Frontiers in Drug Design & Discovery: Volume 11 E-Book
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- Herausgeber: Bentham Science Publishers
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- Serie: Frontiers in Drug Design & Discovery
- Sprache: Englisch
Frontiers in Drug Design & Discovery Volume 11
[Edited for Volume 11]
Frontiers in Drug Design and Discovery is a book series devoted to publishing the latest and the most important advances in drug design and discovery. Eminent scientists have contributed chapters focused on all areas of rational drug design and drug discovery including medicinal chemistry, in-silico drug design, combinatorial chemistry, high-throughput screening, drug targets, and structure-activity relationships. This book series should prove to be of interest to all pharmaceutical scientists who are involved in research in drug design and discovery and who wish to keep abreast of rapid and important developments in the field.
Volume 11 of this series brings together reviews covering immunotherapy of sepsis, new antimalarials, and the medicinal use of onions for respiratory diseases, among other topics.
Topics included in this volume are:
- Heme-oxygenase and autophagy connected as a cytoprotective mechanism: potential therapeutic target
- Development of recombinant therapeutic proteins in animal cells: Challenges and solutions
- Artemisinin Analogues as a Novel Class of Antimalarial Agents: Recent Developments, Current Scenario and Future Perspectives
- The effects of Allium cepa and their derivatives on respiratory diseases and the possible mechanisms of these effects
- Immunotherapy of Sepsis
Audience:
Pharmaceutical scientists, biochemists, researchers in medicine and public health projects
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Seitenzahl: 372
Veröffentlichungsjahr: 2022
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PREFACE
In view of emerging epidemics and infectious diseases as well as the increasing prevalence of metabolic and neurological disorders, it is important to identify new drug targets in order to discover and develop safer and more effective drugs. The 11th volume of Frontiers in Drug Design and Discovery is a collection of five scholarly written reviews, each focused on a specific disease, covering various aspects of development of therapeutics.
Vasconcellos et al have contributed a comprehensive review on the relationship of heme-oxygenase (HO) with autophagy. They have proposed to use this nexus as a drug development strategy against chronic inflammatory and stress related disorders. The chapter by Mufarrege et al discusses the challenges related to the high yield production of biopharmaceuticals in animal cells and related issues, including immunogenicity. Artemisinin has attracted scientific interest as a potent antimalarial in the past few decades. Chaudhary and Tiwari have contributed a comprehensive review on the synthesis of artemisinin peroxide analogues and their antimalarial activities as compared to the parent natural product and other commonly used antimalarial drugs.
In the next chapter, Boskabady et al discuss the effect and mechanisms of action of Allium cepa (onion) extracts, and the well-known phenolic and sulphur phytoconstituents in chronic inflammatory respiratory disorders, both for treatment and prevention. Sepsis remains a major cause of morbidity and mortality. Immunotherapy has therefore been explored extensively as a treatment option for sepsis. Soleimanpour and Dolati have contributed a comprehensive review on this emerging new mode of treatment of sepsis.
We would like to thank all the authors for their excellent contributions. We greatly appreciate the efforts of Miss. Mariam Mehdi (Assistant Manager Publications) and Mr. Mahmood Alam (Director Publications) for the efficient production of this volume. We are confident that this volume will be widely appreciated by the scientific community.
List of Contributors
Heme-oxygenase and Autophagy connected as a Cytoprotective Mechanism: Potential Therapeutic Target
Luiz Ricardo C. Vasconcellos1,Rafael Cardoso Maciel Costa Silva2,Leonardo H. Travassos2,*Abstract
Heme-oxygenase (HO) is an enzyme that catalyzes the main step of heme degradation generating anti-inflammatory end products with protective roles in physiologic and pathological situations. The relevance of HO in inflammatory conditions is well reported through pharmacological and/or genetic modulation, pointing out its importance in several models of stress such as infection, inflammation, and oxidative disturbance. Under the referred situations, another well-known protective process triggered is autophagy, in which defective cytosolic components and organelles are eliminated via lysosomes. Besides its role on organelles and macromolecules recycling, autophagy also contributes to cellular homeostasis by generating the functional blocks required for anabolic reactions. Recently, different studies have demonstrated a link between HO activity and autophagy activation. In this chapter, we would like to draw the reader's attention to the interconnection between HO and autophagy regarding stress response mechanisms, highlighting its importance in homeostasis maintenance that might be useful in the therapy of inflammatory diseases in the future.
