Targeting SARS-CoV-2 by synthetic dual-acting thiol compounds that inhibit Spike/ACE2 interaction and viral protein production.

The SARS-CoV-2 life cycle is strictly dependent on the environmental redox state that influences both virus entry and replication. A reducing environment impairs the binding of the spike protein (S) to the angiotensin-converting enzyme 2 receptor (ACE2), while a highly oxidizing environment is thought to favor S interaction with ACE2. Moreover, SARS-CoV-2 interferes with redox homeostasis in infected cells to promote the oxidative folding of its own proteins. Here we demonstrate that synthetic low molecular weight (LMW) monothiol and dithiol compounds induce a redox switch in the S protein receptor binding domain (RBD) toward a more reduced state. Reactive cysteine residue profiling revealed that all the disulfides present in RBD are targets of the thiol compounds. The reduction of disulfides in RBD decreases the binding to ACE2 in a cell-free system as demonstrated by enzyme-linked immunosorbent and surface plasmon resonance (SPR) assays. Moreover, LMW thiols interfere with protein oxidative folding and the production of newly synthesized polypeptides in HEK293 cells expressing the S1 and RBD domain, respectively. Based on these results, we hypothesize that these thiol compounds impair both the binding of S protein to its cellular receptor during the early stage of viral infection, as well as viral protein folding/maturation and thus the formation of new viral mature particles. Indeed, all the tested molecules, although at different concentrations, efficiently inhibit both SARS-CoV-2 entry and replication in Vero E6 cells. LMW thiols may represent innovative anti-SARS-CoV-2 therapeutics acting directly on viral targets and indirectly by inhibiting cellular functions mandatory for viral replication.

Dithiol Based on l-Cysteine and Cysteamine as a Disulfide-Reducing Agent.

We report the synthesis, chemical properties, and disulfide bond-reducing performance of a dithiol called NACMEAA, conceived as a hybrid of two biologically relevant thiols: cysteine and cysteamine. NACMEAA is conveniently prepared from inexpensive l-cystine in an efficient manner. As a nonvolatile, highly soluble, and neutral compound at physiological pH with the first thiol pKa value of 8.0, NACMEAA is reactive and user-friendly. We also demonstrate that NACMEAA reduces disulfide bonds in GSSG and lysozyme.

Intracellular Redox-Modulated Pathways as Targets for Effective Approaches in the Treatment of Viral Infection.

Host-directed therapy using drugs that target cellular pathways required for virus lifecycle or its clearance might represent an effective approach for treating infectious diseases. Changes in redox homeostasis, including intracellular glutathione (GSH) depletion, are one of the key events that favor virus replication and contribute to the pathogenesis of virus-induced disease. Redox homeostasis has an important role in maintaining an appropriate Th1/Th2 balance, which is necessary to mount an effective immune response against viral infection and to avoid excessive inflammatory responses. It is known that excessive production of reactive oxygen species (ROS) induced by viral infection activates nuclear factor (NF)-kB, which orchestrates the expression of viral and host genes involved in the viral replication and inflammatory response. Moreover, redox-regulated protein disulfide isomerase (PDI) chaperones have an essential role in catalyzing formation of disulfide bonds in viral proteins. This review aims at describing the role of GSH in modulating redox sensitive pathways, in particular that mediated by NF-kB, and PDI activity. The second part of the review discusses the effectiveness of GSH-boosting molecules as broad-spectrum antivirals acting in a multifaceted way that includes the modulation of immune and inflammatory responses.

The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker ß-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.

Design, Synthesis, and Biological Activity of Hydrogen Peroxide Responsive Arylboronate Melatonin Hybrids.

