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Secretome analysis can be described as a subset of proteomics studies consisting in the analysis of the molecules secreted by cells or tissues. Dengue virus (DENV) infection can lead to a broad spectrum of clinical manifestations, with the severe forms of the disease characterized by hemostasis abnormalities and liver injury. The hepatocytes are a relevant site of viral replication and a major source of plasma proteins. Until now, we had limited information on the small molecules secreted by hepatic cells after infection by DENV. In the present study, we analysed a fraction of the secretome of mock- and DENV-infected hepatic cells (HepG2 cells) containing molecules with < 10 kDa, using different proteomic approaches. We identified 175 proteins, with 57 detected only in the samples from mock-infected cells, 59 only in samples from DENV-infected cells, and 59 in both conditions. Most of the peptides identified were derived from proteins larger than 10 kDa, suggesting a proteolytic processing of the secreted molecules. Using in silico analysis, we predicted consistent differences between the proteolytic processing occurring in mock and DENV-infected samples, raising, for the first time, the hypothesis that differential proteolysis of secreted molecules would be involved in the pathogenesis of dengue. Biological significance Since the liver, one of the targets of DENV infection, is responsible for producing molecules involved in distinct biological processes, the identification of proteins and peptides secreted by hepatocytes after infection would help to a better understanding of the physiopathology of dengue. Proteomic analyses of molecules with < 10 kDa secreted by HepG2 cells after infection with DENV revealed differential proteolytic processing as an effect of DENV infection. [ABSTRACT FROM AUTHOR]

Assembly of Photosystem (PS) II in plants has turned out to be a highly complex process which, at least in part, occurs in a sequential order and requires many more auxiliary proteins than subunits present in the complex. Owing to the high evolutionary conservation of the subunit composition and the three-dimensional structure of the PSII complex, most plant factors involved in the biogenesis of PSII originated from cyanobacteria and only rarely evolved de novo. Furthermore, in chloroplasts the initial assembly steps occur in the non-appressed stroma lamellae, whereas the final assembly including the attachment of the major LHCII antenna proteins takes place in the grana regions. The stroma lamellae are also the place where part of PSII repair occurs, which very likely also involves assembly factors. In cyanobacteria initial PSII assembly also occurs in the thylakoid membrane, in so-called thylakoid centres, which are in contact with the plasma membrane. Here, we provide an update on the structures, localisations, topologies, functions, expression and interactions of the low molecular mass PSII subunits PsbY, PsbW and the auxiliary factors HCF136, PsbN, TerC and ALB3, assisting in PSII complex assembly and protein insertion into the thylakoid membrane.

Assembly of Photosystem (PS) II in plants has turned out to be a highly complex process which, at least in part, occurs in a sequential order and requires many more auxiliary proteins than subunits present in the complex. Owing to the high evolutionary conservation of the subunit composition and the three-dimensional structure of the PSII complex, most plant factors involved in the biogenesis of PSII originated from cyanobacteria and only rarely evolved de novo. Furthermore, in chloroplasts the initial assembly steps occur in the non-appressed stroma lamellae, whereas the final assembly including the attachment of the major LHCII antenna proteins takes place in the grana regions. The stroma lamellae are also the place where part of PSII repair occurs, which very likely also involves assembly factors. In cyanobacteria initial PSII assembly also occurs in the thylakoid membrane, in so-called thylakoid centers, which are in contact with the plasma membrane. Here, we provide an update on the structures, localisations, topologies, functions, expression and interactions of the low molecular mass PSII subunits PsbY, PsbW and the auxiliary factors HCF136, PsbN, TerC and ALB3, assisting in PSII complex assembly and protein insertion into the thylakoid membrane. [ABSTRACT FROM AUTHOR]

Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) is an efficient way of separating low molecular mass proteins. However, the standard system is quite complicated and specifically may not be useful when the separated proteins are to be recovered from the gel for quantitative analysis. Here, we describe a simplified system whereby these smaller proteins can be resolved in comparatively low percentage gels which have high compatibility with modern detectors such as UV and inductively coupled plasma-mass spectrometry (ICP-MS).