Nitric Oxide Binding Geometry in Heme-Proteins: Relevance for Signal Transduction.

Nitric oxide (NO) synthesis, signaling, and scavenging is associated to relevant physiological and pathological events. In all tissues and organs, NO levels and related functions are regulated at different levels, with heme proteins playing pivotal roles. Here, we focus on the structural changes related to the different binding modes of NO to heme-Fe(II), as well as the modulatory effects of this diatomic messenger on heme-protein functions. Specifically, the ability of heme proteins to bind NO at either the distal or proximal side of the heme and the transient interchanging of the binding site is reported. This sheds light on the regulation of O2 supply to tissues with high metabolic activity, such as the retina, where a precise regulation of blood flow is necessary to meet the demand of nutrients.

Eye Diseases: When the Solution Comes from Plant Alkaloids.

Plants are an incredible source of metabolites showing a wide range of biological activities. Among these, there are the alkaloids, which have been exploited for medical purposes since ancient times. Nowadays, many plant-derived alkaloids are the main components of drugs used as therapy for different human diseases. This review deals with providing an overview of the alkaloids used to treat eye diseases, describing the historical outline, the plants from which they are extracted, and the clinical and molecular data supporting their therapeutic activity. Among the different alkaloids that have found application in medicine so far, atropine and pilocarpine are the most characterized ones. Conversely, caffeine and berberine have been proposed for the treatment of different eye disorders, but further studies are still necessary to fully understand their clinical value. Lastly, the alkaloid used for managing hypertension, reserpine, has been recently identified as a potential drug for ameliorating retinal disorders. Other important aspects discussed in this review are different solutions for alkaloid production. Given that the industrial production of many of the plant-derived alkaloids still relies on extraction from plants, and the chemical synthesis can be highly expensive and poorly efficient, alternative methods need to be found. Biotechnologies offer a multitude of possibilities to overcome these issues, spanning from genetic engineering to synthetic biology for microorganisms and bioreactors for plant cell cultures. However, further efforts are needed to completely satisfy the pharmaceutical demand.

Ubiquitin proteasome system and glaucoma: A survey of genetics and molecular biology studies supporting a link with pathogenic and therapeutic relevance.

Glaucoma represents a group of progressive neurodegenerative diseases characterized by the loss of retinal ganglion cells (RGCs) and their axons with subsequent visual field impairment. The disease develops through largely uncharacterized molecular mechanisms, that are likely to occur in different localized cell types, either in the anterior (e.g., trabecular meshwork cells) or posterior (e.g., Muller glia, retinal ganglion cells) segments of the eye. Genomic and preclinical studies suggest that glaucoma pathogenesis may develop through altered ubiquitin (Ub) signaling. Ubiquitin conjugation, referred to as ubiquitylation, is a major post-synthetic modification catalyzed by E1-E2-E3 enzymes, that profoundly regulates the turnover, trafficking and biological activity of the targeted protein. The development of new technologies, including proteomics workflows, allows the biology of ubiquitin signaling to be described in health and disease. This post-translational modification is emerging as a key role player in neurodegeneration, gaining relevance for novel therapeutic options, such as in the case of Proteolysis Targeting Chimeras technology. Although scientific evidence supports a link between Ub and glaucoma, their relationship is still not well-understood. Therefore, this review provides a detailed research-oriented discussion on current evidence of Ub signaling in glaucoma. A review of genomic and genetic data is provided followed by an in-depth discussion of experimental data on ASB10, parkin and optineurin, which are proteins that play a key role in Ub signaling and have been associated with glaucoma.

Novel frontiers in neuroprotective therapies in glaucoma: Molecular and clinical aspects.

In the last years, neuroprotective therapies have attracted the researcher interests as modern and challenging approach for the treatment of neurodegenerative diseases, aimed at protecting the nervous system from injuries. Glaucoma is a neurodegenerative disease characterized by progressive excavation of the optic nerve head, retinal axonal injury and corresponding vision loss that affects millions of people on a global scale. The molecular basis of the pathology is largely uncharacterized yet, and the therapeutic approaches available do not change the natural course of the disease. Therefore, in accordance with the therapeutic regimens proposed for other neurodegenerative diseases, a modern strategy to treat glaucoma includes prescription of drugs with neuroprotective activities. With respect to this, several preclinical and clinical investigations on a plethora of different drugs are currently ongoing. In this review, first, the conceptualization of the rationale for the adoption of neuroprotective strategies for retina is summarized. Second, the molecular aspects highlighting glaucoma as a neurodegenerative disease are reported. In conclusion, the molecular and pharmacological properties of most promising direct neuroprotective drugs used to delay glaucoma progression are examined, including: neurotrophic factors, NMDA receptor antagonists, the α2-adrenergic agonist, brimonidine, calcium channel blockers, antioxidant agents, nicotinamide and statins.