Stimulus-responsive cleavage reactions have found broad use to direct drug release at a particular target disease area. Increased levels of reactive oxygen species (ROS) have been associated with the development and progression of cancer and several other disease states, motivating the development of drug conjugates that can undergo a chemoselective ROS-triggered release. Melatonin (MLT) and the reactive electrophile p-benzoquinone methide ( p-QM) have evidenced either cytoprotective or cytotoxic effects in biological systems, depending on the dose, cellular targets, and time of exposure. In this study, we report the synthesis and biological activity of two MLT derivatives linked to ROS-responsive arylboronate triggers (P1 and P2), which can be activated by endogenously generated hydrogen peroxide (H2O2) to release MLT, or 5-methoxytryptamine (5-MeOT), and p-QM-intermediates. Their H2O2-induced activation mechanism was studied by HPLC-DAD-MS. P1, which rapidly releases MLT and p-QM, was able to strongly induce the Nrf2 antioxidant signaling pathway, but was ineffective to provide protection against H2O2-mediated oxidative damage. By contrast, P1 exhibited strong toxic effects in HeLa cancer cells, without causing significant toxicity to normal NCTC-2544 cells. Similar, although more limited, effects were exerted by P2. In both cases, cytotoxicity was accompanied by depletion of cellular glutathione (GSH), probably as a consequence of p-QM release, and increased ROS levels. A role for MLT in toxicity was also observed, suggesting that the P1 released products, MLT and p-QM, contributed additively to promote cell death.

The dual role of mitochondrial superoxide in arsenite toxicity: Signaling at the boundary between apoptotic commitment and cytoprotection.

Arsenite toxicity is in numerous cellular systems dependent on the formation of reactive oxygen and or nitrogen species. This is also true in U937 cells in which the metalloid selectively promotes the formation of mitochondrial superoxide (mitoO2-) rapidly converted to diffusible H2O2. We tested the hypothesis that, under the same conditions, mitoO2- also mediates the triggering of a parallel survival signaling. We found that a low concentration of the metalloid causes an early activation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), and a downstream signaling leading to enhanced GSH biosynthesis, via a mechanism sensitive to various treatments/strategies selectively preventing mitoO2- formation. Under the same conditions, the toxic effects mediated by arsenite, leading to delayed mitochondrial permeability transition (MPT)-dependent apoptosis, were also prevented. Additional studies revealed remarkable similarities in the kinetics of mitoO2- formation, MPT induction, Nrf2 activation and GSH biosynthesis, prior to the onset of apoptosis in a small portion of the cells. Importantly, mitoO2- formation, as well as the ensuing toxic events, were significantly potentiated and anticipated under conditions associated with inhibition of de novo GSH biosynthesis triggered by the metalloid through Nrf2 activation. We conclude that, in the arsenite toxicity paradigm under investigation, mitoO2- represents the only trigger of two opposite pathways leading to activation of the Nrf2 signaling and/or to a MPT-dependent apoptotic death. The first pathway, through enhanced GSH biosynthesis, mitigates the extent of further mitoO2- formation, thereby limiting and delaying an otherwise rapid and massive apoptotic death.

A biotechnological approach for the production of new protein bioplastics.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.

Gastric cancer cell types display distinct proteasome/immunoproteasome patterns associated with migration and resistance to proteasome inhibitors.

PURPOSE: Gastric cancers (GC) display histological and molecular differences. This heterogeneity has limited the development of new therapeutic strategies which requires the identification of the molecular players involved in GC pathogenesis and the investigation of their responsiveness to drugs. Several proteasome subunits have been identified as prognostic markers in GC and their role studied by gene knockdown. However, proteasomes are multi-subunit protein complexes co-existing in multiple forms with distinct activity/specificity and ability to change in response to inhibitors. Information on the role of different proteasome particles in cancer and their relevance as therapeutic targets is limited. METHODS: Based on this evidence, subunit assembly into proteasome complexes and activity were investigated by native PAGE followed by immunoblotting, and by using fluorogenic substrates, respectively. RESULTS: Here we show that GC cell lines with epithelial and/or diffuse Lauren's histotype express different levels of immunoproteasome subunits and equal amounts of constitutive counterparts. Immunoproteasome subunits were highly expressed and preferentially assembled into 19S capped complexes in diffuse-type cells, where most of the activity was catalyzed by the 26S and 30S particles. In epithelial cells, activity appeared equally distributed between 19S- and 11S-capped proteolytic particles. This proteasome pattern was associated with higher resistance of diffuse-type cells to proteasome inhibition. Immunoproteasome inhibition by ONX 0914 did not influence cell viability but affected metastatic cell migration. CONCLUSIONS: These results suggest that pharmacological inhibition of the immunoproteasome may be useful in treating metastatic gastric cancers.

Nrf2-Mediated Pathway Activated by Prunus spinosa L. (Rosaceae) Fruit Extract: Bioinformatics Analyses and Experimental Validation.