HEME-OXYGENASE
Heme-oxygenases (HOs) are conserved enzymes found in almost all kingdoms [1]. They catalyze the oxidative cleavage of intracellular heme, allowing the tetra-
pyrrole backbone for disposal or metabolic usage [2]. HO uses reduced ferredoxin or cytochrome p450 reductase as an electron source to cleave heme and generate biliverdin (which can be further reduced to bilirubin by biliverdin reductase), carbon monoxide (CO), and ferrous iron (Fe+2) [3] (Fig. 1). In humans, there are two isoforms characterized: HO-1 and HO-2 [4]. HO-1 is expressed in practically all tissues and cells submitted to a plethora of insults or different sorts of stress [5]. It suffers transcriptional level regulation, and although HO-1 can be phosphorylated by the Serine/Threonine kinase Akt/PKB, this post-translational modification does not seem to affect HO-1 activity [6]. HO-2 is expressed constitutively in different organs, including liver, kidney, gut, and vasculature, with the highest expression on the brain, central nervous system, and testes [7]. In contrast to HO-1, HO-2 suffers post-transcriptionally regulation through phosphorylation mediated by casein kinase 2 [8]. Unlike what is seen for HO-1, the broadly expressed HO-2 activity does not seem to be induced by external factors. In addition, HO-2 heme-affinity and its reaction speed are three times lower and one-tenth of the HO-1, respectively [9]. Studies suggest that HO-2 can be essential for neurotransmission and vascular homeostasis because its by-product CO acts as a neurotransmitter and promote vasodilatation through direct signalling in endothelial cells [5].
Fig. (1)) Heme-oxygenase activity and by-products. Created with BioRender.com.In mammals, the most studied isoform is HO-1, first described in 1968 and is also known as heat shock protein 32 [2]. HO-1 expression is affected by different transcription factors. These transcription factors bind to regulatory sequences in the HO-1 5’-promoter region [10-13], promoting HO-1 expression. The major transcription factors known to induce HO-1 are; specificity protein 1 (Sp1) regulated by phosphatidylinositol 3-kinase (PI3K); Hypoxia-inducible factor-1 (HIF-1); JunB and JunD; Nuclear Factor Kappa B (NF-κB); peroxisome proliferator-activated receptors (PPARγ) regulated by mitogen-activated protein kinase (MAPK); and nuclear factor erythroid 2-related factor2 (Nrf2). Nrf2 binds to enhancers termed antioxidant response elements (ARE) on the promoter region of HO-1 [14]. In the steady-state, Nrf2 is sequestered in the cytoplasm and targeted for proteasomal degradation via Kelch-like ECH-associated protein-1 (Keap-1) [15]. Furthermore, ARE is repressed by basic leucine zipper heterodimers, formed by either BTB domain and CNC homolog (Bach)-1 or Bach2 and musculoaponeurotic fibrosarcoma family (Maf) members [16]. Such complex regulation suggests the importance of HO-1 in different stress response mechanisms. In addition to its enzymatic activity, identifying a nuclear-truncated HO-1 (without the C-terminal domain), which seems to be enzymatically inactive, suggests a role of HO-1 in gene regulation [17]. It is believed that nuclear HO-1 upregulates genes associated with cytoprotection against oxidative stress and can enhance DNA repair [18].
It is important to consider that HO-1 mediates only part of the effects that confers cytoprotection. Many other crucial proteins and physiological mechanisms must be present, so the conjunction of factors could generate the proper response associated with adaptation and survival. In general, HO-1 is associated with the antioxidant response mechanism that counteracts the role of reactive oxygen species (ROS) on cell death and inflammation. Although many studies described several of these protective effects mediated by HO-1, the scavenging of free iron (generated through HO activity) has been described as crucial for the referred beneficial effects [19, 20]. Free iron binds to iron regulatory protein (IRP) and interrupts IRP association with transcripts involved in iron metabolism, allowing their translation [21]. In addition, HO-1 is associated with iron efflux and the exportation of iron-binding protein, reducing this critical source of intracellular reactive oxygen species (ROS) [22]. However, exacerbated expression of HO-1 is associated with cytotoxic effects mediated by uncontrolled iron levels [23].
