Impact of Ubiquitination Signaling Pathway Modifications on Oral Carcinoma

Among intra-cellular homeostasis mechanisms, ubiquitination plays a critical role in protein metabolism regulation by degrading proteins via activating a broad spectrum of ubiquitin chains. In fact, ubiquitination and sumoylation signaling pathways are characterized by increased complexity regarding the molecules and their interactions. The Ubiquitin-Proteasome System (Ub-PS) recognizes and targets a broad spectrum of protein substrates. Ubiquitin conjugation modifies each substrate protein determining its biochemical fate (degradation). A major functional activity of Ub-PS is autophagy mechanism regulation. Interestingly, Ub-PS promotesall stages of bulk autophagy (initiation, execution, and termination). Autophagy is a crucial catabolic process that provides protein degradation and for this reason the interaction with Ub-PS is crucial. Furthermore, ubiquitination controls and regulates specific types of protein targets. Ub-PS is also involved in oxidative cellular stress and DNA damage response. Additionally, the functional role of Ub-PS in ribosome machinery regulation seems to be crucial. Concerning carcinogenesis, Ub-PS is involved in malignant disease development and progression by negatively affecting the corresponding TGF-B-, MEEK/MAPK/ERK-JNK- dependent signaling pathways. In the current review article, we describe the role of Ub-PSbiochemicalmodifications and alterations in oral squamous cell carcinoma (OSCC).

Point Mutations at a Key Site Alter the Cytochrome P450 OleP Structural Dynamics

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

The Role of Chaperone-Mediated Autophagy in Hepatitis C Virus-Induced Pathogenesis

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.

Silica/OCP Affects the Viability of Osteoblasts through ROS-Induced Autophagy

Objective. To explore the effects of silicone gel nanoparticles modified with octacalcium phosphate on the surface (silica/OCP) polymer drugs on the proliferation of osteoblasts and autophagy. Method. Silica/OCP was prepared in vitro, and the quality of the sample preparation was tested through characterization experiments. The osteoblast cell line (hFOB1.19) was treated with silica/OCP, autophagy inhibitor (3-methyladenine (3-MA)), and silica/OCP+3-MA, respectively. The proliferation of hFOB1.19 cells was detected through the methylthiazolyldiphenyl-tetrazolium bromide (MTT) kit. Flow cytometry was used to detect the cell apoptosis. The change in protein beclin1 and P62 expression in hFOB1.19 cells was observed in Western blot. An ROS detection kit was used to detect the content of reactive oxygen species in hFOB1.19 cells. Results. Silica/OCP was a sphere with a particle size of 50 nm to 130 nm and had an OCP phase in electron projection microscopy and X-ray diffraction techniques. The results indicated that OCP successfully modified silica and the material was successfully prepared. An MTT kit and flow cytometry test showed that the cell viability of the cells treated with silica/OCP increased significantly ( P < 0.05 ), and the intracellular apoptosis phenomenon was significantly decreased ( P < 0.05 ) compared to the control group. Moreover, the inhibition of cell viability and promotion of apoptosis caused by the autophagy inhibitor 3-MA can be rescued. Western blotting demonstrated that the protein level of beclin1 in osteoblasts reached the highest after six hours of treatment with silica/OCP, and the protein level of p62, the substrate protein of autophagy, reached the lowest. At the same time, treatment of cells with the autophagy inhibitor 3-MA and silica/OCP+3-MA found that the protein levels of beclin1 and p62 in the silica/OCP+3-MA group were adjusted back compared to the 3-MA group. After adding the autophagy inhibitor, the reactive oxygen content in the cell was significantly increased ( P < 0.05 ) in the silica/OCP group. In the presence of intracellular reactive oxygen inhibitors catalase and silica/OCP, the cell viability of osteoblasts was significantly lower than that of the silica/OCP group but significantly higher than that of the silica/OCP+3-MA group. The apoptosis level of the silica/OCP+catalase group was also significantly lower than that of the silica/OCP+3-MA group ( P < 0.05 ) but was significantly higher than that of the silica/OCP group ( P < 0.05 ). Conclusion. Silica/OCP nanoparticles can upregulate the level of autophagy in osteoblasts and promote the proliferation of osteoblasts.

The Biological Function and Roles in Phytohormone Signaling of the F-Box Protein in Plants

The ubiquitin–proteasome pathway (UPP) is an important protein degradation pathway that can participate in the regulation of the physiological process of organisms by specifically removing abnormal peptides and degrading cell regulators. UPP mainly involves three enzymes, among which the E3 ubiquitin ligase function is central to UPP. E3 ubiquitin ligases can recruit substrate protein for ubiquitination, and they have various forms. Among them, the Skp1–Cul1–F-box (SCF) complex is the most representative member of the cullin RING ubiquitin ligases type in RING-domain E3 ligases, being mainly composed of Cullin 1, Skp1, Rbx1, and F-box proteins. The F-box protein is the key component for SCF to perform specific functions. The F-box protein is one of the largest protein families in plants, and its family members are involved in the regulation of many key physiological processes, such as growth and development of plants and the response to external stimuli. Herein, we briefly review the structure, classification, function, and hormone signaling pathways of F-box proteins.

Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble

Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps.

