RESUMO
Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4â min to 21.9â min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.
Assuntos
Aminopeptidases , Dipeptidil Peptidases e Tripeptidil Peptidases , Lipofuscinoses Ceroides Neuronais , Proteínas , Serina Proteases , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/isolamento & purificação , Aminopeptidases/metabolismo , Animais , Células CHO , Clonagem Molecular , Cricetulus , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/isolamento & purificação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Terapia de Reposição de Enzimas , Estabilidade Enzimática , Humanos , Linfócitos , Mutação , Lipofuscinoses Ceroides Neuronais/tratamento farmacológico , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Cultura Primária de Células , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , Serina Proteases/química , Serina Proteases/genética , Serina Proteases/isolamento & purificação , Serina Proteases/metabolismo , Tripeptidil-Peptidase 1RESUMO
During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site. In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5'-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.
Assuntos
Capsídeo/metabolismo , Genoma Viral/genética , Vírus de RNA/genética , RNA de Cadeia Dupla/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas do Capsídeo/genética , Humanos , RNA de Cadeia Dupla/metabolismo , RNA Viral/genética , Vírion/genética , Vírion/metabolismoRESUMO
Partitiviruses constitute one of the nine currently recognized families of viruses with encapsidated, double-stranded (ds)RNA genomes. The partitivirus genome is bisegmented, and each genome segment is packaged inside a separate viral capsid. Different partitiviruses infect plants, fungi, or protozoa. Recent studies have shed light on the three-dimensional structures of the virions of three representative fungal partitiviruses. These structures include a number of distinctive features, allowing informative comparisons with the structures of dsRNA viruses from other families. The results and comparisons suggest several new conclusions about the functions, assembly, and evolution of these viruses.