SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substrate-specific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.
Abstract Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.
Enzymatic digestion of membrane-bound proteins is a sensitive procedure for obtaining internal sequence data of proteins that either have a blocked amino terminus or require two or more stretches of sequence data for DNA cloning or confirmation of protein identification. Since the final step of protein purification is usually SDS-PAGE, electroblotting to either PVDF or nitrocellulose is the simplest and most common procedure for recovering protein free of contaminants (SDS, acrylamide, and so forth) with a high yield. The first report for enzymatic digestion of a nitrocellulose-bound protein for internal sequence analysis was by Aebersold et al. in 1987, with a more detailed procedure later reported by Tempst et al. in 1990 (1, 2). Basically, these procedures first treated the nitrocellulose-bound protein with PVP-40 (polyvinyl pyrrolidone, M r 40,000) to prevent enzyme adsorption to any remaining nonspecific protein binding sites on the membrane, washed extensively to remove excess PVP-40, and the sample was enzymatically digested at 37°C overnight. Attempts with PVDF-bound protein using the above procedures (3, 4) give poor results and generally require >25 μg of protein. PVDF is preferred over nitrocellulose because it can be used for a variety of other structural analysis procedures, such as amino-terminal sequence analysis and amino acid analysis. In addition, peptide recovery from PVDF-bound protein is higher, particularly from higher retention PVDF (ProBlott, Westran, Immobilon Psq). Finally, PVDF-bound protein can be stored dry as opposed to nitrocellulose, which must remain wet during storage and work up to prevent losses during digestion.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
To start to understand the role of chromatin structure in regulating transcription in trypanosomes, we analyzed covalent modifications on the four core histones of Trypanosoma brucei . We found unusually few modifications in the N‐terminal tails, which are abundantly modified in other organisms and whose sequences, but not composition, are highly divergent in trypanosomes. In contrast, the C‐terminal region of H2A appears to be hyper‐acetylated. Surprisingly, the N‐terminal alanines of H2A, H2B, and H4, were mono‐methylated, a modification that has not been described previously for histones. Possible functions and evolutionary explanations for these unusual histone modifications are discussed.
The development of high-performance organic electrodes for potassium-ion batteries (KIBs) is attracting interest due to their sustainability and low costs. However, the electrolyte systems and moieties that generally proved to be successful in high-performance Li-ion batteries have found relatively little success in KIBs. Herein, two alkynyl-based covalent organic frameworks (COFs) containing 1,3,5-tris(arylethynyl)benzene (TAEB) and dehydrobenzoannulene (DBA) units are utilized as bulk anode materials for KIBs in a localized high-concentration electrolyte. TAEB-COF provides a high capacity value of 254.0 mAh g–1 at ∼100% efficiency after 300 cycles, and DBA-COF 3 provides a capacity of 76.3 mAh g–1 with 98.7% efficiency after 300 cycles. DFT calculations suggest that the alkynyl units of TAEB-COF facilitate the binding of K-ions through both enthalpic and geometric driving forces, leading to high reversible capacities.
