Homocysteinylation and Sulfhydration in Diseases

Abstract

Homocysteine (Hcy) is an important intermediate in methionine metabolism and generation of one-carbon units, and its dysfunction is associated with many pathological states. Although Hcy is a non-protein amino acid, many studies have demonstrated protein-related homocysteine metabolism and possible mechanisms underlying homocysteinylation. Homocysteinylated proteins lose their original biological function and have a negative effect on the various disease phenotypes. Hydrogen sulfide (H₂S) has been recognized as an important gaseous signaling molecule with mounting physiological properties. H₂S modifies small molecules and proteins via sulfhydration, which is supposed to be essential in the regulation of biological functions and signal transduction in human health and disorders. This review briefly introduces Hcy and H₂S, further discusses pathophysiological consequences of homocysteine modification and sulfhydryl modification, and ultimately makes a prediction that H₂S might exert a protective effect on the toxicity of homocysteinylation of target protein via sulfhydration. The highlighted information here yields new insights into the role of protein modification by Hcy and H₂S in diseases.

1. INTRODUCTION

Homocysteine (Hcy) is considered to be a common intermediate at the crossing point of two important intracellular metabolic pathways. Hcy participates in the folate cycle with vitamin B₁₂, providing a one-carbon unit for nucleotide and amino acid metabolism. Another pathway is sulfur-containing amino acid metabolism, producing methionine (Met), cysteine (Cys), and even homocysteinylated proteins. Nevertheless, the available source of Hcy in human beings comes from Met, the essential amino acid from diet. In addition, the process of Hcy linking to the target protein means protein homocysteinylation (homocysteine modification). There are two ways of protein homocysteinylation: the isopeptide bond to lysine (Lys) residues (named N-homocysteinylated-protein, N-hcy-protein) [1, 2] or the disulfide bond to Cys residues (named S-homocys- teinylated-protein, S-Hcy-protein) [3]. Homocysteine thiolactone (HTL), as the intermediate product in homocysteinylation and only from Hcy, contributes to the protein N-homocysteinylation being the most distinguishing feature for Hcy in comparison to other amino acids. Conversely, the protein S-homocysteinylation is not specific for Hcy because corresponding S-thiolated proteins are produced by the formation of a disulfide bond, with or without Hcy [4]. N-homocysteiny- lation is a new protein modification, which may have a negative effect on the structure and function of the protein, leading to the onset of human disease [5]. Deficiencies of congenital or acquired factors in Hcy metabolism result in the accumulation of N-Hcy-proteins associated with cardiovascular and neurodegenerative diseases [6].

Hydrogen sulfide (H₂S) is an emerging member of the gasotransmitter family. H₂S was once regarded as a poisonous gas mainly present in the atmosphere, but currently, it is known to be synthesized effectively from Cys in mammals [7]. Based on the fact that nitric oxide (NO) transmits signals via covalent modification of Cys in the target protein (nitrosylation), H₂S also modifies Cys through transforming the -SH group of target protein into the -SSH group, called sulfhydration (sulfhydryl modification). While only a small proportion of proteins are nitrosylated, up to 50% of proteins are sulfhydrated in the hepatic tissue [8]. Sulfhydration is a reversible process in the thioredoxin system, like nitrosylation [9]. Sulfhydration is a highly prevalent and abundant protein modification; thus, it participates in a number of pathophysiological processes related to the vascular system and nervous system [7].

Both homocysteine and sulfhydryl modification are relevant to Cys as well as the metabolism of other sulfur-containing amino acids. However, there is a dearth of knowledge regarding the relationship between homocysteinylation and sulfhydration and even the role of these modifications in various disorders. A review of the literature was performed, and relevant publications were identified by searching the following databases: PubMed, Web of Science, and China National Knowledge Infrastructure. Keywords included homocysteine, homocysteinylation, hydrogen sulfide, sulfhydration, and protein modification. 136 articles were screened for relevance to the objective of this manuscript, which included one book. This article provides a review of the literature to highlight the importance of homocysteinylation and sulfhydration in different pathological states, for instance, cardiovascular and neurologic diseases.

