) spectroscopy to investigate GPCRs, providing fundamental insights into GPCR conformational changes, allostery, dynamics, and other facets of GPCR function are challenging to study using other structural techniques. Despite these advantages, NMR-based studies of GPCRs are few relative to the number of published structures, due in part to the challenges and limitations of NMR for the characterization of large membrane proteins. Several studies have circumvented these challenges using a variety of isotopic labeling strategies, including side chain derivatization and metabolic incorporation of NMR-active nuclei. In this chapter, we provide an overview of different isotopic labeling strategies and describe an in-depth protocol for the expression, purification, and NMR studies of the chemokine GPCR atypical chemokine receptor 3 (ACKR3) via 13CH3-methionine incorporation. The goal of this chapter is to provide a resource to the GPCR community for those interested in pursuing NMR studies of GPCRs.


Asunto(s)
Isótopos de Carbono/química , Espectroscopía de Resonancia Magnética/métodos , Receptores CXCR/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Coloración y Etiquetado , Vitamina U/metabolismo , Animales , Línea Celular , Humanos , Soluciones
6.
J Leukoc Biol ; 104(2): 359-374, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29873835

RESUMEN

From an individual bacterium to the cells that compose the human immune system, cellular chemotaxis plays a fundamental role in allowing cells to navigate, interpret, and respond to their environments. While many features of cellular chemotaxis are shared among systems as diverse as bacteria and human immune cells, the machinery that guides the migration of these model organisms varies widely. In this article, we review current literature on the diversity of chemoattractant ligands, the cell surface receptors that detect and process chemotactic gradients, and the link between signal recognition and the regulation of cellular machinery that allow for efficient directed cellular movement. These facets of cellular chemotaxis are compared among E. coli, Dictyostelium discoideum, and mammalian neutrophils to derive organizational principles by which diverse cell systems sense and respond to chemotactic gradients to initiate cellular migration.


Asunto(s)
Quimiotaxis/fisiología , Animales , Humanos , Transducción de Señal/fisiología
7.
Sci Signal ; 10(471)2017 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-28325822

RESUMEN

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein-dependent and ß-arrestin-dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein-dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance-based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical "two-site model" for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.


Asunto(s)
Quimiocina CXCL12/química , Multimerización de Proteína , Receptores CXCR4/química , Transducción de Señal , Secuencia de Aminoácidos , Línea Celular Tumoral , Movimiento Celular/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutación , Unión Proteica , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Arrestina beta 2/genética , Arrestina beta 2/metabolismo
8.
Biochem Pharmacol ; 114: 53-68, 2016 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-27106080

RESUMEN

Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions.


Asunto(s)
Quimiocinas/metabolismo , Modelos Moleculares , Receptores de Quimiocina/metabolismo , Transducción de Señal/inmunología , Animales , Sitios de Unión , Humanos , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína
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