The Insulin-Degrading Enzyme from Structure to Allosteric Modulation: New Perspectives for Drug Design.

The insulin-degrading enzyme (IDE) is a Zn2+ peptidase originally discovered as the main enzyme involved in the degradation of insulin and other amyloidogenic peptides, such as the ß-amyloid (Aß) peptide. Therefore, a role for the IDE in the cure of diabetes and Alzheimer's disease (AD) has been long envisaged. Anyway, its role in degrading amyloidogenic proteins remains not clearly defined and, more recently, novel non-proteolytic functions of the IDE have been proposed. From a structural point of view, the IDE presents an atypical clamshell structure, underscoring unique enigmatic enzymological properties. A better understanding of the structure-function relationship may contribute to solving some existing paradoxes of IDE biology and, in light of its multifunctional activity, might lead to novel therapeutic approaches.

Targeting immunoproteasome in neurodegeneration: A glance to the future.

The immunoproteasome is a specialized form of proteasome equipped with modified catalytic subunits that was initially discovered to play a pivotal role in MHC class I antigen processing and immune system modulation. However, over the last years, this proteolytic complex has been uncovered to serve additional functions unrelated to antigen presentation. Accordingly, it has been proposed that immunoproteasome synergizes with canonical proteasome in different cell types of the nervous system, regulating neurotransmission, metabolic pathways and adaptation of the cells to redox or inflammatory insults. Hence, studying the alterations of immunoproteasome expression and activity is gaining research interest to define the dynamics of neuroinflammation as well as the early and late molecular events that are likely involved in the pathogenesis of a variety of neurological disorders. Furthermore, these novel functions foster the perspective of immunoproteasome as a potential therapeutic target for neurodegeneration. In this review, we provide a brain and retina-wide overview, trying to correlate present knowledge on structure-function relationships of immunoproteasome with the variety of observed neuro-modulatory functions.

Citicoline Eye Drops Protect Trabecular Meshwork Cells from Oxidative Stress Injury in a 3D In Vitro Glaucoma Model.

Intraocular pressure (IOP) is considered an important modifiable risk factor for glaucoma, which is known as the second leading cause of blindness worldwide. However, lowering the IOP is not always sufficient to preserve vision due to other non-IOP-dependent mechanisms being involved. To improve outcomes, adjunctive therapies with IOP-independent targets are required. To date, no studies have shown the effect of citicoline on the trabecular meshwork (TM), even though it is known to possess neuroprotective/enhancement properties and multifactorial mechanisms of action. Given that reactive oxygen species seem to be involved in glaucomatous cascade, in this present study, an advanced millifluidic in vitro model was used to evaluate if citicoline could exert a valid TM protection against oxidative stress. To this end, the cellular behavior, in terms of viability, apoptosis, mitochondrial state, senescence and pro-inflammatory cytokines, on 3D human TM cells, treated either with H2O2 alone or cotreated with citicoline, was analyzed. Our preliminary in vitro results suggest a counteracting effect of citicoline eye drops against oxidative stress on TM cells, though further studies are necessary to explore citicoline's potential as a TM-target therapy.

A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose.

BACKGROUND: Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. RESULTS: By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkBα (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1ß and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S α4 subunit. CONCLUSIONS: This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis.

Modulation of the 20S Proteasome Activity by Porphyrin Derivatives Is Steered through Their Charge Distribution.