In our previous studies, Prunus spinosa fruit (PSF) ethanol extract was showed to exert antioxidant, antimicrobial, anti-inflammatory and wound healing activities. In the present study, an integrated bioinformatics analysis combined with experimental validation was carried out to investigate the biological mechanism(s) that are responsible for the reported PSF beneficial effects as an antioxidant during a pro-inflammatory TLR4 insult. Bioinformatics analysis using miRNet 2.0 was carried out to address which biological process(es) the extract could be involved in. In addition, Chemprop was employed to identify the key targets of nuclear receptor (NR) signaling and stress response (SR) pathways potentially modulated. The miRNet analysis suggested that the PSF extract mostly activates the biological process of cellular senescence. The Chemprop analysis predicted three possible targets for nine phytochemicals found in the extract: (i) ARE signaling, (ii) mitochondrial membrane potential (MMP) and (iii) p53 SR pathways. The PSF extract antioxidant effect was also experimentally validated in vitro using the human monocyte U937 cell line. Our findings showed that Nrf2 is modulated by the extract with a consequent reduction of the oxidative stress level. This was confirmed by a strong decrease in the amount of reactive oxygen species (ROS) observed in the PSF-treated cells subjected to lipopolysaccharide (LPS) (6 h treatment, 1 µg/mL). No visible effects were observed on p53 and MMP modulation.

Unique toxin profile of a Mediterranean Ostreopsis cf. ovata strain: HR LC-MS(n) characterization of ovatoxin-f, a new palytoxin congener.

Currently, the benthic dinoflagellate Ostreopsis cf. ovata represents a serious concern to human health in the whole Mediterranean basin due to the production of palytoxin congeners, a putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d/-e), listed among the most potent marine toxins. High resolution liquid chromatography-mass spectrometry (HR LC-MS) based investigation of a North Western Adriatic strain of Ostreopsis cf. ovata collected at Portonovo (Italy) in 2008 is reported herein. Toxin profile was different from those previously reported for other O. cf. ovata, both qualitatively and quantitatively. For the first time, ovatoxin-a did not dominate the toxin profile, and a new palytoxin congener, here named ovatoxin-f, was detected. Ovatoxin-f and its elemental formula present C(2)H(4) more than ovatoxin-a. HR CID MS(n) experiments allowed us to restrict structural differences between ovatoxin-a and -f to the region between C-95 and C-102, a region not previously been described to be modified in other palytoxins. Ovatoxin-f represents the major component of the toxin profile of the analyzed strain accounting for 50% of the total toxin content, while ovatoxin-a, the dominant toxin in most of the Mediterranean O. cf. ovata strains we have analyzed so far, is the second major component of the toxin profile (23%). Thus, the presence of ovatoxin-f should be taken into account when monitoring programs for palytoxin-like compounds in microalgae and/or seawater are carried out.

Development and in vitro characterization of a humanized scFv against fungal infections.

The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against ß-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind ß-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.

3ß-Hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (SEC-B) Induces Proinflammatory Activation of Human Endothelial Cells Associated with Nitric Oxide Production and Endothelial Nitric Oxide Synthase/Caveolin-1 Dysregulation.

Oxysterols are a family of 27-carbon cholesterol oxidation derivatives found in low-density lipoproteins (LDLs) and atherosclerotic plaques where they trigger several biological responses involved in the initiation and progression of atherosclerosis. Several pieces of evidence suggest that oxysterols contribute to endothelial dysfunction (ED) due to their ability to alter membrane fluidity and cell permeability leading to inflammation, oxidative stress and apoptosis. The present study aimed to investigate the molecular events occurring in human microvascular endothelial cells (HMEC-1) in response to autoxidation-generated 3ß-hydroxy-5ß-hydroxy-B-norcholestane-6ß-carboxaldehyde (SEC-B) exposure. Our results highlight that SEC-B rapidly activates HMEC-1 by inducing oxidative stress, nitric oxide (NO) production and pro-inflammatory cytokine release. Exposure to SEC-B up to 24 h results in persistent accumulation of the vasodilator NO paralleled by an upregulation of the endothelial nitric oxide synthase (eNOS) enzyme and downregulation of Caveolin-1 (Cav-1) protein levels. Moreover, reduced expression and extracellular release of the vasoconstrictor factor endothelin-1 (ET-1) are observed. Furthermore, SEC-B stimulates the expression of the cytokines interleukin-6 (IL-6) and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). This proinflammatory state leads to increased monocyte recruitment on activated HMEC-1 cells. Our findings add new knowledge on the role of SEC-B in ED and further support its potential implication in atherosclerosis.

