Building an Understanding of Proteostasis in Reproductive Cells: The Impact of Reactive Carbonyl Species on Protein Fate

LOX enzymatic activity

LOXs are a family of non-heme iron dioxygenases that catalyze the oxidation of PUFA (and occasionally sterol) substrates into an array of metabolites including hydroxyeicosatetraenoic acids (HETEs) derived from AA, leukotrienes (LTs), and lipoxins (LXs) (Fig. 2A). LOX isoforms are often classified based on their positional specificity for oxygenation (Shi et al., 2020). In mammalian cells, carbon atoms at the 5-, 12-, and 15-positions are thought to serve as the primary oxidation sites of AA, with these reactions catalyzed by 5-LOX, 12-LOX, and 15-LOX enzymes, respectively (Wang et al., 2019). Here, we will solely focus on the products of 5-LOX-catalyzed AA metabolism; however, the same general principles apply for 12- and 15-LOX enzymes. The insertion of molecular oxygen at carbon atom 5 in AA generates 5-hydroperoxyeicosatetraenoic acid (5-HPETE), which is either reduced by peroxidase enzymes into 5-HETE or, alternatively, converted to bioactive lipid mediators such as LTs and LXs (Wang et al., 2021).

This insertion of oxygen at one end of AA does not impinge on the reactive pentadiene system at the other end, thus consecutive oxygenation at the 5- and 15-carbons can yield 5,15-diHETEs (Tejera et al., 2012). Downstream of these events, 5-oxo-eicosatetraenoic acid can also be synthesized via (i) enzymatic or nonenzymatic dehydration of 5-HPETE or (ii) 5-hydroxyeicosanoid dehydrogenase-catalyzed oxidation of 5-HETE (Powell and Rokach, 2015). In a similar manner, LOX enzymes can metabolize LA into hydroperoxyoctadecadienoic acids (HPODEs), hydroxyoctadecadienoic acids, dihydroxyoctadecadienoic acids, and oxooctadecadienoic acids (Kirpich et al., 2016).

The mechanisms through which these oxidative intermediates are metabolized into RCS are not yet fully understood. One proposed mechanism is the lipoxygenase–hydroperoxide lyase (LOX-HPL) pathway in which lipid hydroperoxides (i.e., LA-derived 9- and 13-HPODEs) serve as the primary substrates for RCS formation. In plants, for instance, 9-HPODE is subjected to protonation, rearrangement (C-C to C-O), and subsequent cleavage by 9-HPL (CYP74). This reaction yields two products: (i) 9-oxonanoic acid (an ω-oxo acid) and (ii) 3Z-nononeal (a volatile aldehyde). 3Z-nononeal is rapidly oxidized by alkenal oxygenase to form 4-hydroperoxynonenal (4HPNE), an immediate precursor of 4HNE. Analogous reactions are theorized to facilitate the conversion of hydroperoxides from other ω-6 PUFAs, specifically AA-derived 11- and 15-HPETEs to 4HPNE, which can be further reduced to 4HNE by cellular peroxygenases (Fig. 2) (Ayala et al., 2014; Noordermeer et al., 2001; Noordermeer et al., 2000; Schneider et al., 2001; Spickett, 2013).

COX enzymatic activity

COXs, also known as prostaglandin G/H synthases, are rate-limiting enzymes involved in the conversion of AA into prostaglandins (PGs) and later, through additional mechanisms, thromboxanes (TXs) (Fig. 2B). There are two distinct COX isoforms: COX1 is constitutively expressed in most cells and is the dominant source of prostanoids (active lipid mediators; i.e., PGs and TXs) that maintain tissue homeostasis (Ding et al., 2003). On the contrary, COX2 is an inducible isoform with its expression stimulated by growth factors, cytokines, and tumor promoters (Wang et al., 2021). Given this, COX2 is hypothesized to play a central role in prostanoid formation in inflammation and various pathological disease states (Rouzer and Marnett, 2009; Wang et al., 2021).

Possessing two separate but linked active sites, COX enzymes catalyze the bis-dioxygenation and subsequent reduction of AA to PGG₂ and PGH₂ (Rouzer and Marnett, 2009). These PGs serve as substrates for metabolism by a series of downstream enzymes to generate a family of prostanoids (Ding et al., 2003; Wang et al., 2021), including PGE₂, PGI₂, PGD₂, PGF₂, and TXA₂ (Cao and Prescott, 2002; FitzGerald and Patrono, 2001; Funk, 2001; Hata and Breyer, 2004; Samuelsson et al., 1978; Smith et al., 2000; Wlodawer and Samuelsson, 1973). MDA can be generated as a by-product of thromboxane synthase-catalyzed decomposition of PGH₂ to TXA₂ (Hecker et al., 1987).

CYP enzymatic activity

CYP enzymes are a superfamily of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, including the metabolism of AA (Fig. 2C). The CYP family can be divided into two subgroups according to their mechanism of action, those being CYP epoxygenases (i.e., CYP2J and 2C) and CYP ω-hydroxylases (i.e., CYP4A and 4F) (Spector et al., 2004; Wang et al., 2021). The epoxygenase activity of CYP enzymes catalyzes insertion of an oxygen atom on a carbon attached to one of the double bonds of AA. This double bond is subsequently reduced to form AA epoxides, also referred to as epoxyeicosatrienoic acids (EETs). Epoxidation can occur at any of the four double bonds of AA, giving rise to four unique regioisomers: 5,6-EET, 8,9-EET, 11,12-EET, and, the dominant form, 14,15-EET (Spector, 2009). The resultant EETs are further metabolized, mainly by soluble epoxide hydrolase, into corresponding dihydroxyepoxyeicosatrienoic acids or diols (Chacos et al., 1983; Moghaddam et al., 1996; Zeldin et al., 1995; Zeldin et al., 1993).