The other HO-1 by-products, CO and biliverdin (bilirubin), exert antioxidant effects through different mechanisms. HO-1 activity is the source of 80-85% of the body’s bilirubin and almost all endogenous CO generated [24]. Bilirubin showed superior antioxidant activity compared to biliverdin in a cell-free system [25]. Jansen and collaborators also demonstrated that biliverdin reductase is important for the cytoprotective role of HO-1 in a model of lipopolysaccharide (LPS) induced ROS in endothelial cells. Moreover, bilirubin act as a potent antioxidant molecule protecting HeLa cells, neuronal cells, vascular smooth muscle cells, and endothelial cells from H2O2 and enzymatically derived ROS-mediated cytotoxic effects [26, 27]. These results support that bilirubin generation is an essential part of the protective antioxidant role associated with HO-1 induction. CO also exerts cytoprotective effects by inhibiting tumor necrosis factor (TNF) mediated cell death in mouse fibroblasts and endothelial cells [28]. However, even these “protective” by-products can be detrimental, after exceeding some level, mediating cell injury through the inverse effect, exceeding oxidative stress [29]. Besides these quantitative opposing effects associated with HO-1 expression, recent reviews discussed the HO-1 sub-cellular localization as an important issue to its role on cell physiology. Mitochondrial and endoplasmic reticulum forms of HO-1 can be both detrimental and beneficial to the cells, depending on the circumstances and expression level [30]. For example, CO production and a decrease in heme levels mediated by mitochondrial HO-1 activity can regulate enzymes from the electron transport chain, inhibiting its activity and generating mitochondrial ROS-induced pathways [31]. So, to characterize the subcellular activity site and the HO-1 expression levels seems crucial to understand its protective role in different models.
HO-1 mediates key events in different organs, keeping body homeostasis, especially after insults. Several studies described a vital role of HO-1 in vascular homeostasis, promoting angiogenesis and vasodilatation [32]. Moreover, HO-1 genetic ablation is related to several abnormalities in mice serving as a crucial animal model to elucidate HO-1 role on different diseases [33]. Some of the abnormalities associated with HO-1 deficiency include decreased life span, with features such as anemia, defective iron recycling, increased serum ferritin and progressive chronic inflammatory disease [34, 35]. HO-1 knockout mice also present enlargement of the spleen, progressing to fibrosis and atrophy. Kovtunovych and collaborators [36] demonstrated that the reduced number of splenic and hepatic macrophages was due to heme toxicity after senescent red blood cells phagocytosis. The failure of a crucial part of the RBCs turnover was associated with increased hemolysis and heme levels on plasma. In these circumstances, heme might be a player in the increased inflammatory settings and hyperlipidemia (possibly mediated by oxidative damage). Furthermore, hemolysis and increased heme levels could be responsible for iron deposition on hepatocytes and proximal tubular epithelial cells of the kidney, as an abnormal compensatory mechanism of heme degradation through HO-2 enzymatic activity in these cells.
The chronic inflammatory state developed in HO-1 deficient mice supports the different anti-inflammatory roles associated with HO-1 expression [37]. HO-1 can counteract inflammation in multiple ways, like degrading heme (a prototypic danger-associated molecular pattern-DAMP), restraining cell death, providing a negative feedback after pattern recognition receptors (PRRs) sensing, regulating antigen presentation by DCs and T cell activation [38], and mediating part of the effects induced by anti-inflammatory mediators [39]. Thus, HO-1 can act as an important inhibitor of inflammatory responses and consequent secondary tissue damage.
HO-1 genetic absence was reported in different infant patients [40]. The patients presented several abnormalities, including asplenia, growth retardation, anemia, iron deposition, endothelial cell damage (as evidenced by elevated thrombomodulin and von Willebrand factor on plasma), and vulnerability to stressful injury recapitulating many features presented by HO-1 knockout mice. Altogether, these observations highlight the importance of studies regarding the mechanisms behind the HO-1 effect in systemic homeostasis.
AUTOPHAGY
Autophagy (from Greek self-eating) is a well-conserved catabolic mechanism in eukaryotes that involves the degradation and recycling of cytoplasmic macromolecules and organelles through lysosomal proteolysis (Fig. 2) [41]. This process is essential for physiological homeostasis as proposed by studies employing autophagy-deficient mice, which present an accumulation of dysfunctional organelles and cytoplasmic aggregates and reduced survival under starvation [42, 43]. In this context, autophagy-deficient mice develop neurodegeneration associated with cytoplasmic aggregates accumulation that increases with aging [44]. Moreover, autophagy has essential functions in cell signalling and influences cell death, inflammatory responses, and degradation of pathogens (termed xenophagy) [45]. Like HO-1 induction, autophagy is an adaptive process induced by numerous stress stimuli, like ROS, nutrient deprivation, hypoxia, infection, among others [46].