Electroacupuncture Attenuates Inflammatory Pain by Activating AMPK/autophagy and Inhibiting iNOS and IL-1β in Inflamed Tissues

BackgroundElectroacupuncture (EA) produces analgesic effects on inflammatory pain partially via activating adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway in the spinal cord. However, it is unclear whether EA activates AMPK in peripheral tissues in inflammatory pain. This study was aimed at determining whether EA promotes autophagy by activating AMPK to inhibit the expression of inflammatory mediators IL-1β and iNOS in inflamed skin tissues. MethodsIn CFA-induced inflammatory pain in mice, mechanical allodynia and thermal hyperalgesia were tested 2 hours after EA treatment. The AMPK antagonist Compound C was injected intraperitoneally 30 minutes before EA treatment. The analgesic effects of AMPK activator 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) were determined and its effects on autophagy, IL-1β and iNOS expression were detected. Also, the effects of the autophagy inhibitor 3-methyladenine (3-MA) on EA analgesia and iNOS/IL-1β expression in inflamed skin tissues were examined. The phosphorylation of AMPK (Thr172) and total AMPK proteins, LC3BII/I, autophagy substrate protein p62, IL-1β and iNOS were detected using Western blotting. Co-labeling of macrophages (CD68) with IL-1β and iNOS was detected using immunofluorescence. In addition, after NR8383 macrophages were treated with CFA, the effects of AICAR and Compound C on autophagy were determined using stubRFP-sensGFP-LC3 Lentivirus..ResultsEA reduced CFA-induced inflammatory pain, activated AMPK and autophagy, and inhibited iNOS and IL-1β expression in inflamed skin tissues. AICAR also attenuated CFA-induced hyperalgesia, promoted autophagy and inhibited iNOS and IL-1β expression in vivo and in vitro. In addition, the AMPK inhibitor Compound C reversed the effect of EA on autophagy. Pretreatment with 3-MA, an inhibitor of autophagy, inhibited the effect of EA on inflammatory pain and expression of iNOS and IL-1β in inflamed skin tissues. ConclusionsEA treatment alleviated inflammatory pain by activation of AMPK, enhancing autophagy, and inhibiting iNOS and IL-1β expression in the inflamed skin tissues.

The role of calcium binding to the EF-hand-like motif in bacterial solute-binding protein for alginate import

Gram-negative Sphingomonas sp. A1 incorporates acidic polysaccharide alginate into the cytoplasm via a cell-surface alginate-binding protein (AlgQ2)-dependent ATP-binding cassette transporter (AlgM1M2SS). We investigated the function of calcium bound to the EF-hand-like motif in AlgQ2 by introducing mutations at the calcium-binding site. The X-ray crystallography of the AlgQ2 mutant (D179A/E180A) demonstrated the absence of calcium binding and significant disorder of the EF-hand-like motif. Distinct from the wild-type AlgQ2, the mutant was quite unstable at temperature of strain A1 growth, although unsaturated alginate oligosaccharides stabilized the mutant by formation of substrate/protein complex. In the assay of ATPase and alginate transport by AlgM1M2SS reconstructed in the liposome, the wild-type and mutant AlgQ2 induced AlgM1M2SS ATPase activity in the presence of unsaturated alginate tetrasaccharide. These results indicate that the calcium bound to EF-hand-like motif stabilizes the substrate-unbound AlgQ2 but is not required for the complexation of substrate-bound AlgQ2 and AlgM1M2SS.

SUMOylation of Arginyl tRNA Synthetase Modulates the Drosophila Innate Immune Response

SUMO conjugation of a substrate protein can modify its activity, localization, interaction or function. A large number of SUMO targets in cells have been identified by Proteomics, but biological roles for SUMO conjugation for most targets remains elusive. The multi-aminoacyl tRNA synthetase complex (MARS) is a sensor and regulator of immune signaling. The proteins of this 1.2 MDa complex are targets of SUMO conjugation, in response to infection. Arginyl tRNA Synthetase (RRS), a member of the sub-complex II of MARS, is one such SUMO conjugation target. The sites for SUMO conjugation are Lys 147 and 383. Replacement of these residues by Arg (RRSK147R,K383R), creates a SUMO conjugation resistant variant (RRSSCR). Transgenic Drosophila lines for RRSWT and RRSSCR were generated by expressing these variants in a RRS loss of function (lof) animal, using the UAS-Gal4 system. The RRS-lof line was itself generated using CRISPR/Cas9 genome editing. Expression of both RRSWT and RRSSCR rescue the RRS-lof lethality. Adult animals expressing RRSWT and RRSSCR are compared and contrasted for their response to bacterial infection by gram positive M. luteus and gram negative Ecc15. We find that RRSSCR, when compared to RRSWT, shows modulation of the transcriptional response, as measured by quantitative 3′ mRNA sequencing. Our study uncovers a possible non-canonical role for SUMOylation of RRS, a member of the MARS complex, in host-defense.

Novel cryo-EM structure of an ADP-bound GroEL–GroES complex

The GroEL–GroES chaperonin complex is a bacterial protein folding system, functioning in an ATP-dependent manner. Upon ATP binding and hydrolysis, it undergoes multiple stages linked to substrate protein binding, folding and release. Structural methods helped to reveal several conformational states and provide more information about the chaperonin functional cycle. Here, using cryo-EM we resolved two nucleotide-bound structures of the bullet-shaped GroEL–GroES1 complex at 3.4 Å resolution. The main difference between them is the relative orientation of their apical domains. Both structures contain nucleotides in cis and trans GroEL rings; in contrast to previously reported bullet-shaped complexes where nucleotides were only present in the cis ring. Our results suggest that the bound nucleotides correspond to ADP, and that such a state appears at low ATP:ADP ratios.