Alternatively, CYP ω-hydroxylases mediate the metabolism of fatty acids via the addition of a hydroxyl group to the ω or ω-1 carbon atom. Notably, ω-hydroxylation of AA yields several metabolic products, including 19- and 20-HETEs (Ni and Liu, 2021). In a similar manner, CYPs can hydroxylate the three bisallylic carbons of AA to generate 7-, 10-, and 13-HETEs. The latter compounds are unstable in acidic conditions and can rearrange to give conjugated diene HETEs. 13-HETE undergoes molecular rearrangement into 11- and 15-HETEs, whereas 10-HETE can be converted into 8- and 12-HETEs (Brash et al., 1995; Powell and Rokach, 2015).

Ultimately, members of the LOX, COX, and CYP families function both independently and in combination to generate short-chain RCS, such as 4HNE, MDA, and ACR. While the male germline is known to possess many of the same peroxidative enzymes detected in somatic cells, including COX1 and COX2 (Matzkin et al., 2010; Perrotta et al., 2012), their role in germ cells is yet to be fully elucidated. Current knowledge surrounding the role of RCS in the male germline will be discussed later in this review (see the RCS in the Context of Reproduction section). In addition to the enzymatic mechanisms discussed previously, RCS may also be generated via nonenzymatic pathways, as reviewed by Ayala et al. (2014) and Spickett (2013).

4HNE reactivity

4HNE is a highly toxic molecule containing three functional groups that contribute to its high reactivity (Fig. 3A). The first of these is a carbon–carbon double bond (alkene; C = C, C2/C3) that can be a target for irreversible Michael additions (protein carbonylation) or can undergo reduction or epoxidation. The second is an aldehyde (carbonyl; C = O) group, which can react with thiol substrates (organosulfur compound; R–SH) to yield an acetal/thioacetal or can be a target for Schiff base formation, oxidation or reduction. Finally, 4HNE harbors a secondary alcohol (hydroxyl group attached to a saturated carbon atom, which is bonded to two alkyl groups; R–CHOH–R′) that can be oxidized to a ketone (Ayala et al., 2014; Bilska-Wilkosz et al., 2022; Schaur, 2003; Schaur et al., 2015).

MDA reactivity

MDA is a small and highly abundant three-carbon molecule that, due to its pH-dependent tautomeric properties, exists in several different forms in aqueous solution (Esterbauer et al., 1991). At neutral pH, the dominant form is the relatively unreactive enolate anion. At lower pH, such as that induced under stress conditions, MDA exists in equilibrium between the highly reactive protonated enol aldehyde (β-hydroxyacrolein) and dialdehyde forms (Fig. 3B). The β-hydroxyacrolein form contains an α,β-unsaturated carbonyl group that can be targeted to Michael addition and Schiff base reactions (Ayala et al., 2014; Morales and Munné-Bosch, 2019; Weber et al., 2004). Contrastingly, the dialdehyde form is characterized by two carbonyl groups, joined by the common group, R–CHO, consisting of a carbonyl center bonded to hydrogen. These carbonyl groups are able to form two Schiff bases (Jové et al., 2020).

ACR reactivity

ACR (2-propen-1-al) is the simplest and most reactive of the α,β-unsaturated aldehydes (Esterbauer et al., 1991; Witz, 1989). Chemically, ACR is a type-2 alkene composed of a polarizable alkene bond (C1/C2, C = C) in close proximity to a carbonyl group (C = O) (Fig. 3C). The resultant structure forms a conjugated system that contains mobile outer shell π-electrons. Typically, alkene functional groups are electron rich. In ACR, however, the combination of π-electron mobility and electron-withdrawing capacity of the carbonyl group creates a strong electron deficiency at the terminal β-carbon (LoPachin et al., 2009). In a similar manner to the previously discussed RCS, these alkene and carbonyl functional groups can be targeted for Michael additions and Schiff base reactions, respectively.

Ultimately, these functional groups afford RCS the ability to form covalent bonds, via Michael additions and Schiff bases, with nucleophilic (electron-rich) sites on biological targets, such as DNA, lipids, and proteins. The resultant RCS-macromolecule adducts can elicit a variety of physiological and pathological effects, either through direct (impaired substrate function) or indirect (cell signaling) mechanisms. Most commonly, RCS adduction leads to the inhibition of cellular processes and eventual cytotoxicity (Beauchamp et al., 1985; Esterbauer et al., 1991; Kehrer and Biswal, 2000; LoPachin and Gavin, 2014; Witz, 1989).

4HNE-protein adducts

4HNE is able to modify proteins through the covalent adduction of nucleophilic amino acid residues, specifically deprotonated side chains of Cys, His, and Lys (Andringa et al., 2014; Ayala et al., 2014; Dalleau et al., 2013; Doorn and Petersen, 2002; Mihalas et al., 2017; Peters et al., 2020; Pizzimenti et al., 2013; Sayre et al., 2006; Wall et al., 2014). Depending on the functional group involved in this reaction, 4HNE-mediated protein modification can yield a variety of adducts. For instance, the addition of thiol or amino compounds on the electrophilic β-carbon (carbon of the double bond) of 4HNE produces the corresponding Michael adduct. The preference of substrates for Michael addition (from highest to lowest) is Cys>>His>Lys (Fig. 4A) (Doorn and Petersen, 2002).

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FIG. 4.

4HNE adduction of protein amino acid residues through Michael addition and Schiff base formation. 4HNE can modify protein substrates through the covalent adduction of nucleophilic amino acid residues, specifically deprotonated side chains of Cys, His, Lys, and less commonly, Arg. Depending on the functional group involved, 4HNE-mediated protein modification can yield a variety of adducts through Michael addition (A, B) and Schiff base (C, D) reactions. (A) The general mechanism through which Michael adducts are formed. Initially, protein-bound amino acid residues (denoted as XH, where X = S, N and the imidazole group for Cys, His, and Lys, respectively) attack the β-carbon (alkene group; C = C) of an α,β-unsaturated carbonyl compound. The nucleophilic residue forms a covalent bond with the β-carbon, resulting in the formation of an adduct. (B) 4HNE-mediated modification of protein-bound Cys, His, and Lys residues to form irreversible Michael adducts (1,4-addition). These adducts can exist in the reduced form or may undergo an additional cyclization step to form a hemiacetal derivative. (C) The general mechanism through which Schiff base reactions occur. Initially, nucleophilic amino acid residues attack the carbonyl group of an aldehyde or ketone. This results in the formation of a covalent bond between the nitrogen atom of the nucleophile and the carbon atom of the carbonyl group. The oxygen atom of the carbonyl group loses a proton, whereas the nitrogen atom of the amino acid residue gains a proton. The reaction yields a carbinolamine intermediate, which subsequently undergoes rearrangement to form a Schiff base (C = N) and water (H₂O). (D) Schiff base reactions (1,2-addition) between 4HNE and protein-bound His, Lys, and Arg residues. Arg, arginine; Cys, cysteine; His, histidine; Lys, lysine. Created with ChemDraw and BioRender.com