There are three types of autophagy: chaperone-mediated autophagy (CMA), microautophagy, and macroautophagy. Microautophagy is based on the direct interaction of macromolecules with lysosomal membrane proteins and subsequent internalization and degradation [47]. CMA requires the activity of a chaperone protein termed HSC70 (heat shock cognate 71kDa protein) that recognizes a five amino acid motif (KFERQ) in the target macromolecule and mediates lysosomal internalization and degradation through the interaction between HSC70 and lysosomal membrane protein (LAMP2A) [47]. Macroautophagy is based on cytosolic engulfment into double-membrane structures (termed autophagosomes) which are addressed to lysosomes for degradation. It stands for a more complex and tightly regulated process that governs macromolecules and whole organelles lysosomal degradation and is commonly referred to as autophagy. Following the literature, from now on, we refer to autophagy considering macroautophagy.
Fig. (2)) Autophagic flux scheme. Created with BioRender.com.Autophagy is a multistep process that requires several proteins for the characteristic double-membrane autophagosome formation, including the autophagy-related genes (ATGs). It consists of elongation and closure for a double membrane structure formation, trafficking, and fusion to the lysosome in a so-called maturation process where the process terminates (Fig. 2) [48]. The early steps depend on coordinated events and signalling pathways guided by proper localization of core autophagy protein complexes. The number of proteins involved in the control of autophagy increases every year, with more detailed studies linking autophagy and several other pathways. Nevertheless, the central molecular mechanisms of autophagy triggering are well established, and for its accomplishment, two ubiquitin-like conjugation systems are required in autophagosome biogenesis: the ATG12 and ATG8 conjugation systems, which are described in details below.
Autophagosome Biogenesis
The first step required is the generation of a double-membrane structure termed phagophore (Fig. 2). The origin of the membrane used to make the phagophore might differ for each situation in which autophagy is triggered and are still under debate, though some candidate sources have been reported as endoplasmic reticulum, Golgi apparatus, mitochondria, and plasma membrane [49, 50]. Despite the intense debate about the origin of the phagophore membrane [51], the mechanisms triggered during autophagy are clear, as stated below.
A cluster of proteins termed Unc51-like kinase (ULK) complex, composed of the serine/threonine-protein kinase ULK1, ATG13, FIP200 (FAK family kinase-interacting protein of 200 kDa), and ATG101, is the first protein complex considered the primary regulator of autophagosome generation [52]. In short, in resting state pathways triggering cell growth activate the mechanistic target of rapamycin (mTOR) kinase, which inhibits ULK1 and consequently the autophagosome formation, whilst under poor energy and low nutrient status, mTOR kinase ceases ULK1 and the AMP kinase (AMPK) repression leading to ULK1 autophosphorylation, followed by ATG13 and FIP200 phosphorylation, also essential for autophagosome promotion [52]. The activated ULK1 complex phosphorylates and activates several proteins leading to the formation and activation of class III PI3K complex (PI3KC3) (composed by the catalytic subunit vacuolar protein sorting 34 (VPS34), general vesicular transport factor p115, beclin-1, and ATG14L) on the autophagosome membrane resulting in a PI3P decorated membrane. In fact, the PI3KC3 recruitment to the phagophore membrane depends on ULK1 complex activation. The PI3P is essential for the recruitment of effector proteins involved in autophagosome formation and closure, being PI3K inhibitors broadly used to block autophagy induction (Fig. 2) [53]. After PI3P generation in the membrane, anchor proteins from WD-repeat protein interacting with the phosphoinositide family (WIPI1 and WIPI2) act as scaffold effector proteins recruiting ATG16L1 as one example of their action [54].
