No abstract

White (WAT) and brown (BAT) adipose tissue are tissues of energy storage and energy dissipation, respectively. Experimental evidence suggests that brown and white preadipocytes are differentially determined, but so far not much is known about the genetic control of this determination process. The aim of this study was to identify differentially expressed genes involved in brown and white preadipocyte development. Using representational difference analysis (cDNA RDA) and DNA microarray screening, we identified four genes with higher expression in white preadipocytes (three different complement factors and ␦-6 fatty acid desaturase) and seven genes with higher expression levels in brown preadipocytes, of which three are structural genes implicated in cell adhesion and cytoskeleton organization (fibronectin, ␣-actinin-4, metargidin) and four that might function in gene transcription and protein synthesis (vigilin, necdin, snRNP polypeptide A, and a homolog to human hepatocellular carcinomaassociated protein). The expression profile of these genes was analyzed during preadipocyte differentiation, upon ␤-adrenergic stimulation, and in WAT and BAT tissue in vivo compared with references genes such as peroxisome proliferatoractivated receptor-␥ (PPAR␥), uncoupling protein 1 (UCP1), cytochrome c oxidase. adipocyte differentiation; thermogenesis; preadipocyte marker genes; uncoupling protein; cDNA representational-difference analysis; DNA microarray analysis WHITE AND BROWN ADIPOSE TISSUES represent counter actors in energy partitioning, channeling lipid energy either to accumulation in white fat (WAT) or to oxidation, i.e., dissipation in brown fat (BAT) a highly thermogenic tissue (23). Throughout the last years considerable progress has been made in elucidating the molecular mechanisms of adipocyte differentiation which involves sequential activation of numerous transcription factors from several families like different members of the CCAAT/enhancer binding proteins (C/ EBP) and peroxisome proliferator-activated receptors (PPAR) (1,15,30,44,52). However, most of these studies focused on differentiation of white preadipocytes using established white preadipocyte cell lines such as 3T3-L1 and 3T3-F442A cells (37). One of the remaining questions is how and at which stage of development the differentiation of BAT vs. WAT is regulated, of which very little is currently known. Brown and white adipocytes show distinct morphological and biochemical phenotypes in vivo (9). When differentiated in vitro, brown adipocytes show a higher respiratory capacity than white adipocytes and express the BAT specific uncoupling protein 1 (UCP1), which is considered to be a marker for brown adipocytes (21). It is still not clear whether BAT and WAT derive from the same adipose precursor cells or arise independently from distinct mesenchymal stem cells (44), although recently PGC-1, a coactivator of PPAR␥, has been identified, which induces genes important in the development of brown adipocyte phenotype (41).We have performed parallel culture...

Ubiquitylation is a complex posttranslational protein modification and deregulation of this pathway has been associated with different human disorders. Ubiquitylation comes in different flavors: Besides mono-ubiquitylation, ubiquitin chains of various topologies are formed on substrate proteins. The fate of ubiquitylated proteins is determined by the linkage-type of the attached ubiquitin chains, however, the underlying mechanism is poorly characterized. Herein, we describe a new method based on codon expansion and click-chemistry-based polymerization to generate linkage-defined ubiquitin chains that are resistant to ubiquitin-specific proteases and adopt native-like functions. The potential of these artificial chains for analyzing ubiquitin signaling is demonstrated by linkage-specific effects on cell-cycle progression.

Herein we describe a simple protocol for the efficient generation of site-specific ubiquitin-protein conjugates using click chemistry. By using two different methods to expand the genetic code, the two bio-orthogonal functionalities that are necessary for Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), an alkyne and an azide, are co-translationally incorporated into the proteins of interest with unnatural amino acids. Protein ubiquitylation is subsequently carried out with the purified proteins in vitro by CuAAC. In addition, we provide a protocol for the incorporation of two unnatural amino acids into a single ubiquitin, resulting in a 'bifunctional' protein that contains both an alkyne and an azide functionality, thereby enabling assembly of free ubiquitin chains as well as ubiquitin chains conjugated to a target protein. Our procedure enables the synthesis of nonhydrolyzable ubiquitin-protein conjugates within 1 week (given that the relevant cDNAs are at hand), and it yields conjugates in milligram quantities from 1-liter expression cultures. The approach described herein is faster and less laborious than other methods, and it requires only standard molecular biology equipment. Moreover, the protocol can be readily adapted to achieve conjugation at any site of any target protein, which facilitates the generation of custom-tailored ubiquitin-protein conjugates.

Evidence for Nr4a1 as a cold-induced effector of brown fat thermogenesis.