LDL receptors, caveolae and cholesterol in endothelial dysfunction: oxLDLs accomplices or victims?

Oxidized LDLs (oxLDLs) and oxysterols play a key role in endothelial dysfunction and the development of atherosclerosis. The loss of vascular endothelium function negatively impacts vasomotion, cell growth, adhesiveness and barrier functions. While for some of these disturbances, a reasonable explanation can be provided from a mechanistic standpoint, for many others, the molecular mediators that are involved are unknown. Caveolae, specific plasma membrane domains, have recently emerged as targets and mediators of oxLDL-induced endothelial dysfunction. Caveolae and their associated protein caveolin-1 (Cav-1) are involved in oxLDLs/LDLs transcytosis, mainly through the scavenger receptor class B type 1 (SR-B1 or SCARB1). In contrast, oxLDLs endocytosis is mediated by the lectin-like oxidized LDL receptor 1 (LOX-1), whose activity depends on an intact caveolae system. In addition, LOX-1 regulates the expression of Cav-1 and vice versa. On the other hand, oxLDLs may affect cholesterol plasma membrane content/distribution thus influencing caveolae architecture, Cav-1 localization and the associated signalling. Overall, the evidence indicate that caveolae have both active and passive roles in oxLDL-induced endothelial cell dysfunction. First, as mediators of lipid uptake and transfer in the subendothelial space and, later, as targets of changes in composition/dynamics of plasma membrane lipids resulting from increased levels of circulating oxLDLs. Gaining a better understanding of how oxLDLs interact with endothelial cells and modulate caveolae-mediated signalling pathways, leading to endothelial dysfunction, is crucial to find new targets for intervention to tackle atherosclerosis and the related clinical entities. LINKED ARTICLES: This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Activation of NRF2 and ATF4 Signaling by the Pro-Glutathione Molecule I-152, a Co-Drug of N-Acetyl-Cysteine and Cysteamine.

I-152 combines two pro-glutathione (GSH) molecules, namely N-acetyl-cysteine (NAC) and cysteamine (MEA), to improve their potency. The co-drug efficiently increases/replenishes GSH levels in vitro and in vivo; little is known about its mechanism of action. Here we demonstrate that I-152 not only supplies GSH precursors, but also activates the antioxidant kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (KEAP1/NRF2) pathway. The mechanism involves disulfide bond formation between KEAP1 cysteine residues, NRF2 stabilization and enhanced expression of the γ-glutamil cysteine ligase regulatory subunit. Accordingly, a significant increase in GSH levels, not reproduced by treatment with NAC or MEA alone, was found. Compared to its parent compounds, I-152 delivered NAC more efficiently within cells and displayed increased reactivity to KEAP1 compared to MEA. While at all the concentrations tested, I-152 activated the NRF2 pathway; high doses caused co-activation of activating transcription factor 4 (ATF4) and ATF4-dependent gene expression through a mechanism involving Atf4 transcriptional activation rather than preferential mRNA translation. In this case, GSH levels tended to decrease over time, and a reduction in cell proliferation/survival was observed, highlighting that there is a concentration threshold which determines the transition from advantageous to adverse effects. This body of evidence provides a molecular framework for the pro-GSH activity and dose-dependent effects of I-152 and shows how synergism and cross reactivity between different thiol species could be exploited to develop more potent drugs.

The Synthetic Cannabinoid URB447 Reduces Brain Injury and the Associated White Matter Demyelination after Hypoxia-Ischemia in Neonatal Rats.

The number of functions controlled by the endocannabinoid system in health and disease continues growing over the years. In the brain, these include the modulation of harmful events such as glutamate excitotoxicity, oxidative stress, and inflammation, mainly regulated by activation/blockade of CB1/CB2 cannabinoid receptors. In the present work, we evaluated the capacity of the CB1 antagonist/CB2 agonist synthetic cannabinoid URB447 on reducing neurodegeneration after brain injury. By using a model of hypoxia-ischemia (HI) in neonatal rats, we found that URB447 strongly reduced brain injury when administered before HI. A comparable effect was observed with the CB1 antagonist SR141716A, whereas the CB1 agonist WIN-55,212-2 reduced the effect of URB447. When administered 3 h after HI, which is considered a clinically feasible therapeutic window to treat perinatal brain injury in humans, URB447 reduced neurodegeneration and white matter damage. Markers of astrogliosis and microglial activation also appeared reduced. These results confirm the important role played by the endocannabinoid system in the neurodegenerative process and strongly encourage further research into the mechanisms of URB447-induced neuroprotection.

