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Exp Cell Res ; 357(1): 40-50, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28442266


The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children.

Ácido Aspártico Endopeptidases/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Linhagem Celular , Humanos , Lisossomos/metabolismo , Transporte Proteico , Solubilidade , Rede trans-Golgi/metabolismo
Artigo em Inglês | MEDLINE | ID: mdl-31544130


T-cell activation is mediated by a combination of signals from the antigen receptor (TCR) and co-receptors such as CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death antigen 1 (PD-1), CD28H and others. Each is a member of the CD28 receptor gene family. CD28 sends positive signals that promote T-cell responses, while CTLA-4 and PD-1 limit responses. It is the balance between these positive and negative signals that determines the amplitude and level of T-cell responses. The regulatory role of other family members is also becoming the focus of increasing interest. The function of certain CD28 family members such as CTLA-4 and PD-1 is dependent the expression of CD28. Together, these findings have important implications in generation of immune responses and the application of anti-receptor blocking reagents in immunotherapy.

Biochem Biophys Res Commun ; 433(1): 90-5, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23485461


Sortilin is a transmembrane domain protein that has been implicated in the sorting of prosaposin and other soluble cargo from the Golgi to the lysosomal compartment. While the majority of the receptor is recycled back to the Golgi from endosomes, it is known that upon successive rounds of transport, a proportion of sortilin is degraded in lysosomes. Recently, it was shown that sortilin is palmitoylated and that this post-translational modification prevents its degradation and enables sortilin to efficiently traffic back to the Golgi. Thus palmitoylation can be used to modulate the amount of receptor and hence cargo reaching the lysosome. In this work, we demonstrate that non-palmitoylated sortilin is ubiquitinated and internalized into the lysosomal compartment via the ESCRT pathway for degradation. Furthermore, we identified Nedd4 as an E3 ubiquitin ligase that mediates this post-translational modification. We propose a model where palmitoylation and ubiquitination play opposite roles in the stability and turnover of sortilin and serve as a control mechanism that balances the amount of lysosomal sorting and trafficking in cells.

Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Sequência de Aminoácidos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lipoilação , Lisina/química , Lisossomos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ubiquitina-Proteína Ligases Nedd4 , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
Mol Cell Biol ; 32(10): 1855-66, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22431521


Mutations in the gene encoding CLN5 are the cause of Finnish variant late infantile Neuronal Ceroid Lipofuscinosis (NCL), and the gene encoding CLN5 is 1 of 10 genes (encoding CLN1 to CLN9 and cathepsin D) whose germ line mutations result in a group of recessive disorders of childhood. Although CLN5 localizes to the lysosomal compartment, its function remains unknown. We have uncovered an interaction between CLN5 and sortilin, the lysosomal sorting receptor. However, CLN5, unlike prosaposin, does not require sortilin to localize to the lysosomal compartment. We demonstrate that in CLN5-depleted HeLa cells, the lysosomal sorting receptors sortilin and cation-independent mannose 6-phosphate receptor (CI-MPR) are degraded in lysosomes due to a defect in recruitment of the retromer (an endosome-to-Golgi compartment trafficking component). In addition, we show that the retromer recruitment machinery is also affected by CLN5 depletion, as we found less loaded Rab7, which is required to recruit retromer. Taken together, our results support a role for CLN5 in controlling the itinerary of the lysosomal sorting receptors by regulating retromer recruitment at the endosome.

Endossomos/metabolismo , Lipofuscinoses Ceroides Neuronais/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Complexo de Golgi , Células HeLa , Humanos , Lisossomos/metabolismo , Ligação Proteica , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular/metabolismo