RESUMEN
Point mutations in cysteine string protein-α (CSPα) cause dominantly inherited adult-onset neuronal ceroid lipofuscinosis (ANCL), a rapidly progressing and lethal neurodegenerative disease with no treatment. ANCL mutations are proposed to trigger CSPα aggregation/oligomerization, but the mechanism of oligomer formation remains unclear. Here we use purified proteins, mouse primary neurons and patient-derived induced neurons to show that the normally palmitoylated cysteine string region of CSPα loses palmitoylation in ANCL mutants. This allows oligomerization of mutant CSPα via ectopic binding of iron-sulfur (Fe-S) clusters. The resulting oligomerization of mutant CSPα causes its mislocalization and consequent loss of its synaptic SNARE-chaperoning function. We then find that pharmacological iron chelation mitigates the oligomerization of mutant CSPα, accompanied by partial rescue of the downstream SNARE defects and the pathological hallmark of lipofuscin accumulation. Thus, the iron chelators deferiprone (L1) and deferoxamine (Dfx), which are already used to treat iron overload in humans, offer a new approach for treating ANCL.
Asunto(s)
Proteínas del Choque Térmico HSP40/genética , Proteínas de la Membrana/genética , Lipofuscinosis Ceroideas Neuronales/genética , Mutación Puntual , Agregación Patológica de Proteínas/genética , Animales , Células Cultivadas , Femenino , Células HEK293 , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Quelantes del Hierro/metabolismo , Lipoilación , Proteínas de la Membrana/metabolismo , Ratones , Lipofuscinosis Ceroideas Neuronales/metabolismo , Neuronas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Unión Proteica , Multimerización de ProteínaRESUMEN
Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.
Asunto(s)
Antígenos B7/química , Antígenos B7/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfocitos T/metabolismo , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Antígenos B7/antagonistas & inhibidores , Antígenos B7/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Cristalografía por Rayos X , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Femenino , Histidina/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Linfocitos T/citología , Linfocitos T/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics.
Asunto(s)
Biocatálisis , Reparación del ADN , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Oxigenasas de Función Mixta/metabolismo , Alquilación , Cromatografía Líquida de Alta Presión , Dicroismo Circular , ADN Bacteriano/metabolismo , Proteínas de Escherichia coli/química , Ácidos Cetoglutáricos/metabolismo , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Oxigenasas de Función Mixta/química , Proteínas Mutantes/metabolismo , Mutación/genética , Oxidación-Reducción , Conformación Proteica , Especificidad por Sustrato , Sacarosa/metabolismoRESUMEN
Hepsin is a serine protease that is widely expressed in different tissues and cell types, most prominently in the normal liver and kidney. Overexpression of hepsin has been associated with prostate cancers, ovarian cancers and renal cell carcinomas. The physiological functions of hepsin in normal tissues and tumors are poorly understood. To gain insight into its function in ovarian cancer, we analyzed the expression and subcellular localization of hepsin protein in ovarian cancer cell lines and tumors. We showed that the membrane-associated hepsin protein is present at desmosomal junctions, where it colocalizes with its putative proteolytic substrate hepatocyte growth factor. Consistent with the growing evidence that desmosomal junctions and their constituents play a role in cancer progression, we demonstrated that overexpression of hepsin promotes ovarian tumor growth in a mouse model. The ability of ectopic hepsin to induce tumor growth in mice is abrogated by the mutation of 3 critical residues in the catalytic domain, thus implicating the enzymatic activity of hepsin in promoting tumor progression.