From this point starts the elongation of the autophagosome structure mediated by two ubiquitin-like conjugations. In the first, ATG12 is activated by a C-terminal glycine modification induced by the E1-like enzyme ATG7. Then, ATG10 (an E2-like enzyme) mediates the conjugation between ATG12 and ATG5 [55]. Subsequently, ATG16L1 interacts with ATG12-ATG5 to form the ATG12-ATG5-ATG16L complex, which dimerizes and interacts with the outer part of the autophagosomes [56]. The second ubiquitin-like conjugation step works to modify proteins from ATG8/LC3 family. It is a step broadly monitored to assess autophagy induction by different protocols [57]. In the resting state, LC3 is diffuse in the cytosol. Upon autophagy induction, LC3 undergoes the removal of an arginine residue by the cysteine protease ATG4, which exposes a glycine in the C-terminus of LC3 that is activated by ATG7. Following, the LC3 association with the autophagosome membrane takes place through ATG3 engagement that converts LC3-I freely diffuse form into a membrane-associated form bound to phosphatidylethanolamine (PE), forming the termed LC3-II, a process that is enhanced by ATG5-12 complex (Fig. 2) [58].
Autophagosome Maturation
After the autophagosome formation, the engulfed content travels through the endocytic system within the double membrane and reaches lysosomes [59]. Lysosomes are degradative cellular compartments that contain a large variety of enzymes that act under their relative acidic environment allowing proteins, carbohydrates, nucleic acids, and lipids, present as cargo in autophagosomes, to be degraded [60]. The vesicular trafficking from the phagophore formation until lysosome fusion is orchestrated in a multistep process. The major controllers of the vesicular movement are proteins from the Rab GTPase family, which alternates between active (GTP-bound) and inactive state (GDP-bound) [61]. An important characteristic of Rab proteins is the association with effector proteins which act, depending on each situation, as motor-driven trafficking inducers, vesicle interaction with other compartments, and signaling [61]. The fusion of autophagosomes with lysosomes depends on Rab GTPases and cytoskeleton proteins, which drive autophagosome maturation where the autophagy process terminates. After degradation, autophagic products are exported to the cytosol via transporters present on the lysosomal membrane to be reutilized in anabolic reactions (Fig. 2) [62]. The maturation process is regulated by transcription factors involved in lysosomal biogenesis, such as transcription factor EB (TFEB), and defects in this regard lead to lysosomal dysfunction, inhibiting autophagosome maturation, resulting in autophagosome accumulation in the cytosol [63].
Selective Autophagy
The autophagic degradation of cytosolic components was long considered to be randomly guided as a non-selective process of cargo sequestration. While starvation-induced autophagy seems to be a process involving random cytoplasmic uptake into phagophores, the identification of autophagy adaptor molecules connecting the cargo to ATG8/LC3 proteins that decorate the autophagic membrane, opened a new field for studies in autophagy cargo selectivity [64]. Selective autophagy has been described as an important mechanism for specific cytosolic components clearance, such as protein aggregates and damaged organelles [65, 66]. In autophagy-dependent degradation, the cargo must be exclusively targeted for elimination. For this purpose, these adaptor molecules possess multiple domains, one that must specifically interact with LC3 (with some exceptions, like in the case of ferritinophagy) and others that interact with exposed domains or inserted groups of molecules in the organelles\macromolecules that will be directed to autophagic degradation [64]. Several adaptor proteins for autophagy in mammals have been described so far, and the best characterized are sequestosome-1/p62 (p62); histone deacetylase 6 (HDAC6); optineurin (OPTN); and Neighbor of BRCA1 gene 1 (NBR1) [67, 68]. Besides the LC3 interacting domain, the adaptors might possess a domain that interacts with ubiquitin, unless exception. The presence of these domains allows the adaptor proteins to perform their function, binding to the specific cargo (the majority is ubiquitinated) and delivering it into the autophagosomes. Some domains are found in different adaptor proteins, which confer redundant functions to many of them, a feature that can be observed in genetically deficient mice, which remains viable, although with distinct phenotypes, depending on the autophagy adaptor deleted [69]. Nonetheless, the adaptors are necessary for autophagy regulation in multiple levels, including signalling events, autophagic maturation, and cargo selection and delivery [70].