Following this primary reaction, which confers rotational freedom to the C2–C3 bond, secondary reactions involving the carbonyl and hydroxyl group may occur (Schaur, 2003). Notably, the reaction between the nitrogen atom of primary amines (i.e., amino acids) and the aldehyde group of 4HNE yields a reversible Schiff base (Fig. 4B) (Ayala et al., 2014). His, Lys, and, less commonly, arginine (Arg) act as primary targets for Schiff base formation (Andringa et al., 2014; Isom et al., 2004). Broadly, Michael adducts account for >99% of HNE protein modifications, whereas Schiff base adducts are less prevalent even in the presence of excess HNE (Bruenner et al., 1995; Rauniyar and Prokai, 2009; Uchida and Stadtman, 1992).

The formation of RCS-protein adducts is biologically significant given these compounds can participate in secondary deleterious reactions, notably, intramolecular and/or intermolecular protein cross-linking (Ayala et al., 2014; Bilska-Wilkosz et al., 2022; Petersen and Doorn, 2004). Mechanistically, 4HNE covalently cross-links proteins through sequential 1,4 Michael addition and 1,2 Schiff base reactions between residues on the same or two independent polypeptide chains (Carini et al., 2004; Cohn et al., 1996; Siegel et al., 2007). These protein cross-linking events can have detrimental consequences for protein function, cell survival, and overall organism health (see the Biological consequences of protein carbonylation section).

As mentioned previously, RCS are constantly being produced at basal concentrations as a by-product of lipid peroxidation. At these physiological levels, three major detoxification pathways are responsible for the enzymatic conversion of 4HNE into less reactive chemical species and the maintenance of steady-state concentrations (Zhong and Yin, 2015). These mechanisms involve (i) conjugation with GSH, which occurs spontaneously or can be catalyzed by glutathione S-transferases, (ii) oxidation by aldehyde dehydrogenase (ALDH), and (iii) reduction of ALDH intermediates by aldo-keto reductases (AKRs) such as alcohol dehydrogenase. The intermediate products generated by these reactions; 4HNE-GSH, hydroxynonenoic acid, and 1,4-dihydroxynonene, respectively, may be further metabolized or excreted by the cell (Fig. 6A) (Ayala et al., 2014; Castro et al., 2017; Zhong and Yin, 2015). While this detoxification initially limits the availability of 4HNE for macromolecular substrate adduction, these mechanisms become rapidly overwhelmed as the concentration of 4HNE increases. At this point, the cell enters a state of carbonyl stress.

MDA-protein adducts

Despite a relatively low reactivity toward free amino acids, MDA possesses a high affinity for the modification of peptide chains/protein-bound amino acids (Esterbauer et al., 1991). In a similar manner to 4HNE, the two distinct isomeric structures of MDA can modify proteins by (i) Michael addition at the β-carbon of the α,β-unsaturated carbonyl group in the β-hydroxyacrolein isomer or (ii) Schiff base formation at the carbonyl carbon in this isomer or in the dialdehyde (Weber et al., 2004). While the free ɛ-amine group of Lys acts as the primary target of these reactions (Esterbauer et al., 1991; Kikugawa and Beppu, 1987), other amino acid substrates including Arg, His, tyrosine, and methionine may also be altered to some extent (Chio and Tappel, 1969; Jové et al., 2020; Requena et al., 1997).

In addition to modifying nucleophilic groups, MDA can also react with acetaldehyde (a product of MDA metabolism) under conditions of oxidative stress. This reaction yields hybrid protein conjugates termed MDA acetaldehyde adducts (Tuma, 2002; Tuma et al., 2001) that have been shown to be highly immunogenic (Ayala et al., 2014; Duryee et al., 2008; Wållberg et al., 2007; Wang et al., 2012; Wang et al., 2007; Weismann and Binder, 2012).

As mentioned previously, MDA readily forms a reversible Schiff base with the side chain of Lys residues to generate the MDALys adduct; however, Lys-MDA-Lys cross-links may also be produced (Chio and Tappel, 1969; Domingues et al., 2013; Jové et al., 2020; Kikugawa and Beppu, 1987; Miyata et al., 1998; Refsgaard et al., 2000; Requena et al., 1997; Slatter et al., 1998). The molecular consequences of MDALys adduct formation mostly include alterations in physicochemical properties, such as conformation (Thorpe and Baynes, 2003), charge (Mol et al., 2019), hydrophobicity and solubility (Dalfó and Ferrer, 2008), aggregation (Dalfó and Ferrer, 2008; Requena et al., 1997; Vay et al., 2001), loss of enzymatic activity (Domingues et al., 2013; Yarian et al., 2005), and accelerated rate or resistance (in the case of Lys-MDA-Lys cross-links) to proteolysis (Mahmoodi et al., 1995). The mechanisms involved in MDALys adduct and Lys-MDA-Lys cross-link formation, as well as the cytotoxic effects of these molecules have been thoroughly reviewed by Jové et al. (2020).

ACR-protein adducts

In a similar manner to 4HNE and MDA, ACR preferentially targets the nucleophilic side chains of Cys, His, and Lys residues, as well as free N-terminal amino groups (Esterbauer et al., 1991). ACR readily reacts with these amino acid groups via two distinct mechanisms: (i) Schiff base formation at the carbonyl group or (ii) Michael addition at the terminal β-carbon (Cai et al., 2009). The retained aldehyde group in ACR-amino acid Michael addition adducts can further react with additional nucleophilic groups to form intramolecular and/or intermolecular cross-links (Burcham and Pyke, 2006).