I-152, a supplier of N-acetyl-cysteine and cysteamine, inhibits immunoglobulin secretion and plasma cell maturation in LP-BM5 murine leukemia retrovirus-infected mice by affecting the unfolded protein response.

Excessive production of immunoglobulins (Ig) causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR). Hypergammaglobulinemia and lymphadenopathy are hallmarks of murine AIDS that develops in mice infected with the LP-BM5 murine leukemia retrovirus complex. In these mice, Th2 polarization and aberrant humoral response have been previously correlated to altered intracellular redox homeostasis. Our goal was to understand the role of the cell's redox state in Ig secretion and plasma cell (PC) maturation. To this aim, LP-BM5-infected mice were treated with I-152, an N-acetyl-cysteine and cysteamine supplier. Intraperitoneal I-152 administration (30 µmol/mouse three times a week for 9 weeks) decreased plasma IgG and increased IgG/Syndecan 1 ratio in the lymph nodes where IgG were in part accumulated within the ER. PC containing cytoplasmic inclusions filled with IgG were present in all animals, with fewer mature PC in those treated with I-152. Infection induced up-regulation of signaling molecules involved in the UPR, i.e. CHAC1, BiP, sXBP-1 and PDI, that were generally unaffected by I-152 treatment except for PDI and sXBP-1, which have a key role in protein folding and PC maturation, respectively. Our data suggest that one of the mechanisms through which I-152 can limit hypergammaglobulinemia in LP-BM5-infected mice is by influencing IgG folding/assembly as well as secretion and affecting PC maturation.

De-ubiquitylation is the most critical step in the ubiquitin-mediated homeostatic control of the NF-kappaB/IKK basal activity.

The role of ubiquitylation in signal-induced activation of nuclear factor -kappaB (NF-kappaB) has been well established, while its involvement in maintaining NF-kappaB basal activity is less certain. Recent evidences demonstrate that in unstimulated cells, NF-kappaB homeostasis is actually the result of opposing forces: pro-activating activity of the IkappaB Kinase (IKK) and inhibitory activity of the Inhibitor of -kappaB (IkappaB) proteins. It is well known that endogenous de-ubiquitylating mechanisms are less effective on Ub motifs containing UbG76A. Here, we show that overexpression of a ubiquitin (Ub) G76A mutant leads to persistent activation of the IKK/NF-kappaB pathway in the absence of extra-cellular stimuli. In contrast, no effects on NF-kappaB activation were observed upon expression of UbK48R and UbK63R mutants, which are known to impair elongation of Lys(48)- and Lys(63)-linked poly-ubiquitin chains, respectively. Overall, these findings indicate that under basal conditions, the rate of de-ubiquitylation, rather than that of substrate ubiquitylation, is critical for the maintenance of appropriate levels of IKK/NF-kappaB activity.

Proteasome-mediated remodeling of the proteome and phosphoproteome during kiwifruit pollen germination.

