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Increasing evidence indicates that many small secretory preproteins can undergo post-translational translocation across the membrane of the endoplasmic reticulum. Although the cellular machinery involved in post-translational translocation of small secretory preproteins has begun to be elucidated, the intrinsic signals contained within these small secretory preproteins that contribute to their efficient post-translational translocation remain unknown. Here, we analyzed the eukaryotic secretory proteome and discovered the small secretory preproteins tend to have a higher probability to harbor the positive charge in the n-region of the signal peptide (SP). Eliminating the positive charge of the n-region blocked post-translational translocation of newly synthesized preproteins and selectively impaired translocation efficiency of small secretory preproteins. The pathophysiological significance of the positive charge in the n-region of SP was underscored by recently identified preproinsulin SP mutations that impair translocation of preproinsulin and cause maturity onset diabetes of youth (MODY). Remarkably, we have found that slowing the polypeptide elongation rate of small secretory preproteins could alleviate the translocation defect caused by loss of the n-region positive charge of the signal peptide. Together, these data reveal not only a previously unrecognized role of the n-region's positive charge in ensuring efficient post-translational translocation of small secretory preproteins, but they also highlight the molecular contribution of defects in this process to the pathogenesis of genetic disorders such as MODY.

Background: MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA-protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. Results: We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. Conclusions: Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosinebinding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping.

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Aurone glycosides display a variety of biological activities. However, reports about glycosyltransferases (GTs) responsible for aurones glycosylation are limited. Here, the transcriptome-wide discovery and identification of an aurone glycosyltransferase with glycosidase activity is reported. Specifically, a complementary DNA (cDNA), designated as OsUGT1, was isolated from the plant Ornithogalum saundersiae based on transcriptome mining. Conserved domain (CD)-search speculated OsUGT1 as a flavonoid GT. Phylogenetically, OsUGT1 is clustered as the same phylogenetic group with a putative 5,6-dihydroxyindoline-2-carboxylic acid (cyclo-DOPA) 5-O-glucosyltransferase, suggesting OsUGT1 may be an aurone glycosyltransferase. The purified OsUGT1 was therefore used as a biocatalyst to incubate with the representative aurone sulfuretin. In vitro enzymatic analyses showed that OsUGT1 was able to catalyze sulfuretin to form corresponding monoglycosides, suggesting OsUGT1 was indeed an aurone glycosyltransferase. OsUGT1 was observed to be a flavonoid GT, specific for flavonoid substrates. Moreover, OsUGT1 was demonstrated to display transglucosylation activity, transferring glucosyl group to sulfuretin via o-Nitrophenyl-β-d-glucopyranoside (oNP-β-Glc)-dependent fashion. In addition, OsUGT1-catalyzed hydrolysis was observed. This multifunctionality of OcUGT1 will broaden the application of OcUGT1 in glycosylation of aurones and other flavonoids.