As anticipated, the major modification associated with the cargo-adaptor binding is ubiquitination. The ubiquitin-binding system is an ancient process used for protein degradation. Ubiquitin is a small (76 amino acid residues) protein that marks lysine residues from macromolecules for degradation via proteasome or lysosome (via autophagy). The distinction is dictated by cargo shape and interaction with its adaptors [71]. The ubiquitination process involves several mediators, including an activating enzyme (E1), a conjugating enzyme (E2), and one ligase (E3) transferring ubiquitin to the appropriate lysine in the target macromolecule [72]. Thus, ubiquitin is added to specific macromolecules after a series of reactions involving proteins from the ubiquitin system and proteins that act as sensors and signal messengers [72]. For example, mitochondrial degradation via autophagy (termed mitophagy) requires the PTEN induced putative protein kinase 1 (PINK1) stabilization in the outer membrane of mitochondria due to compromised integrity of the mitochondrial membrane [73]. PINK1 phosphorylates Parkin and Miro, allowing stabilization of Parkin on the mitochondrial membrane and immobilization of mitochondria (due to phosphorylation and subsequent degradation of Miro, an essential anchoring protein in the cytoskeleton-motor complex kinesin) [74, 75]. In turn, Parkin mediates ubiquitination of several proteins of the outer mitochondrial membrane, such as Mitofusins and voltage-dependent anion channels, allowing its interaction with the adaptors, p62 and OPTN, addressing it for autophagic degradation [76-78]. In some situations, the adaptor absence restrains the specific cargo degradation, as observed in OPTN deficient mice that accumulate dysfunctional mitochondria. However, concerning p62, its genetic ablation restricts the ubiquitin-positive protein aggregates, indicating the role of this adaptor in aggregates formation and not only in their clearance [79]. This finding was counterintuitive because p62 is a vital adaptor protein used for protein aggregates selection for autophagy degradation [80]. Thus, the referred works show diverse functions for the adaptors, apart from delivering cargo for degradation.
Although increasing knowledge in this field is being added to the literature, the complete mechanism for autophagy selectivity is limited and still needs more studies.
HEME-OXYGENASE AND AUTOPHAGY LINKED FOR PROTECTION
As two conserved and essential stress response mechanisms, HO-1 and autophagy might influence each other as homeostatic mechanisms triggered by cells [81]. Furthermore, some studies have demonstrated the influence of the HO-1 system in autophagy induction and vice versa, showing that the combined action of both might be an important protective mechanism triggered in several pathologic events. Below we refer to some of them.
Ischemia and reperfusion (IR) is a pathologic condition that causes extensive cellular damage due to transient hypoxia exacerbated by the rapid return of the blood flow [82]. A link between HO-1 induction and autophagy in protection against IR injury has been demonstrated [83]. In this study, the use of an HO-1 activity inhibitor blocked autophagy and aggravated the disease. Similarly, in a study investigating the recovery after liver transplantation, HO-1 promoted liver protection to ischemia-reperfusion injury due to autophagy induction [84]. Although, both studies point to HO-1 and autophagy for protection the interaction of both needs to be further investigated.
Sepsis is characterized by a systemic immunological response to infection that can evolve to multi-organ dysfunction leading to severe prognosis and death, is a significant cause of mortality in critically ill patients [85]. In a murine model of sepsis using cecal ligation puncture, hepatic cell death was abrogated by HO-1 dependent autophagy mediated by MAPK activation [86]. As this model consists of polymicrobial disseminated infection with microbial components recognition leading to shock, it can be mimicked, in a more simplistic way, by using LPS as a microbial component triggering the inflammatory response. In the referred study, LPS induced HO-1 expression in a Toll-like receptor (TLR4)-dependent manner indicating that this mechanism seems to be an important part of a negative feedback loop to restrain both inflammation and macrophage activation [87]. Still, in an I/R model, baicalein treatment showed protective effects against liver damage by HO-1-dependent autophagy activation. In this study, HO-1 suppression blocks the protective effects of that drug [88].
In a model to study the role of podocytes in diabetic nephropathy progression, the impact of HO-1 activation in autophagy protective effects in situations of high glucose-induced apoptosis was evaluated [89]. This work demonstrated HO-1 requirement for autophagy induction under high glucose situations and for cytoprotective effects [89]. In addition, they demonstrated that the interaction between HO and autophagy is done by AMPK activation that could be one of the molecular mechanisms linking both [89]. Although this mechanism has been described, it needs to be further appreciated in other models to determine the exact interplay between HO and autophagy.
The reports in this section highlight the interaction between autophagy and the HO-system; nonetheless, the mechanisms of autophagy and HO-system interactions are not fully described yet.
HO Products Promoting Autophagy
Although it does not discard the direct interaction between HO and proteins involved in autophagy, some studies regarding HO involvement in autophagy induction point out for HO end-products as autophagy activators. In the sections below, we summarise the known mechanism of autophagy induction mediated by the three products of HO reaction: CO, iron, and bilirubin/biliverdin.