Furthermore, evidence suggests that several molecules of ACR can react with the same residue, thus forming complex adducts such as N^ɛ-(3-formyl-3,4-dehydropiperidino)lysine (FDP-Lys) (Uchida et al., 1998) and N^ɛ-(3-methylpyridinium)lysine (Furuhata et al., 2003). These complex and highly stable adducts can further perpetuate the damaging effects of ACR. By way of example, FDP-Lys adducts generated in the ACR-modified protein can function as thiol-reactive electrophiles that covalently bind to the free sulfhydryl group (Cys residues) in key substrates such as GSH. Together, these data indicate that elevated production of ACR and its protein adducts during conditions of oxidative stress can further potentiate a state of redox imbalance via the depletion of GSH (Furuhata et al., 2002).

4HNE as a signaling molecule

Initially, proteotoxic stress, defined as the accumulation of misfolded protein species, elicits adaptive responses in cells aimed at restoring proteostasis, thus promoting cell survival during conditions of elevated stress (Brancolini and Iuliano, 2020). At low levels, 4HNE can play a physiologically significant role in the regulation of central signaling pathways (Forman et al., 2008; Jaganjac et al., 2012) and cellular processes (Shoeb et al., 2014). This is largely attributed to the activation of proteins involved in signal transduction and gene expression, including transcription factors, receptors, kinases, and phosphatases (Ayala et al., 2014; Ullery and Marnett, 2012). By way of example, nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor involved in the regulation of several genes that contain antioxidant-responsive elements.

Under physiological conditions, NRF2 is sequestered in the cytoplasm by the repressor protein, Kelch-like ECH-associated protein 1 (KEAP1). In the absence of excess oxidant molecules, NRF2 expression is maintained at low levels through a process of ubiquitination and proteasomal degradation. Conversely, under conditions of oxidative stress, KEAP1 is adducted by 4HNE at Cys513 and Cys518, resulting in disrupted binding of the KEAP1-NRF2 complex and reduced degradation of NRF2 (Gao et al., 2020). Adduction of additional cysteine residues in KEAP1, including Cys151, Cys288, Cys226, and Cys368, is similarly thought to be involved in 4HNE-induced activation of the NRF2 signaling pathway (Dinkova-Kostova et al., 2002; McMahon et al., 2010; Parvez et al., 2015).

Free from the inhibition of KEAP1, accumulated cytoplasmic NRF2 translocates into the nucleus and activates the expression of antioxidant-responsive element-containing genes to stimulate antioxidant (i.e., glutathione reductase, glutathione peroxidase, glutaredoxin, peroxiredoxin, and thioredoxin) and cytoprotective defenses (Fig. 5) (Chorley et al., 2012; Cyran and Zhitkovich, 2022; Schmidlin et al., 2019; Thimmulappa et al., 2002). Moreover, NRF2 activation is associated with upregulated expression of AKR and ALDH enzymes, which, as discussed earlier, are capable of detoxifying sublethal levels of 4HNE (Breitzig et al., 2016; Dalleau et al., 2013; Schaur et al., 2015).

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FIG. 5.

4HNE adduction contributes to a loss of protein function in somatic and reproductive cells. At moderate concentrations, 4HNE induces protein denaturation with consequences for secondary and tertiary structures, stability, and function. (A) Physiological conditions: NRF2 is a central transcription factor that regulates the resistance of somatic cells to oxidative stress. Under physiological conditions, NRF2 is sequestered in the cytoplasm by the repressor protein, KEAP1. This protein–protein interaction is stabilized by the presence of two binding motifs located at the N-terminus of NRF2: ETGE and DLG. In the absence of excess oxidant molecules, NRF2 expression is maintained at low levels through a process of ubiquitination and subsequent proteasomal degradation. Thereby, regulation of NRF2 serves to maintain basal levels of antioxidant expression. Pathological conditions: KEAP1 can be adducted by 4HNE (primarily at Cys513 and Cys518) leading to a loss of its repressive activity and subsequent dissociation of the NRF2-KEAP1 complex. Once freed from the inhibition of KEAP1, NRF2 translocates into the nucleus and activates the expression of ARE genes to stimulate antioxidant/cytoprotective defenses. (B) Physiological conditions: Localized within the mitochondrial-rich midpiece of the sperm flagellum, the SDH enzyme complex (complex II) that spans the IMM is known to play a fundamental role in mitochondrial function. The SDH enzyme complex is uniquely responsible for (i) catalyzing the oxidation of succinate into fumarate as part of the TCA cycle and (ii) transferring electrons from succinate to ubiquinone (UQ) in the ETC. Maintenance of these respiration pathways is necessary to support sperm motility and capacitation, as well as oocyte fusion and penetration. Pathological conditions: During conditions of oxidative stress, SDH protein modification by 4HNE leads to a loss of function. This facilitates the leakage of electrons (−) from the ETC, where they subsequently bind to molecular oxygen (O) to generate ROS (i.e., O₂ ^•−), which further perpetuate a state of oxidative stress. The events that follow 4HNE-SDH adduction are characterized by a rapid loss of mitochondrial membrane potential, impaired motility, oxidative DNA damage and fragmentation, as well as decreased cell viability, all of which are associated with infertility phenotypes and poor offspring outcomes. ARE, antioxidant-responsive element; ETC, electron transport chain; IMM, inner mitochondrial membrane; KEAP1, Kelch-like ECH-associated protein 1; NRF2, nuclear factor erythroid 2-related factor 2; O₂ ^•−, superoxide; SDH, succinate dehydrogenase; TCA, tricarboxylic acid. Created with BioRender.com

There is also evidence to suggest that NRF2 facilitates the clearance of potentially cytotoxic misfolded and aggregated proteins species through the targeted activation of ALP (Qin et al., 2019; Wang et al., 2014) and/or UPS-mediated (Pajares et al., 2017) degradation pathways. In this way, 4HNE can positively regulate metabolism and restore proteostasis (Ayala et al., 2014; Dalleau et al., 2013; Gan and Johnson, 2014; Grimsrud et al., 2008; Huang et al., 2012; Ishii et al., 2004; Levonen et al., 2004; Siow et al., 2007; Tanito et al., 2007; Zhang et al., 2010). Unfortunately, the antioxidant capacity of the cell can be rapidly depleted as the concentration and activity of 4HNE increases, thus propagating a state of proteotoxic stress.