Cationic porphyrins exhibit an amazing variety of binding modes and inhibition mechanisms of 20S proteasome. Depending on the spatial distribution of their electrostatic charges, they can occupy different sites on α rings of 20S proteasome by exploiting the structural code responsible for the interaction with regulatory proteins. Indeed, they can act as competitive or allosteric inhibitors by binding at the substrate gate or at the grooves between the α subunits, respectively. Moreover, the substitution of a charged moiety in the peripheral arm with a hydrophobic moiety revealed a "new" 20S functional state with higher substrate affinity and catalytic efficiency. In the present study, we expand our structure-activity relationship (SAR) analysis in order to further explore the potential of this versatile class of 20S modulators. Therefore, we have extended the study to additional macrocyclic compounds, displaying different structural features, comparing their interaction behavior on the 20S proteasome with previously investigated compounds. In particular, in order to evaluate how the introduction of a peptidic chain can affect the affinity and the interacting mechanism of porphyrins, we investigate the MTPyApi, a porphyrin derivatized with an Arg-Pro-rich antimicrobial peptide. Moreover, to unveil the role played by the porphyrin core, this was replaced with a corrole scaffold, a "contracted" version of the tetrapyrrolic ring due to the lack of a methine bridge. The analysis has been undertaken by means of integrated kinetic, Nuclear Magnetic Resonance, and computational studies. Finally, in order to assess a potential pharmacological significance of this type of investigation, a preliminary attempt has been performed to evaluate the biological effect of these molecules on MCF7 breast cancer cells in dark conditions, envisaging that porphyrins may indeed represent a powerful tool for the modulation of cellular proteostasis.

Silybins are stereospecific regulators of the 20S proteasome.

A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently "open gate" mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7" and C-8" of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.

Neuroprotective Effect of Carnosine Is Mediated by Insulin-Degrading Enzyme.

l-Carnosine is an endogenous dipeptide that has high potential for therapeutic purposes, being an antioxidant with metal chelating, anti-aggregating, anti-inflammatory, and neuroprotective properties. Despite its potential therapeutic values, the biomolecular mechanisms involved in neuroprotection are not fully understood. Here, we demonstrate, at chemical and biochemical levels, that insulin-degrading enzyme plays a pivotal role in carnosine neuroprotection.

Insulin-Degrading Enzyme Is a Non Proteasomal Target of Carfilzomib and Affects the 20S Proteasome Inhibition by the Drug.

Carfilzomib is a last generation proteasome inhibitor (PI) with proven clinical efficacy in the treatment of relapsed/refractory multiple myeloma. This drug is considered to be extremely specific in inhibiting the chymotrypsin-like activity of the 20S proteasome, encoded by the ß5 subunit, overcoming some bortezomib limitations, the first PI approved for multiple myeloma therapy which is however burdened by a significant toxicity profile, due also to its off-target effects. Here, molecular approaches coupled with molecular docking studies have been used to unveil that the Insulin-Degrading Enzyme, a ubiquitous and highly conserved Zn2+ peptidase, often found to associate with proteasome in cell-based models, is targeted by carfilzomib in vitro. The drug behaves as a modulator of IDE activity, displaying an inhibitory effect over 10-fold lower than for the 20S. Notably, the interaction of IDE with the 20S enhances in vitro the inhibitory power of carfilzomib on proteasome, so that the IDE-20S complex is an even better target of carfilzomib than the 20S alone. Furthermore, IDE gene silencing after delivery of antisense oligonucleotides (siRNA) significantly reduced carfilzomib cytotoxicity in rMC1 cells, a validated model of Muller glia, suggesting that, in cells, the inhibitory activity of this drug on cell proliferation is somewhat linked to IDE and, possibly, also to its interaction with proteasome.

Structural and (Pseudo-)Enzymatic Properties of Neuroglobin: Its Possible Role in Neuroprotection.

Neuroglobin (Ngb), the third member of the globin family, was discovered in human and murine brains in 2000. This monomeric globin is structurally similar to myoglobin (Mb) and hemoglobin (Hb) α and ß subunits, but it hosts a bis-histidyl six-coordinated heme-Fe atom. Therefore, the heme-based reactivity of Ngb is modulated by the dissociation of the distal HisE7-heme-Fe bond, which reflects in turn the redox state of the cell. The high Ngb levels (~100-200 µM) present in the retinal ganglion cell layer and in the optic nerve facilitate the O2 buffer and delivery. In contrast, the very low levels of Ngb (~1 µM) in most tissues and organs support (pseudo-)enzymatic properties including NO/O2 metabolism, peroxynitrite and free radical scavenging, nitrite, hydroxylamine, hydrogen sulfide reduction, and the nitration of aromatic compounds. Here, structural and (pseudo-)enzymatic properties of Ngb, which are at the root of tissue and organ protection, are reviewed, envisaging a possible role in the protection from neuronal degeneration of the retina and the optic nerve.

