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1.
Genome Res ; 26(2): 183-91, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26755636

RESUMEN

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.


Asunto(s)
Deformidades Congénitas de las Extremidades/genética , Quinasas Quinasa Quinasa PAM/genética , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas , Proteína 9 Asociada a CRISPR , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Cocultivo , Endonucleasas , Exoma , Femenino , Humanos , Escala de Lod , Quinasas Quinasa Quinasa PAM/química , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Mutación Missense , Linaje , Polimorfismo de Nucleótido Simple , Proteínas Quinasas/química , Análisis de Secuencia de ADN
2.
BMC Struct Biol ; 14: 17, 2014 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-24998259

RESUMEN

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder leading to end-stage renal failure in humans. In the PKD/Mhm(cy/+) rat model of ADPKD, the point mutation R823W in the sterile alpha motif (SAM) domain of the protein ANKS6 is responsible for disease. SAM domains are known protein-protein interaction domains, capable of binding each other to form polymers and heterodimers. Despite its physiological importance, little is known about the function of ANKS6 and how the R823W point mutation leads to PKD. Recent work has revealed that ANKS6 interacts with a related protein called ANKS3. Both ANKS6 and ANKS3 have a similar domain structure, with ankyrin repeats at the N-terminus and a SAM domain at the C-terminus. RESULTS: The SAM domain of ANKS3 is identified as a direct binding partner of the ANKS6 SAM domain. We find that ANKS3-SAM polymerizes and ANKS6-SAM can bind to one end of the polymer. We present crystal structures of both the ANKS3-SAM polymer and the ANKS3-SAM/ANKS6-SAM complex, revealing the molecular details of their association. We also learn how the R823W mutation disrupts ANKS6 function by dramatically destabilizing the SAM domain such that the interaction with ANKS3-SAM is lost. CONCLUSIONS: ANKS3 is a direct interacting partner of ANKS6. By structurally and biochemically characterizing the interaction between the ANKS3 and ANKS6 SAM domains, our work provides a basis for future investigation of how the interaction between these proteins mediates kidney function.


Asunto(s)
Repetición de Anquirina , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dicroismo Circular , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Resonancia por Plasmón de Superficie
3.
Monoclon Antib Immunodiagn Immunother ; 38(6): 242-254, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31825302

RESUMEN

Although CD3 T cell redirecting antibodies have been successfully utilized for the treatment of hematological malignancies (blinatumomab), the T cell signaling pathways induced by these molecules are incompletely understood. To gain insight into the mechanism of action for T cell redirection antibodies, we created a novel murine CD3xEpCAM bispecific antibody that incorporates a silent Fc to dissect function and signaling of murine CD8 OT1 T cells upon stimulation. T cell-mediated cytotoxicity, cytokine secretion, expression of activation markers, and proliferation were directly induced in T cells treated with the novel CD3xEpCAM bispecific molecule in vitro in the presence of epithelial cell adhesion molecule (EpCAM) expressing tumor cells. Nanostring analysis showed that CD3xEpCAM induced a gene expression profile that resembled antigen-mediated activation, although the magnitude was lower than that of the antigen-induced response. In addition, this CD3xEpCAM bispecific antibody exhibited in vivo efficacy. This is the first study that investigates both in vitro and in vivo murine CD8 T cell function and signaling induced by a CD3xEpCAM antibody having a silent Fc to delineate differences between antigen-independent and antigen-specific T cell activation. These findings expand the understanding of T cell function and signaling induced by CD3 redirection bispecific antibodies and may help to develop more efficacious CD3 redirection therapeutics for cancer treatment, particularly for solid tumors.


Asunto(s)
Anticuerpos Biespecíficos/inmunología , Complejo CD3/inmunología , Neoplasias/inmunología , Linfocitos T/inmunología , Animales , Complejo CD3/genética , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/genética , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/inmunología , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Ratones , Neoplasias/terapia , Transducción de Señal/inmunología
4.
Structure ; 26(2): 209-224.e6, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29290488

RESUMEN

Head-to-tail polymers of sterile alpha motifs (SAM) can scaffold large macromolecular complexes. Several SAM-domain proteins that bind each other are mutated in patients with cystic kidneys or laterality defects, including the Ankyrin (ANK) and SAM domain-containing proteins ANKS6 and ANKS3, and the RNA-binding protein Bicc1. To address how their interactions are regulated, we first determined a high-resolution crystal structure of a Bicc1-SAM polymer, revealing a canonical SAM polymer with a high degree of flexibility in the subunit interface orientations. We further mapped interactions between full-length and distinct domains of Bicc1, ANKS3, and ANKS6. Neither ANKS3 nor ANKS6 alone formed macroscopic homopolymers in vivo. However, ANKS3 recruited ANKS6 to Bicc1, and the three proteins together cooperatively generated giant macromolecular complexes. Thus, the giant assemblies are shaped by SAM domains, their flanking sequences, and SAM-independent protein-protein and protein-mRNA interactions.


Asunto(s)
Proteínas Portadoras/química , Ciliopatías/metabolismo , Proteínas Nucleares/química , Proteínas de Unión al ARN/química , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Polímeros , Conformación Proteica , Motivo alfa Estéril
5.
Sci Rep ; 7(1): 15521, 2017 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-29138497

RESUMEN

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.


Asunto(s)
Anticuerpos Biespecíficos/genética , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Mutación , Receptores de IgG/química , Proteína Estafilocócica A/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/aislamiento & purificación , Sitios de Unión , Cromatografía de Afinidad , Expresión Génica , Células HEK293 , Semivida , Humanos , Concentración de Iones de Hidrógeno , Fragmentos Fc de Inmunoglobulinas/biosíntesis , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/aislamiento & purificación , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Cinética , Ratones , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas/métodos , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Estructura Secundaria de Proteína , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteína Estafilocócica A/inmunología , Proteína Estafilocócica A/metabolismo
6.
Protein Sci ; 20(10): 1697-706, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21805519

RESUMEN

The sterile alpha motif (SAM) domain is one of the most common protein modules found in eukaryotic genomes. Many SAM domains have been shown to form helical polymer structures suggesting that SAM modules can be used to create large protein complexes in the cell. Because many polymeric SAM domains form heterogenous and insoluble aggregates that are experimentally intractable when isolated, it is likely that many polymeric SAM domains have gone uncharacterized. We, therefore, developed a method to maintain polymeric SAM domains in a soluble form that allowed rapid screening for potential SAM polymers. SAM domains were expressed as fusions to a super-negatively charged green fluorescent protein (negGFP). The negGFP imparts three useful properties to the SAM domains: (1) the charge helps to maintain solubility; (2) the charge leads to reliable migration toward the cathode on native gels; and (3) the fluorescence emission allows visualization in crude extracts. Using the negGFP-SAM fusions, we screened a large library of human SAM domains for polymerization using a native gel screen. A selected set of hSAM domains were then purified and examined for true polymer formation by electron microscopy. In this manner, we identified a set of new potential SAM polymers: ANKS3, Atherin, BicaudalC1, Caskin1, Caskin2, Kazrin, L3MBTL3, L3MBTL4, LBP, LiprinB1, LiprinB2, SAMD8, SAMD9, and STIM2. While further characterization will be necessary to verify that the SAM domains identified here truly form polymers, our results provide a much stronger working hypothesis for a large number of proteins that was possible from sequence analysis alone.


Asunto(s)
Secuencias de Aminoácidos , Proteínas/química , Humanos , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo
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