Proteasome activity is essential for pollen tube emergence and growth; nevertheless, little is known about proteasome function at the molecular level. The objective of this study was to identify molecular targets and pathways which are directly/indirectly controlled by the proteasome during pollen germination. To this aim, changes in the proteome and phosphoproteome of Actinidia pollen, germinated in the presence of the proteasome inhibitor MG132, were investigated. Phosphoproteins were enriched by metal oxide/hydroxide affinity chromatography and phosphopeptides were further isolated using titanium ion (Ti4+) functional magnetic microparticles prior to liquid chromatography-tandem mass spectrometry analysis. Our results show that proteasome inhibition affects the phosphoproteome more profoundly than the proteome. Accordingly, the steady-state abundance of some kinases and phosphatases was changed and/or their phosphorylation status altered. Notably, affected proteins are involved in processes that are fundamental to pollen germination such as cytoskeletal organization, vesicular transport, cell wall synthesis and remodeling, protein synthesis, folding and degradation as well as energetic metabolism. Our data provide a molecular framework for the structural alterations observed when the proteasome is inhibited, contribute to the understanding of how proteasome activity regulates pollen germination, show the cross-talk between phosphorylation and proteasomal degradation and are a resource for further functional analyses. SIGNIFICANCE: Pollen germination and tube growth are fundamental to successful fertilization in seed plants. These events are based on dramatic remodeling and the dismantling of existing programs, which are replaced by new ones. Degradation plays a prominent role in reshaping the protein repertoire, also cross talking with the bulk of post-translational modifications. At present, phosphorylation is the only modification studied in germinating pollen on a large scale. The proteasome has been universally recognized as one of the most important sites for protein degradation and its function has been shown to be essential for pollen tube emergence and elongation. Upon proteasome inhibition structural alterations and dysregulation of pivotal processes governing pollen germination have been described; however, a mechanistic framework for the proteasome function at the molecular level is still lacking. In this investigation we provide the very first view of the global impact of the proteasome in remodeling the proteome and phosphoproteome during germination and tube growth. Our results show how proteasome inhibition alters the levels, and profoundly affects the phosphorylation status of many proteins involved, controlling energetic and synthetic pathways and signaling cascades.

Boosting GSH Using the Co-Drug Approach: I-152, a Conjugate of N-acetyl-cysteine and ß-mercaptoethylamine.

Glutathione (GSH) has poor pharmacokinetic properties; thus, several derivatives and biosynthetic precursors have been proposed as GSH-boosting drugs. I-152 is a conjugate of N-acetyl-cysteine (NAC) and S-acetyl-ß-mercaptoethylamine (SMEA) designed to release the parent drugs (i.e., NAC and ß-mercaptoethylamine or cysteamine, MEA). NAC is a precursor of L-cysteine, while MEA is an aminothiol able to increase GSH content; thus, I-152 represents the very first attempt to combine two pro-GSH molecules. In this review, the in-vitro and in-vivo metabolism, pro-GSH activity and antiviral and immunomodulatory properties of I-152 are discussed. Under physiological GSH conditions, low I-152 doses increase cellular GSH content; by contrast, high doses cause GSH depletion but yield a high content of NAC, MEA and I-152, which can be used to resynthesize GSH. Preliminary in-vivo studies suggest that the molecule reaches mouse organs, including the brain, where its metabolites, NAC and MEA, are detected. In cell cultures, I-152 replenishes experimentally depleted GSH levels. Moreover, administration of I-152 to C57BL/6 mice infected with the retroviral complex LP-BM5 is effective in contrasting virus-induced GSH depletion, exerting at the same time antiviral and immunomodulatory functions. I-152 acts as a pro-GSH agent; however, GSH derivatives and NAC cannot completely replicate its effects. The co-delivery of different thiol species may lead to unpredictable outcomes, which warrant further investigation.

A negative feedback mechanism links UBC gene expression to ubiquitin levels by affecting RNA splicing rather than transcription.

UBC gene plays a critical role in maintaining ubiquitin (Ub) homeostasis. It is upregulated under stress conditions, and herein we report that it is downregulated upon Ub overexpression. Downregulation occurs in a dose-dependent manner, suggesting the existence of a fine-tuned Ub sensing mechanism. This "sensor" requires a conjugation competent ubiquitin to detect Ub levels. Searching the sensor among the transcription factors involved in basal and stress-induced UBC gene expression was unsuccessful. Neither HSF1 and HSF2, nor Sp1 and YY1 are affected by the increased Ub levels. Moreover, mutagenesis of their binding sites in the UBC promoter-driven reporter constructs does not impair the downmodulation effect. Epigenetic studies show that H2A and H2B ubiquitination within the UBC promoter region is unchanged upon ubiquitin overexpression. Noteworthy, quantification of nascent RNA molecules excludes that the downmodulation arises in the transcription initiation step, rather pointing towards a post-transcriptional mechanism. Indeed, a significantly higher fraction of unspliced UBC mRNA is detected in ubiquitin overexpressing cells, compared to empty vector transfected cells. Our findings suggest how increasing cellular ubiquitin levels may control the expression of UBC gene by negatively affecting the splicing of its pre-mRNA, providing a straightforward feedback strategy for the homeostatic control of ubiquitin pools.