Protein misfolding and loss of function

At moderate levels, 4HNE induces protein denaturation with consequences for secondary and tertiary structures, stability, and function (Alviz-Amador et al., 2019; Fritz et al., 2012; Perluigi et al., 2012; Smathers et al., 2012). Examples of 4HNE-induced loss of protein function in the context of somatic biology are presented in Figure 5A. These intrinsic changes typically manifest in the form of misfolded and/or nonfunctional proteins. Even a low level of misfolded proteins can be detrimental to the cell, with some studies suggesting that the presence of one metastable or misfolded protein can destabilize additional unrelated proteins within the proteome (Brettschneider et al., 2015; Gidalevitz et al., 2009; Gidalevitz et al., 2006; Taylor and Dillin, 2011). This phenomenon, termed as “bystander” mechanism, has been observed as a consequence of the high amyloid burden in neurodegenerative disorders, such as Alzheimer's disease (Xu et al., 2013).

The accumulation and subsequent aggregation of these misfolded protein species not only impair normal cellular function but may elicit cytotoxicity, ultimately leading to the induction of cell death via apoptotic, necrotic, or ferroptotic pathways (Ayala et al., 2014; Haeri and Knox, 2012). To combat the accumulation of these potentially cytotoxic non-native species, key effectors of the proteostasis network are responsible for the inhibition or reversal of protein misfolding/aggregation, or alternatively, direct terminally misfolded proteins toward a range of degradation pathways (Klaips et al., 2017; Tittelmeier et al., 2020). Most notably, a diverse and ubiquitous class of proteins, termed molecular chaperones, recognizes and transiently stabilizes hydrophobic motifs exposed on the surface of misfolded proteins (Stirling et al., 2003). A mere binding activity is considered a holdase function that does not require ATP. This holdase activity temporarily stabilizes misfolded protein species, thereby preventing (i) their association with other unstable proteins, (ii) protein aggregation, and/or (iii) aberrant degradation.

However, protein folding and refolding often rely on an ATP-dependent cycle that facilitates repeated binding and release of the same or different chaperones and/or co-chaperones (Brehme et al., 2014; Bukau et al., 2000; Cho et al., 2015; Liberek et al., 2008; Mayer and Bukau, 2005; Preissler and Deuerling, 2012; Reichmann and Suss, 2015; Tittelmeier et al., 2020). Despite these investments into the maintenance of proteostasis, the accumulation of misfolded protein species is further compounded by the fact that molecular chaperones, including heat shock protein 70 (HSP70), heat shock protein 90 (HSP90), and the protein disulfide isomerase family, are directly targeted by 4HNE (Castro et al., 2017).

For example, 4HNE-mediated modification of heat shock protein A2 (HSPA2), the inducible variant of HSP70, significantly inhibits the protein refolding ability of this chaperone in vitro. This inhibitory effect has been indirectly linked to the covalent adduction of Cys267 in the ATPase domain of HSPA2 (Carbone et al., 2004). Given that the affinity of the HSP70 family for protein substrates is a function of nucleotide (i.e., ATP or ADP) presence at the ATPase domain (Fink, 1999), it follows that modification of this site can alter the state of nucleotide binding and, ultimately, reduces the affinity of HSPA2 for its substrate (Carbone et al., 2004).

In addition to directly modifying proteins, carbonyl-induced oxidative stress may also lead to ATP depletion as observed in bacterial cells subjected to HOCl stress (Hannum et al., 1995; Ulfig and Leichert, 2021). Ultimately, depletion of cellular ATP leads to the inactivation of essential proteases and ATP-dependent chaperone systems (i.e., HSP70/HSP40) that typically protect against oxidative stress-induced unfolding and protein aggregation (Khor et al., 2004; Winter et al., 2005). To any extent, the incapacitation of these remodeling pathways contributes significantly to the accumulation of misfolded and potentially cytotoxic protein species that are either degraded or go on to form aggregates.

Protein degradation

The targeted removal of carbonylated proteins can occur through several proteolytic mechanisms. While we exclusively discuss pathways associated with the UPS here, the proteostasis network also employs the ALP, which digests modified and/or misfolded proteins. The 20S ubiquitin-independent proteasome is known to catalyze the degradation of misfolded proteins based on its ability to detect exposed hydrophobic amino acids and aromatic residues (Grimsrud et al., 2008; Grune and Davies, 2003; Grune et al., 1997).

As mentioned previously, RCS adduction induces several physiochemical changes within the target protein, one of those being an increase in surface hydrophobicity (Thorpe and Baynes, 2003). This occurs when hydrophobic moieties from within the protein core are exposed on the surface via oxidative rearrangement (partial unfolding) of secondary and tertiary protein structure (Grune and Davies, 2003; Grune et al., 1997). Such changes render RCS-modified proteins more susceptible to degradation than their non-oxidized counterparts (Castro et al., 2017; Dukan et al., 2000; Grune et al., 2004; Grune et al., 2003; Huang et al., 1995; Maisonneuve et al., 2008).

Alternatively, RCS-modified proteins may also serve as targets for preferential ubiquitination (compared with their native counterparts), with this modification tagging them for degradation either by the 26S ubiquitin-dependent proteasome (Botzen and Grune, 2007) or by a lysosome and ubiquitin-dependent but proteasome-independent pathway (Marques et al., 2004). In a similar way to ubiquitination, it has been postulated that carbonylation itself may tag proteins for degradation (Dukan et al., 2000; Grune et al., 2003; Maisonneuve et al., 2008). Illustrative of this phenomenon, key examples of somatic and reproductive-related protein degradation pathways induced by 4HNE adduction are depicted in Figure 6.

