Joseph, S. R. | bioRxiv Science

bioRxiv2020

Germline ERBB3 mutation in familial non-small cell lung carcinoma: expanding the role of the ErbB family in oncogenesis.

BackgroundLung cancer is the commonest cause of cancer deaths worldwide. Although strongly associated with smoking, predisposition to lung cancer is also heritable with multiple common risk variants identified. Rarely, dominantly inherited non-small-cell lung cancer (NSCLC) has been reported due to somatic mutations in EGFR/ErbB1 and ERBB2. MethodsGermline exome sequencing was performed in a multi-generation family with autosomal dominant NSCLC, including an affected child. Tumour samples were also sequenced. Full-length wild-type (wtErbB3) and mutant ERBB3 (mutErbB3) constructs were transfected into HeLa cells. Protein expression, stability, and sub-cellular localisation were assessed; and cellular proliferation, pAkt/Akt, and pERK levels were determined. ResultsA novel germline variant in ERBB3 (c.1946T>G: p.Iso649Arg), coding for receptor tyrosineprotein kinase erbB-3 (ErbB3), was identified, with appropriate segregation. There was no loss-of-heterozygosity in tumour samples. Both wtErbB3 and mutErbB3 were stably expressed. MutErbB3-transfected cells demonstrated an increased ratio of the 80kD form (which enhances proliferation) compared to the full-length (180kD) form. MutErbB3 and wtErbB3 had similar punctate cytoplasmic localisation pre- and post-EGF stimulation; however, EGFR levels decreased faster post-stimulation in mutErbB3-transfected cells, suggesting more rapid processing of the mutErbB3/EGFR heterodimer. Cellular proliferation was increased in mutErbB3-transfected cells compared to wtErbB3 transfection. MutErbB3-transfected cells also showed decreased pAkt/tAkt ratios and increased pERK/tERK 30 minutes post-stimulation compared to wtErbB3 transfection, demonstrating altered signalling pathway activation by mutErbB3. Cumulatively, these results support this mutation as tumorogenic. ConclusionsThis is the first reported family with a germline ERBB3 mutation causing heritable NSCLC, furthering understanding of the ErbB family pathway in oncogenesis.

genomics↗

bioRxiv2017

MS Western, a method of multiplexed absolute protein quantification is a practical alternative to western blotting

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the sub-femtomole level in whole cell or tissue lysates without metabolic or chemical labelling and without using specific antibodies. MS Western relies upon GeLC-MS/MS and quantifies proteins by in-gel co-digestion with an isotopically labelled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

biochemistry↗