The role of sarcosine metabolism in prostate cancer progression.

Metabolomic profiling of prostate cancer (PCa) progression identified markedly elevated levels of sarcosine (N-methyl glycine) in metastatic PCa and modest but significant elevation of the metabolite in PCa urine. Here, we examine the role of key enzymes associated with sarcosine metabolism in PCa progression. Consistent with our earlier report, sarcosine levels were significantly elevated in PCa urine sediments compared to controls, with a modest area under the receiver operating characteristic curve of 0.71. In addition, the expression of sarcosine biosynthetic enzyme, glycine N-methyltransferase (GNMT), was elevated in PCa tissues, while sarcosine dehydrogenase (SARDH) and pipecolic acid oxidase (PIPOX), which metabolize sarcosine, were reduced in prostate tumors. Consistent with this, GNMT promoted the oncogenic potential of prostate cells by facilitating sarcosine production, while SARDH and PIPOX reduced the oncogenic potential of prostate cells by metabolizing sarcosine. Accordingly, addition of sarcosine, but not glycine or alanine, induced invasion and intravasation in an in vivo PCa model. In contrast, GNMT knockdown or SARDH overexpression in PCa xenografts inhibited tumor growth. Taken together, these studies substantiate the role of sarcosine in PCa progression.

Characterization of LINE-1 ribonucleoprotein particles.

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1-encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.

Selective coactivator interactions in gene activation by SREBP-1a and -1c.

Requisite levels of intracellular cholesterol and fatty acids are maintained in part by the sterol regulatory element binding proteins (SREBPs). Three major SREBP isoforms exist; SREBP-1a and SREBP-1c are expressed from overlapping mRNAs, whereas SREBP-2 is encoded by a separate gene. The active forms of SREBP-1a and SREBP-1c differ only at their extreme N termini; SREBP-1c lacks 28 aa present in SREBP-1a and instead contains 4 unique aa of its own. While the SREBP-1a and -1c isoforms differentially activate transcription, the molecular basis of this difference is unknown. Here we define the differences between these proteins that confer the enhanced activity of SREBP-1a and demonstrate that this enhancement is a direct result of its avid binding to the coactivator CREB binding protein (CBP) and the mammalian mediator complex. While previous work determined that the C/H1 zinc finger and KIX domains of CBP bind to SREBP-1a, we provide evidence that the interaction with C/H1 is important for gene activation. We further show that the association between the activation domain of SREBP-1 and mediator is through aa 500 to 824 of DRIP150. Finally, we demonstrate the recruitment of mediator to an SREBP-responsive promoter in a sterol-dependent manner.

A YY1-binding site is required for accurate human LINE-1 transcription initiation.

The initial step in Long Interspersed Element-1 (LINE-1) retrotransposition requires transcription from an internal promoter located within its 5'-untranslated region (5'-UTR). Previous studies have identified a YY1 (Yin Yang 1)-binding site as an important sequence in LINE-1 transcription. Here, we demonstrate that mutations in the YY1-binding site have only minor effects on transcription activation of the full-length 5'-UTR and LINE-1 mobility in a single round cultured cell retrotransposition assay. Instead, these mutations disrupt proper initiation of transcription from the +1 site of the 5'-UTR. Thus, we propose that the YY1-binding site functions as a component of the LINE-1 core promoter to direct accurate transcription initiation. Indeed, this sequence may explain the evolutionary success of LINE-1 by enabling full-length retrotransposed copies to undergo autonomous retrotransposition in subsequent generations.