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FIG. 6.

4HNE adduction marks somatic and reproductive proteins for preferential degradation via proteasomal pathways. To combat the accumulation of misfolded protein species, key effectors of the proteostasis network are responsible for the targeted removal of carbonylated proteins. (A) Physiological conditions: ADH is a key oxidoreductase enzyme involved in the conversion of aldehydes (i.e., 4HNE) to less reactive alcohol species. At low concentrations, both 4HNE and its GSH conjugate (4HNE-GSH) can be reduced via the activity of ADH into DHN or GSH-DHN. This represents one of three detoxification pathways responsible for the maintenance of steady-state 4HNE concentrations. Pathological conditions: 4HNE has been shown to regulate equine liver ADH activity in a concentration-dependent manner, with these effects attributed to 4HNE-induced conformational change of two Cys residues (Cys46 and Cys111) at the zinc-binding domain of this protein. Evidently, at low concentrations (2-molar excess), 4HNE-ADH adduction accelerates the rate at which ADH is ubiquitinated and subsequently degraded by the 26S proteasome. Alternatively, at higher concentrations (100-molar excess), the extent of 4HNE modification appears to have an inhibitory effect on ADH degradation (not depicted). Ultimately, altered levels of ADH may potentially impair the detoxification capacity of the cell. (B) Physiological conditions: In humans, the critical interaction between the sperm and the oocyte is largely mediated by the regulated expression of multimeric zona pellucida receptor complexes on the sperm surface. One such complex is composed of (i) SPAM1, a hyaluronidase primarily involved in cumulus cell matrix dispersal, (ii) ARSA, an enzyme implicated in adhesion to sulfated ligands adorning the zona pellucida, and (iii) HSPA2 that coordinates receptor complex formation and remodeling during capacitation. Pathological conditions: 4HNE adduction is correlated with HSPA2 ubiquitination and subsequent degradation through proteasomal pathways, thus contributing to a significant reduction in HSPA2 abundance in developing germ cells. This loss of HSPA2-mediated human sperm–egg interaction in vitro is likely due to the attenuation of HSPA2 chaperoning activity and the resultant failure to correctly position oocyte receptors (i.e., SPAM1 and ARSA) on the anterior surface of the sperm head. Ultimately, dysregulation of HSPA2 is associated with reduced sperm quality and poor fertility outcomes following the use of artificial reproductive technologies. ADH, alcohol dehydrogenase; ARSA, arylsulfatase A; DHN, 1,4-dihydroxynonene; GSH, glutathione; HSPA2, heat shock protein A2; SPAM1, sperm adhesion molecule 1. Created with BioRender.com

To the detriment of the cell, the efficiency of proteolytic machinery rapidly declines during aging as the concentration of intracellular RCS increases, thus contributing to the accumulation of misfolded proteins and the perpetuation of proteotoxicity. This decline in proteolytic function may result from the direct modification of proteases by RCS, or, alternatively, the indirect inhibition of proteolytic enzymes by the carbonylated proteins they intend to degrade (see the Protein aggregation section) (Smerjac and Bizzozero, 2008). For instance, several studies suggest that members of the UPS, including the 20S and 26S proteosome, serve as targets for modification by 4HNE (Bardag-Gorce et al., 2005; Castro et al., 2017; Ferrington and Kapphahn, 2004; Grune and Davies, 2003; Kessova and Cederbaum, 2005; Okada et al., 1999; Vieira et al., 2000). In this way, RCS-mediated inhibition of protein degradation machinery increases the number of misfolded species available to participate in cross-linking and/or aggregation events.

Protein aggregation

While moderately carbonylated proteins are susceptible to misfolding and degradation, heavily carbonylated proteins have a propensity to form high-molecular-weight aggregates (Gonos et al., 2018; Smerjac and Bizzozero, 2008). Protein aggregates are a continuum of misfolded structures that range from cytotoxic globular oligomers, which retain some solubility, to more advanced amyloid fibrils or diffuse amorphous aggregates, which are largely insoluble (Adamcik and Mezzenga, 2018; Breydo and Uversky, 2015; Chen et al., 2017; Luo et al., 2014; Wu et al., 2022; Wu et al., 2021; Wu et al., 2020). As mentioned earlier, aggregates are capable of inhibiting the proteasome whose function is to degrade them, thus conferring resistance to proteolysis and further accelerating the rate of aggregate formation (Castro et al., 2017).

For instance, 4HNE modification of neuronal proteins, such as amyloid beta peptide (Aβ) and α-synuclein (α-Syn), can promote intermolecular cross-linking, oligomerization, and the subsequent formation of protofibrillar aggregates that can inhibit the proteasome (Bae et al., 2013; Shringarpure et al., 2000). Mechanistically, the soluble oligomeric forms of Aβ and α-Syn have been shown to inhibit the 20S proteasome through allosteric stabilization of a closed substrate gate conformation, consequently blocking substrate translocation into the catalytic degradation chamber. While the ability to degrade proteins is reduced as oligomers begin to accumulate, the level of proteasomal impairment continues to increase exponentially as more proteins accumulate and oligomerize (Fig. 7A) (Thibaudeau et al., 2018). The accumulation of protein oligomers and/or aggregates can perturb organelle and cellular functions, trigger cell death pathways, and ultimately, lead to a decline in the overall well-being of an organism (Iuchi et al., 2021).

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FIG. 7.

