Faculty Bibliography

Identification of proteins by mass spectrometry is crucial for better understanding of many biological, biochemical, and biomedical processes. Here we describe two methods for the identification of electroblotted proteins by on-membrane digestion prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.

OBJECTIVES: Vitiligo commonly presents in children, with half of all cases developing before 20 years of age. Although studies have characterized differences between pediatric and adult vitiligo, little is known about vitiligo presenting in early childhood. The purpose of this study was to compare clinical features of early-onset (<3 years old) and later-onset (3-18 years old) childhood vitiligo. METHODS: This retrospective case series examined patients given a diagnosis of vitiligo in a pediatric dermatology practice at an academic medical center from 1990 to 2014. Characteristics of the early- and later-onset groups were compared by chi2 and t test for categorical and continuous variables, respectively. RESULTS: Of the 208 children in the study, 31 had early-onset and 177 had later-onset disease. Early-onset vitiligo was associated with higher percentages of body surface area involvement and increased rates of disease progression during an average 1.9 years of follow-up. There were no significant differences between the 2 groups in repigmentation, vitiligo type, halo nevi, gender ratio, or personal and family history of autoimmune diseases. LIMITATIONS: This was a retrospective, single-institution study. CONCLUSION: Patients given a diagnosis of vitiligo at younger ages tend to have more extensive and progressive disease.

Mutations in the gene for the latent transforming growth factor beta binding protein 4 (LTBP4) cause autosomal recessive cutis laxa type 1C. To understand the molecular disease mechanisms of this disease, we investigated the impact of LTBP4 loss on transforming growth factor beta (TGFbeta) signaling. Despite elevated extracellular TGFbeta activity, downstream signaling molecules of the TGFbeta pathway, including pSMAD2 and pERK, were down-regulated in LTBP4 mutant human dermal fibroblasts. In addition, TGFbeta receptors 1 and 2 (TGFBR1 and TGFBR2) were reduced at the protein but not at the ribonucleic acid level. Treatment with exogenous TGFbeta1 led to an initially rapid increase in SMAD2 phosphorylation followed by a sustained depression of phosphorylation and receptor abundance. In mutant cells TGFBR1 was co-localized with lysosomes. Treatment with a TGFBR1 kinase inhibitor, endocytosis inhibitors or a lysosome inhibitor, normalized the levels of TGFBR1 and TGFBR2. Co-immunoprecipitation demonstrated a molecular interaction between LTBP4 and TGFBR2. Knockdown of LTBP4 reduced TGFbeta receptor abundance and signaling in normal cells and supplementation of recombinant LTBP4 enhanced these measures in mutant cells. In a mouse model of Ltbp4 deficiency, reduced TGFbeta signaling and receptor levels were normalized upon TGFBR1 kinase inhibitor treatment. Our results show that LTBP4 interacts with TGFBR2 and stabilizes TGFbeta receptors by preventing their endocytosis and lysosomal degradation in a ligand-dependent and receptor kinase activity-dependent manner. These findings identify LTBP4 as a key molecule required for the stability of the TGFbeta receptor complex, and a new mechanism by which the extracellular matrix regulates cytokine receptor signaling.