CO as a Prototype Autophagy Inducer
CO is one of the HO end-products characterized as a toxic gas because of its high affinity for hemoglobin, competing with oxygen when in high concentrations. Conversely, when in lower quantities, it has been reported as a candidate therapeutical molecule by conferring protection against a myriad of cellular stresses due to anti-inflammatory and anti-apoptotic aspects [90]. The mechanism behind the referred protection is not fully characterized yet, however. In addition, some studies investigated the role of autophagy in CO protective effects.
CO has been considered a potential therapeutic molecule to be used against lung injury in animal models [91]. In an in vivo model of acute lung injury, CO treatment via inhalation showed cytoprotective effects that were autophagy-mediated. Autophagy was induced as a homeostatic mechanism against mitochondrial ROS generated by CO in the regimen administrated. In this study, inhaled CO induced autophagy for up to 72h in mice [92].
Another way to deliver CO for therapeutic purposes is via a CO-releasing molecule (CORM), which delivers CO as an effective alternative to the gas and has been broadly applied to investigate its therapeutic potential. In a seminal yeast model-based study, CORM treatment prevented oxidative damage associated with cell death via autophagy induction [93]. CO-induced autophagy, under physiological conditions, was necessary for partial protection against oxidative stress-induced cell death in yeast. These cytoprotective aspects of CO treatment were reproduced in primary culture astrocytes upon oxidative stress. Again, the protective effect of CO treatment was totally dependent on autophagy in astrocytes [93]. This work indicates that CO can trigger autophagy in physiological conditions.
In another model using CORM, it was evaluated the involvement of autophagy after hypoxia-induced apoptosis in islet β cells. In this regard, CORM treatment suppressed apoptosis, inflammation, and endoplasmic reticulum stress, abrogating insulin resistance in vitro. In addition, CO exposure after graft transplantation attenuated both inflammation, and cell death in diabetic non-obese mice. All these suppressive effects were positively regulated by autophagy inducers and hindered when autophagy was blocked, proposing the connection between CO benefits and autophagy induction [94].
Another study highlighted the role of mitophagy in the neurological improvement and increased survival related to CORM treatment in a cardiac arrest (CA) murine model [95]. Mitochondrial dysfunction has an essential role in the pathogenesis of ischemia-reperfusion (IR) induced after CA, and such dysfunction is also present in neurological injury in these CA models. CO treatment reduced the mitochondrial dysfunction observed, increasing mitophagy via accumulation of PINK1 and Parkin in the mitochondria, targeting damaged mitochondria for degradation via lysosome. Therefore, in this model, CO treatment impacted mitochondrial properties via reduction of its dysfunction and recycling of damaged mitochondria, contributing to neuron homeostasis [95].
In a detailed report, the mechanism behind CO protection was deeply investigated after liver injury. In this study, CORM increased the nuclear translocation of TFEB (important for lysosomal and mitochondrial biogenesis) through augmentation of protein kinase R-like endoplasmic reticulum kinase (PERK) and calcineurin phosphatase activities, and cytosolic Ca+2 peak. Through this mechanism, CO leads to mitophagy via PERK phosphorylation and TFEB nuclear translocation, both required for CO therapeutic effects in a liver injury model [96]. In this way, CO effects are associated with mitochondrial clearance via lysosomes in a mitophagy process (Fig. 3). So, CO positive therapeutical effects might be associated with the removal of dysfunctional mitochondria via mitophagy and CO-mediated TFEB activation might be involved in mitochondrial biogenesis to keep cellular homeostasis after mitochondria replacement [96] (Fig. 3).
Fig. (3)) Mechanism of action of autophagy induced by carbon monoxide. Created with BioRender.com.Iron as an Autophagy Inducer
Iron is a bona fide essential micronutrient for all living organisms with essential physiological roles, acting as a cofactor for several biochemical processes involved in cell proliferation and reduction-oxidation reactions [97]. Mammals absorb iron mainly from their diet in two forms: derived from heme-hemoproteins or derived from non-heme molecules. Heme consists of more than 95% of functional iron stores in mammals, and to become available in the iron labile pool, and it needs to be degraded via the HO system [98].
