The ubiquitin–proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1–Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin–proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.

Autophagy, the intracellular lysosomal degradation process plays a pivotal role in podocyte homeostasis in diabetic kidney disease (DKD). Lysosomal function, autophagic activity, and their actions were investigated in vitro and in vivo. We found that LC3-II- and p62-positive vacuoles accumulated in podocytes of patients with DKD. Moreover, we found that advanced glycation end products (AGEs) could increase the protein expression of LC3-II and p62 in a dose- and time-dependent manner in cultured podocytes. However, the mRNA expression of LC3B, Beclin-1 or ATG7, as well as the protein level of Beclin-1 or ATG7 did not change significantly in the AGE-treated cells compared with that in control groups, suggesting that AGEs did not induce autophagy. In addition, AGEs led to an increase in the number of autophagosomes but not autolysosomes, accompanied with a failure in lysosomal turnover of LC3-II or p62, indicating that the degradation of autophagic vacuoles was blocked. Furthermore, we observed a dramatic decrease in the enzymatic activities, and the degradation of DQ-ovalbumin was significantly suppressed after podocytes were treated with AGEs. Plasma-irregular lysosomal-associated membrane protein 1 granules accompanied with the diffusion of cathepsin D expression and acridine orange redistribution were observed in AGE-treated podocytes, indicating that the lysosomal membrane permeability was triggered. Interestingly, we also found that AGEs-induced autophagic inhibition and podocyte injury were mimicked by the specific lysosomotropic agent, l-leucyl-l-leucine methyl ester. The exacerbated apoptosis and Rac-1-dependent actin-cytoskeletal disorganization were alleviated by an improvement in the lysosomal-dependent autophagic pathway by resveratrol plus vitamin E treatment in AGE-treated podocytes. However, the rescued effects were reversed by the addition of leupeptin, a lysosomal inhibitor. It suggests that restoring lysosomal function to activate autophagy may contribute to the development of new therapeutic strategies for DKD.

The reversible post-translational modification (PTM) of eukaryotic proteins by ubiquitin (Ub) regulates key cellular processes including protein degradation and gene transcription. Studies of the mechanistic roles for protein ubiquitylation require quantities of homogenously modified substrates that are typically inaccessible from natural sources or by enzymatic ubiquitylation in vitro. Therefore, we developed a facile and scalable methodology for site-specific chemical ubiquitylation. Our semisynthetic strategy utilized a temporary ligation auxiliary, 2-(aminooxy)ethanethiol, to direct ubiquitylation at specific lysines in peptide substrates. Mild reductive removal of the auxiliary after ligation yielded ubiquitylated peptides with the native isopeptide linkage. Alternatively, retention of the ligation auxiliary yielded protease-resistant analogues of ubiquitylated peptides. Importantly, our strategy was fully compatible with protein sulfhydryl groups, as demonstrated by the synthesis of peptides modified by the human small ubiquitin-related modifier 3 (SUMO-3) protein.

The complex of the HIV TAR RNA with the viral regulatory protein Tat is of considerable interest, but the plasticity of this interaction has made it impossible so far to establish the structure of that complex. In order to explore a new approach to obtain structural information on protein-RNA complexes, we performed 13C/15N-19F REDOR NMR experiments in the solid state on TAR bound to a peptide comprising the RNA-binding section of Tat. A critical arginine in the peptide was uniformly 13C and 15N labeled and 5-fluorouridine was incorporated at the U23 position of TAR. REDOR irradiation resulted in dephasing of the 13C and 15N resonances, indicating proximity of the U23(5F)-C and U23(5F)-N spin pairs. Best fits to the REDOR data shows the U23(5F)-C distances and the U23(5F)-N distances are in good agreement with the distances obtained from solution NMR structures of partial complexes of Tat with TAR. These results demonstrate that it is possible to study protein-RNA complexes using solid-state REDOR NMR measurements, adding to a growing list of solid state techniques for studying protein-nucleic acid complexes.

Formation of the complex between human immunodeficiency virus type-1 Tat protein and the transactivation response region (TAR) RNA is vital for transcriptional elongation, yet the structure of the Tat-TAR complex remains to be established. The NMR structures of free TAR, and TAR bound to Tat-derived peptides have been obtained by solution NMR, but only a small number of intermolecular NOEs could be identified unambiguously, preventing the determination of a complete structure. Here we show that a combination of multiple solid state NMR REDOR experiments can be used to obtain multiple distance constraints from (15)N to (13)C spins within the backbone and side chain guanidinium groups of arginine in a Tat-derived peptide, using (19)F spins incorporated into the base of U23 in TAR and (31)P spins in the P22 and P23 phosphate groups. Distances between the side chain of Arg52 and the base and phosphodiester backbone near U23 measured by REDOR NMR are comparable to distances observed in solution NMR-derived structural models, indicating that interactions of TAR RNA with key amino acid side chains in Tat are the same in the amorphous solid state as in solution. This method is generally applicable to other protein-RNA complexes where crystallization or solution NMR has failed to provide high resolution structural information.

No abstract

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program “Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)”. We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T1ρ relaxation times for 13C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T1ρ times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 μs.

Intrinsic motions may allow HIV-1 transactivation response (TAR) RNA to change its conformation to form a functional complex with the Tat protein, which is essential for viral replication. Understanding the dynamic properties of TAR necessitates determining motion on the intermediate nanosecond-to-microsecond time scale. To this end, we performed solid-state deuterium NMR line-shape and T relaxation-time experiments to measure intermediate motions for two uridine residues, U40 and U42, within the lower helix of TAR. We infer global motions at rates of ∼10 s in the lower helix, which are much slower than those in the upper helix (∼10 s), indicating that the two helical domains reorient independently of one another in the solid-state sample. These results contribute to the aim of fully describing the properties of functional motions in TAR RNA.