Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.     

High-Fat Diet Leads to Reduced Protein O-GlcNAcylation and Mitochondrial Defects Promoting the Development of Alzheimer’s Disease Signatures

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer’s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.     

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca²⁺ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α₁α₂ loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α₂ and α₃-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α₂-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein–protein interactions.     

Adenosine Signaling in Mast Cells and Allergic Diseases

Adenosine is a nucleoside involved in the pathogenesis of allergic diseases. Its effects are mediated through its binding to G protein-coupled receptors: A1, A2a, A2b and A3. The receptors differ in the type of G protein they recruit, in the effect on adenylyl cyclase (AC) activity and the downstream signaling pathway triggered. Adenosine can produce both an enhancement and an inhibition of mast cell degranulation, indicating that adenosine effects on these receptors is controversial and remains to be clarified. Depending on the study model, A1, A2b, and A3 receptors have shown anti- or pro-inflammatory activity. However, most studies reported an anti-inflammatory activity of A2a receptor. The precise knowledge of the adenosine mechanism of action may allow to develop more efficient therapies for allergic diseases by using selective agonist and antagonist against specific receptor subtypes.     

Solubility and Aggregation of Selected Proteins Interpreted on the Basis of Hydrophobicity Distribution

Protein solubility is based on the compatibility of the specific protein surface with the polar aquatic environment. The exposure of polar residues to the protein surface promotes the protein’s solubility in the polar environment. The aquatic environment also influences the folding process by favoring the centralization of hydrophobic residues with the simultaneous exposure to polar residues. The degree of compatibility of the residue distribution, with the model of the concentration of hydrophobic residues in the center of the molecule, with the simultaneous exposure of polar residues is determined by the sequence of amino acids in the chain. The fuzzy oil drop model enables the quantification of the degree of compatibility of the hydrophobicity distribution observed in the protein to a form fully consistent with the Gaussian 3D function, which expresses an idealized distribution that meets the preferences of the polar water environment. The varied degrees of compatibility of the distribution observed with the idealized one allow the prediction of preferences to interactions with molecules of different polarity, including water molecules in particular. This paper analyzes a set of proteins with different levels of hydrophobicity distribution in the context of the solubility of a given protein and the possibility of complex formation.     

Genome-Wide Analysis of the Late Embryogenesis Abundant (LEA) and Abscisic Acid-, Stress-, and Ripening-Induced (ASR) Gene Superfamily from Canavalia rosea and Their Roles in Salinity/Alkaline and Drought Tolerance

Canavalia rosea (bay bean), distributing in coastal areas or islands in tropical and subtropical regions, is an extremophile halophyte with good adaptability to seawater and drought. Late embryogenesis abundant (LEA) proteins typically accumulate in response to various abiotic stresses, including dehydration, salinity, high temperature, and cold, or during the late stage of seed development. Abscisic acid-, stress-, and ripening-induced (ASR) genes are stress and developmentally regulated plant-specific genes. In this study, we reported the first comprehensive survey of the LEA and ASR gene superfamily in C. rosea. A total of 84 CrLEAs and three CrASRs were identified in C. rosea and classified into nine groups. All CrLEAs and CrASRs harbored the conserved motif for their family proteins. Our results revealed that the CrLEA genes were widely distributed in different chromosomes, and all of the CrLEA/CrASR genes showed wide expression features in different tissues in C. rosea plants. Additionally, we introduced 10 genes from different groups into yeast to assess the functions of the CrLEAs/CrASRs. These results contribute to our understanding of LEA/ASR genes from halophytes and provide robust candidate genes for functional investigations in plant species adapted to extreme environments.     

The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment

Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.     

Discovery–Versus Hypothesis–Driven Detection of Protein–Protein Interactions and Complexes

Protein complexes are the main functional modules in the cell that coordinate and perform the vast majority of molecular functions. The main approaches to identify and quantify the interactome to date are based on mass spectrometry (MS). Here I summarize the benefits and limitations of different MS-based interactome screens, with a focus on untargeted interactome acquisition, such as co-fractionation MS. Specific emphasis is given to the discussion of discovery- versus hypothesis-driven data analysis concepts and their applicability to large, proteome-wide interactome screens. Hypothesis-driven analysis approaches, i.e., complex- or network-centric, are highlighted as promising strategies for comparative studies. While these approaches require prior information from public databases, also reviewed herein, the available wealth of interactomic data continuously increases, thereby providing more exhaustive information for future studies. Finally, guidance on the selection of interactome acquisition and analysis methods is provided to aid the reader in the design of protein-protein interaction studies.