Due to its ability to generate reactive oxygen species, iron availability needs to be appropriately compartmentalized to exert its important physiological roles. With this purpose, protective mechanisms must be present in sites where eventually high iron levels are found in the body [99]. One of such mechanisms is intracellular iron storage mediated by a multicomplex protein termed ferritin (Ft). Ferritin is an iron-binding protein consisting of heavy and light chain polypeptides bound in a 24 subunits composition that can store up to 4500 iron atoms [100]. Due to its ferroxidase activity, Ft converts Fe+2 into Fe+3 (the considered inert state) in which iron is sequestered within the Ft mineral core, restraining the reactive oxygen species generation. Ft suffers both transcriptional and post-transcriptional expression regulation. Its transcription is controlled by NF-κB, transcription factor nuclear factor E2- related factor-2 (Nrf2) and by Bach1 [101]. Like most iron-related genes, it suffers post-transcriptional regulation by IRP, which binds to Ft mRNA, inhibiting its translation under low iron levels.
In contrast, in situations of intracellular iron accumulation, IRP interaction with Ft mRNA is repressed, allowing Ft mRNA to be translated [101]. In addition, Ft expression is regulated by a mechanism that involves its degradation via lysosome. This process is induced under low intracellular iron levels addressing Ft for lysosomal degradation to make iron available through a recycling process called ferritinophagy [102]. This occurs via selective autophagy guided by nuclear receptor co-activator 4 (NCOA4), essential for cellular iron homeostasis [102]. In this regard, Ft clearance, via lysosome, under low iron levels can be considered an iron-regulated autophagy process, even though it seems to be a process exclusive for Ft degradation (Fig. 4) [102]. Moreover, ferritinophagy has been regarded as a mechanism to induce cytotoxicity due to increased iron availability that triggers ferroptosis (an iron-mediated cell death) [103].
Fig. (4)) The dichotomy of iron cellular levels that triggers autophagy. Created with BioRender.com.It has been demonstrated the ability of oxidative stress-mediated by heme to induce protein aggregation which is dependent on autophagy for degradation. In this context, iron is the minimal molecular requirement to induce oxidative damage and protein aggregation, consequently [104]. In fact, heme degradation via HO-1 seems to be essential for protein aggregates induction and the absence of Ft, to stock the iron generated increases the formation of the aggregates. In addition, the treatment with deferoxamine (an iron chelator) abrogates this phenomenon. This work raised the possibility that HO-1 participates in both protein aggregates formation and in the autophagy induction for their elimination from cytosol under heme stimulus (Fig. 4). Nevertheless, the mechanism behind the referred phenomenon is not well established, and the HO-1 and autophagy interaction upon heme stimulus needs to be further investigated [104].
Recently, a detailed study has elucidated the mechanisms behind iron-mediated autophagy. They employed a model of chronic iron overload in vitro using skeletal muscle cell line and in vivo to evaluate the relationship between iron overload and insulin sensitivity [105]. They have demonstrated that iron induces autophagy as an acute response to iron overload, a state that progresses to dysfunctional autophagolysosomes accumulation in chronic high iron levels. This occurs due to impairment of tuberous sclerosis complex (TSC2) repression mediated by Akt, which stops Rheb-mediated activation of mTORC1, resulting in long-term autophagic-lysosome regeneration (ALR) disruption. In this way, autophagy is transiently induced early, as an acute response to iron, while long-term iron exposition leads to autophagy defect through an ALR defect that impacts autophagosomes and lysosomes replacement and, consequently, insulin signalling (Fig. 4) [105].
Biliverdin/bilirubin and Autophagy
The interplay between autophagy and biliverdin/bilirubin has not been extensively studied. Though, bilirubin absence, through genetic deletion of biliverdin reductase A (BVR-A) that converts biliverdin into bilirubin, is associated with increased neuronal damage and reduced autophagy [106]. The absence of BVR-A leads to increased mTORC1 levels in the cerebral cortex of mice. The mechanisms involved in BVR-A impact on the AMPK-mTORC1 axis were not elucidated, and it is not clear whether it was the result of bilirubin absence or due to BVR-A interaction with other proteins through its different motifs or both. On the other hand, as previously discussed, exacerbated levels of bilirubin can lead to neuronal damage. Autophagy is crucial as a stress response mechanism in these settings, preventing cell death [107]. Autophagy induction by bilirubin occurs in a PKC/calcium signaling-dependent manner and culminates in reduced HO-1 expression, providing an important negative feedback loop to bilirubin-generated oxidative damage [107]. Further studies are necessary to better comprehend the interplay between bilirubin/biliverdin and autophagy, but the circumstances and levels of bilirubin/biliverdin greatly impact it.