4HNE-mediated modification drives protein aggregation in somatic and reproductive cells. While moderately carbonylated proteins are susceptible to misfolding and degradation, heavily carbonylated proteins have a propensity to form oligomers and/or aggregates. (A) Physiological conditions: α-Syn is a neuronal protein that primarily localizes within the presynaptic terminal. While the physiological roles of α-Syn remain elusive, it is understood that this protein plays a key role in the regulation of synaptic vesicle trafficking and subsequent neurotransmitter (i.e., dopamine) release. In this context, membrane-bound α-Syn has been shown to promote synaptic vesicle clustering and SNARE complex assembly (interaction between v-SNARE and t-SNARE) at the presynaptic plasma membrane. Pathological conditions: During periods of elevated oxidative stress and aging, 4HNE modification of α-Syn promotes intermolecular cross-linking, oligomerization, and the subsequent formation of protofibrillar aggregates. To the detriment of the cell, oligomeric α-Syn can block the 20S proteasome, thus impairing the capacity of the cell to degrade misfolded and/or aggregated proteins, including α-Syn itself. The resultant accumulation of α-Syn oligomers, protofibrils, and fibrils can contribute to both protein and cellular toxicity, a hallmark in synucleinopathies such as Parkinson's disease. (B) Physiological conditions: AKAP4 is the most abundant constitutive protein of the fibrous sheath, which extends along the principle-piece of the sperm flagellum. Functionally, AKAP4 is responsible for recruiting cAMP-dependent PKA to the fibrous sheath and acts as a subcellular scaffold to tether PKA within the immediate proximity of its substrates. Following release from its repressor (PKA-R), PKA functions to catalyze the phosphorylation of proteins involved in the development, activation, and maintenance of sperm motility and capacitation. Pathological conditions: 4HNE-AKAP4 adduction has been shown to induce (1) aberrant degradation of AKAP4 via nonclassical pathways resulting in decreased protein expression and (2) an increased degree of AKAP4 aggregation. In this way, the modification of AKAP4 is associated with a significant reduction in activity, thus contributing to lower levels of PKA and phosphorylation in mature sperm. This negatively impacts the motility profile of capacitated human sperm (i.e., altered velocity parameters and reduced progressive motility) as well as the ability of these cells to undergo the acrosome reaction. α-Syn, α-synuclein; AKAP4, A-kinase anchor protein 4; PKA, protein kinase A. Created with BioRender.com

In the context of human health, aberrant protein aggregation is a common feature associated with the development and/or progression of serious pathological conditions, collectively termed protein misfolding diseases (PMDs). Upward of 30 PMDs have been identified to date, including diverse systemic disorders such as type 2 diabetes mellitus (Mukherjee et al., 2015), cystic fibrosis (Fraser-Pitt and O'Neil, 2015), and certain types of cancer (Li et al., 2022). The most prevalent and increasingly common classes of PMDs are neurodegenerative proteinopathies (i.e., Alzheimer's disease, Parkinson's disease, and Creutzfeldt–Jakob disease) that arise from protein misfolding/aggregation in the central nervous system.

It is important to note that PMDs are typically defined by the misfolding of one or several distinct aggregation-prone protein(s). For example, neuropathological protein deposits are predominantly composed of Aβ and tau in Alzheimer's disease (Hardy and Higgins, 1992; Iqbal et al., 2005; Tiwari et al., 2019), α-Syn in Parkinson's disease (Atik et al., 2016; Stefanis, 2012; Yoo et al., 2023), and TAR DNA-binding protein 43 in multiple disorders such as frontotemporal dementia and amyotrophic lateral sclerosis (Jo et al., 2020; Steinacker et al., 2019). Identification of the individual protein(s) responsible for these disorders makes it easier to track disease and develop target-based treatments.

SDH-4HNE adduction

Mitochondria play a fundamental role in energy production, redox equilibrium, calcium regulation, and apoptotic pathways, all of which are necessary to support sperm motility, capacitation, hyperactivation, and the acrosome reaction, as well as oocyte fusion and penetration (Boguenet et al., 2021). Given the aforementioned functions, it follows that mitochondrial dysfunction is linked to a decline in sperm quality and/or infertility (Durairajanayagam et al., 2021). In fact, recent comparative proteomic studies indicate that proteins associated with mitochondrial oxidative phosphorylation (OXPHOS) and the tricarboxylic acid (TCA) cycle are dysregulated in sperm samples from asthenozoospermia patients (characterized by a reduced or complete absence of motility) compared with fertile men (Amaral et al., 2014; Nowicka-Bauer et al., 2018).

Among the identified mitochondrial proteins, SDH activity has been positively correlated with sperm function and fertility (Woodhouse et al., 2022). Located in the inner mitochondrial membrane, the SDH enzyme complex plays a unique dual role in respiration, (i) catalyzing the oxidation of succinate into fumarate in the TCA cycle (Cecchini, 2003; Miyadera et al., 2003) and (ii) transferring electrons from succinate to ubiquinone in the electron transport chain as part of the OXPHOS pathway (Cecchini, 2003; Miyadera et al., 2003; Sun et al., 2005).

During conditions of elevated oxidative stress, 4HNE adducts to SDH proteins localized within the mitochondria-rich midpiece of the sperm flagellum (Aitken et al., 2012b). This finding is consistent with previous studies (in rodents) that have identified mitochondrial SDH as a target for 4HNE modification in the central nervous system (Picklo et al., 1999) and the diabetic heart (Lashin et al., 2006). This indicates that SDH proteins may be conserved targets of 4HNE adduction in the body, and their adduction may be an early indication of pathology. In human spermatozoa, 4HNE-SDH adduction results in protein loss of function and widespread mitochondrial dysfunction.

Notably, 4HNE-mediated disruption of SDH activity facilitates the leakage of electrons from the electron transport chain, subsequently increasing ROS production and activating apoptotic pathways, thus perpetuating a state of oxidative stress (Fig. 5B). The cascade of events that follow 4HNE-SDH adduction can be characterized by a rapid loss of mitochondrial membrane potential, impaired motility, oxidative DNA damage and fragmentation, as well as a profound loss of cell viability (Aitken et al., 2012b), all of which are associated with infertility phenotypes and poor offspring outcomes (Aitken and De Iuliis, 2010; Aitken et al., 2011; Espinoza et al., 2009; Koppers et al., 2008; Mitchell et al., 2011).