At the Cutting Edge against Cancer: A Perspective on Immunoproteasome and Immune Checkpoints Modulation as a Potential Therapeutic Intervention.

Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers.

Proteasome inhibition by bortezomib parallels a reduction in head and neck cancer cells growth, and an increase in tumor-infiltrating immune cells.

Head and neck cancer (HNC) has frequently an aggressive course for the development of resistance to standard chemotherapy. Thus, the use of innovative therapeutic drugs is being assessed. Bortezomib is a proteasome inhibitor with anticancer effects. In vitro antitumoral activity of Bortezomib was investigated employing human tongue (SCC-15, CAL-27), pharynx (FaDu), salivary gland (A-253) cancer cell lines and a murine cell line (SALTO-5) originated from a salivary gland adenocarcinoma arising in BALB-neuT male mice transgenic for the oncogene neu. Bortezomib inhibited cell proliferation, triggered apoptosis, modulated the expression and activation of pro-survival signaling transduction pathways proteins activated by ErbB receptors and inhibited proteasome activity in vitro. Intraperitoneal administration of Bortezomib delayed tumor growth of SALTO-5 cells transplanted in BALB-neuT mice, protracted mice survival and adjusted tumor microenvironment by increasing tumor-infiltrating immune cells (CD4+ and CD8+ T cells, B lymphocytes, macrophages, and Natural Killer cells) and by decreasing vessels density. In addition, Bortezomib modified the expression of proteasome structural subunits in transplanted SALTO-5 cells. Our findings further support the use of Bortezomib for the treatment of HNC and reveal its ineffectiveness in counteracting the activation of deregulated specific signaling pathways in HNC cell lines when resistance to proteasome inhibition is developed.

Dexamethasone Downregulates Autophagy through Accelerated Turn-Over of the Ulk-1 Complex in a Trabecular Meshwork Cells Strain: Insights on Steroid-Induced Glaucoma Pathogenesis.

Steroid-induced glaucoma is a severe pathological condition, sustained by a rapidly progressive increase in intraocular pressure (IOP), which is diagnosed in a subset of subjects who adhere to a glucocorticoid (GC)-based therapy. Molecular and clinical studies suggest that either natural or synthetic GCs induce a severe metabolic dysregulation of Trabecular Meshwork Cells (TMCs), an endothelial-derived histotype with phagocytic and secretive functions which lay at the iridocorneal angle in the anterior segment of the eye. Since TMCs physiologically regulate the composition and architecture of trabecular meshwork (TM), which is the main outflow pathway of aqueous humor, a fluid which shapes the eye globe and nourishes the lining cell types, GCs are supposed to trigger a pathological remodeling of the TM, inducing an IOP increase and retina mechanical compression. The metabolic dysregulation of TMCs induced by GCs exposure has never been characterized at the molecular detail. Herein, we report that, upon dexamethasone exposure, a TMCs strain develops a marked inhibition of the autophagosome biogenesis pathway through an enhanced turnover of two members of the Ulk-1 complex, the main platform for autophagy induction, through the Ubiquitin Proteasome System (UPS).

Citicoline in Ophthalmological Neurodegenerative Disease: A Comprehensive Review.

Cytidine 5'-diphosphocholine has been widely studied in systemic neurodegenerative diseases, like Alzheimer's disease, Parkinson's disease, and brain ischemia. The rationale for the use of citicoline in ophthalmological neurodegenerative diseases, including glaucoma, anterior ischemic optic neuropathy, and diabetic retinopathy, is founded on its multifactorial mechanism of action and the involvement in several metabolic pathways, including phospholipid homeostasis, mitochondrial dynamics, as well as cholinergic and dopaminergic transmission, all being involved in the complexity of the visual transmission. This narrative review is aimed at reporting both pre-clinical data regarding the involvement of citicoline in such metabolic pathways (including new insights about its role in the intracellular proteostasis through an interaction with the proteasome) and its effects on clinical psychophysical, electrophysiological, and morphological outcomes following its use in ophthalmological neurodegenerative diseases (including the results of the most recent prospective randomized clinical trials).