Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5NC genotyping kit, based on sequence analysis of the 5 noncoding region (5 NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5 NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.Hepatitis C virus (HCV) is considered the major cause of posttransfusion non-A, non-B hepatitis. The viral genome, a positive-sense single-stranded RNA of about 9,400 nucleotides (5), is characterized by a high genetic heterogeneity like other RNA viruses. HCV isolates show four levels of genetic variability: types, subtypes, isolates, and quasispecies (3). An HCV genotype is therefore used with the histological results from liver biopsy and viral load for counseling individual patients about the risk-benefit ratio of therapy (17,21,22). HCV genotypes are distributed differently depending on geography and the etiology of infection (15,25). For the purpose of nomenclature, it has been proposed that HCV be classified into types, corresponding to the main branches in the phylogenetic tree, and subtypes, corresponding to the more related sequences within the major groups (23,24). HCV genotypes can be established by methods based on PCR typing and/or serological typing (2, 4, 16). The high degree of conservation in the 5Ј noncoding regions (5Ј NCR) has made it the target of choice for reverse transcriptase PCR-based detection assays. Moreover, several PCR typing methods, such as reverse dot blot (26), restriction fragment length polymorphism (10), cleavase fragment length polymorphism, dideoxy fingerprinting, heteroduplex mobility analysis (28), and hybridization to genotype-specific probes (18), exploit sequence-based differences and/or differences in secondary structure of the 5Ј NCR for HCV genotyping (9). Nucleotide sequence analysis is the reference method for identifying different genotypes of HCV (9, 13). However, because this method is expensive and timeconsuming and requires special equipment for sequencing, it has been restricted to the research setting and considered impractical for large clinical studies. A standardized sequencing assay has recently been developed for routine determination of HCV genotypes. In order to determine whether direct sequencing could be a routine tool for the determination of hepatitis C virus genotype, we assessed this first commercial type of HCV genotyping method (Trugene 5ЈNC HCV genot...

The three human Tribbles (TRIB) pseudokinases have been implicated in a plethora of signaling and metabolic processes linked to cancer initiation and progression and can potentially be used as biomarkers of disease and prognosis. While their modes of action reported so far center around protein–protein interactions, the comprehensive profiling of TRIB interactomes has not been reported yet. Here, we have developed a robust mass spectrometry (MS)-based proteomics approach to characterize Tribbles’ interactomes and report a comprehensive assessment and comparison of the TRIB1, -2 and -3 interactomes, as well as domain-specific interactions for TRIB3. Interestingly, TRIB3, which is predominantly localized in the nucleus, interacts with multiple transcriptional regulators, including proteins involved in gene repression. Indeed, we found that TRIB3 repressed gene transcription when tethered to DNA in breast cancer cells. Taken together, our comprehensive proteomic assessment reveals previously unknown interacting partners and functions of Tribbles proteins that expand our understanding of this family of proteins. In addition, our findings show that MS-based proteomics provides a powerful tool to unravel novel pseudokinase biology.

Aberrant expression or activity of proteins are amongst the best understood mechanisms that can drive cancer initiation and progression, as well as therapy resistance. TRIB3, a member of the Tribbles family of pseudokinases, is often dysregulated in cancer and has been associated with breast cancer initiation and metastasis formation. However, the underlying mechanisms by which TRIB3 contributes to these events are unclear. In this study, we demonstrate that TRIB3 regulates the expression of PPARγ, a transcription factor that has gained attention as a potential drug target in breast cancer for its antiproliferative actions. Proteomics and phosphoproteomics analyses together with classical biochemical assays indicate that TRIB3 interferes with the MLL complex and reduces MLL-mediated H3K4 trimethylation of the PPARG locus, thereby reducing PPARγ mRNA expression. Consequently, the overexpression of TRIB3 blunts the antiproliferative effect of PPARγ ligands in breast cancer cells, while reduced TRIB3 expression gives the opposite effect. In conclusion, our data implicate TRIB3 in epigenetic gene regulation and suggest that expression levels of this pseudokinase may serve as a predictor of successful experimental treatments with PPARγ ligands in breast cancer.