HSPA2-4HNE adduction

HSPA2 is a testis-enriched member of the HSP70 chaperone family implicated in a variety of cellular processes including protection of the proteome from stress (Filipczak et al., 2012), protein (re)folding and disaggregation (Mayer and Bukau, 2005), transmembrane transport of client proteins (Dun et al., 2012, Huszar et al., 2000) and the assembly/dissociation of multimeric protein complexes (Redgrove et al., 2011). Within the human testis, HSPA2 is expressed at two discrete stages; (1) during meiosis as a component of the synaptonemal complex, and (2) during spermiogenesis/sperm maturation, where it is considered to provide a robust discriminative index of fertilizing potential (Ergur et al., 2002, Huszar et al., 2006, Huszar et al., 2000, Motiei et al., 2013). In addition to its generic chaperoning roles, HSPA2 is also involved in several testis-specific functions, including regulation of germ cell differentiation (Eddy, 1999), the replacement of histones by protamines during nuclear compaction (Govin et al., 2006) and priming of the sperm surface for zona pellucida recognition and binding (Bromfield et al., 2015, Nixon et al., 2015, Redgrove et al., 2011, Redgrove et al., 2013, Redgrove et al., 2012).

Defective sperm-zona pellucida interaction is a major contributor to male infertility (Liu and Baker, 2003). Studies by Redgrove et al. (2012) revealed that in human spermatozoa these interactions are largely mediated by the regulated expression of multimeric zona pellucida-receptor complexes on the sperm surface. The most extensively studied of these complexes is constituted by; (1) sperm adhesion molecule 1 (SPAM1), a hyaluronidase primarily involved in cumulus cell matrix dispersal (Kimura et al., 2009, Lathrop et al., 1990), (2) arylsulfatase A (ARSA), an enzyme implicated in adhesion to sulphated ligands adorning the zona pellucida (Carmona et al., 2002, Tantibhedhyangkul et al., 2002), and (3) HSPA2 which coordinates receptor complex formation and remodeling during capacitation (Redgrove et al., 2012).

Given this regulatory role, it follows that 4HNE-mediated modification of HSPA2 causes a reduction in human sperm–egg interaction in vitro, likely due to the attenuation of HSPA2 chaperoning activity and the resultant failure to correctly position oocyte receptors (i.e. SPAM1 and ARSA) on the anterior surface of the sperm head (Bromfield et al., 2015). Furthermore, 4HNE-adduction is correlated with HSPA2 ubiquitination and subsequent degradation through proteasomal pathways, thus contributing to a significant reduction in HSPA2 abundance in developing germ cells (Fig. 6B). Ultimately, dysregulation of HSPA2 is associated with oligozoospermia (low sperm count) (Cedenho et al., 2006, Lima et al., 2006), poor sperm quality (Ayaz et al., 2018) and reduced levels of zona binding (Huszar et al., 2000), as well as impaired fertility and adverse pregnancy outcomes following in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI) (Ergur et al., 2002, Huszar et al., 1992).

AKAP4-4HNE adduction

AKAP4 is the most abundant constitutive protein of the sperm fibrous sheath (Brown et al., 2003), a unique cytoskeletal structure surrounding the axoneme and outer dense fibers that extend along the principle-piece of the flagellum (Eddy et al., 2003). Functionally, AKAP4 is responsible for recruiting cAMP-dependent protein kinase A (PKA) to the fibrous sheath (Miki et al., 2002) and acts as a subcellular scaffold to tether PKA within the immediate proximity of its enzymatic substrates (Brown et al., 2003). In this way, AKAP4-coordinated compartmentalization of PKA serves to regulate the specificity of signal transduction pathways and metabolic processes that support the development, activation, and maintenance of sperm motility and capacitation (Luconi et al., 2011; Sergeant et al., 2019).

The importance of AKAP4 for reproductive success is eloquently highlighted by gene ablation studies in which male mice lacking precursor (proAKAP4) and mature AKAP4 expression are rendered infertile due to abnormal sperm morphology (i.e., aberrant fibrous sheath formation and a shorted flagella) and a loss of progressive motility (Fang et al., 2019; Miki et al., 2002). Recent studies have reported the application of affinity-based isolation techniques coupled with mass spectrometry to characterize the 4HNE adductome of oxidatively stressed human spermatozoa (Baker et al., 2015). Among the dominant targets identified was AKAP4, with these data revealing at least three AKAP4 peptides that harbor either one or two 4HNE-modified residues following exogenous treatment with the aldehyde (Baker et al., 2015). Further validation of these data confirmed that both the precursor and mature forms of AKAP4 are conserved targets for 4HNE adduction in post-meiotic germ cells (round spermatids) and mature mouse and human spermatozoa (Nixon et al., 2019).

Evidently, 4HNE modification is associated with a substantial reduction in proAKAP4 and AKAP4 levels, although this loss appears to be independent of conventional proteolytic degradation pathways. A concomitant increase in the degree of proAKAP4 and AKAP4 aggregation was similarly observed in germ cells following 4HNE treatment. While the mechanisms are not yet fully understood, it has been theorized that 4HNE-induced aggregation of these proteins may reduce their activity and thus affect the efficacy with which they are recovered from cell lysates (Nixon et al., 2019).

As in somatic cells, RCS-mediated protein modification in the germline can have detrimental consequences for both protein and cell function. Taking proAKAP4 and AKAP4 for example, Nixon et al. (2019) observed that 4HNE-induced aggregation and/or nonclassical degradation of these proteins is correlated with reduced levels of PKA and tyrosine kinase phosphorylation in mature human spermatozoa. These data suggest that 4HNE can dysregulate capacitation-associated phosphorylation, potentially through attenuation of the signaling framework assembled around the AKAP4 scaffold.

Downstream of these signaling events, 4HNE negatively impacts the motility profile of capacitated human spermatozoa (i.e., altered velocity parameters and reduced progressive motility) as well as the ability of these cells to undergo the acrosome reaction (Fig. 7B) (Nixon et al., 2019). As a caveat for the interpretation of these data, it is important to consider that alternate proteins involved in capacitation signaling (PKA), motility (dynein, outer dense fiber protein 1), and membrane remodeling (HSPA2) can also serve as primary targets for 4HNE modification (Baker et al., 2015). The broad-spectrum of 4HNE targets makes it difficult to differentiate the downstream consequences of proAKAP4 and AKAP4 adduction from those elicited by the modification of other proteins.