2. HOMOCYSTEINE

Hcy represents a rather critical part in sulfur-containing amino acid metabolism, together with Cys and Met, which may have a significant impact on the beginning of life on earth, as the origin of coded peptide synthesis [10, 11]. The first sulfur-containing amino acid, disulfide cystine, was originally discovered from a urinary calculus in 1810. Later, Cys was recognized as a major constituent of proteins [12]. The new sulfur-containing amino acid Met was isolated from casein in 1922 [13]. The following study has demonstrated the existence and specific chemical structure of Met [14]. The third sulfur-containing amino acid Hcy, a homologue of Cys, is detected by the chemical decomposition of methionine with sulfuric acid [15]. Subsequent studies have demonstrated the physiological role of Hcy in the Cys and Met metabolic process by the classical transsulfuration and remethylation pathways, respectively [16]. Cys and Met can be used for coding genes, then incorporated into protein by the ribosome to take part in protein transcription and translation processes, whereas Hcy generally is not involved in the genetic code and ribosomal protein biosynthesis, regarded as a non-proteinogenic and auxiliary amino acid [17]. Recently, there has been increasing interest in the role of Hcy as a risk factor for several diseases due to its possible effects on neurons, including augmenting of oxidative stress [18], calcium influx [19], amyloid-β toxicity [20], activation of excitotoxicity [21], and caspase-dependent apoptosis [22]. Especially, it is likely that protein homocysteinylation is the possible mechanism underlying the pathological consequences of elevated Hcy levels.

2.1. Homocysteine Metabolism

Hcy, a result of transmethylation reaction in the cellular metabolism, is produced from Met in the body, whereas Met is from dietary protein, released in the alimentary canal, absorbed by the enterocyte, and transported to different organs via blood circulation [6]. Met is metabolized to S-adenosylmethionine (AdoMet or SAM) under the condition of Met S-adenosyltransferase (MAT) and ATP, which can offer activated methyl group for the biological methylated process [23]. The transmethylation reaction of converting AdoMet to S-adenosylhomocysteine (AdoHcy or SAH) leads to the formation of Hcy, which is the primary source of Hcy in the body [24].

Previous studies have clarified that Hcy metabolism is established and maintained stably through several fundamental pathways in mammals (Fig. 1) [25-27]. Hcy is remethylated to Met under the influence of Met synthase (MS), occurring in each organ of our body. The enzymeization of MS in the 5-methyltetrahydrofolate pathway also requires two cofactors: methyltetrahydrofolate reductase (MTHFR) and vitamin B₁₂, involved in the one-carbon metabolism pathway of Hcy. Another remethylation pathway is that betaine-Hcy methyltransferase (BHMT) transfers the methyl group from betaine to Hcy to form Met [28]. Moreover, Hcy is transsulfurated to Cys by two enzymes, cystathionine β-synthase (CBS) and cystathionine γ–lyase (CSE), with the requirement of vitamin B₆. The transsulfuration by CBS and CSE only occurs in the liver [29], kidneys [30], and brain tissues [31]. Additionally, Hcy metabolizes HTL through methionyl-tRNA synthetase (MetRS), which occurs in each organ of our body [32, 33]. HTL is then synthesized to N-Hcy-protein, a homocysteine modification of the protein. Nonetheless, hydrolysis of HTL by paraoxonase 1 (PON1) into Hcy protects against the accumulation of N-Hcy-protein in human beings. Cytoplasmic bleomycin hydrolase (BLMH) works like HTL [34], as well as mitochondrial biphenol hydrolase-like enzyme (BPHL), but the latter only works in vitro [35]. Apart from these, Hcy is converted to AdoHcy via the hydrolysis of S-adenosylhomocysteine hydrolase (SAHH). Furthermore, Hcy reacts with the sulfhydryl group of protein to form disulfide, for example, Cys-S-S-Cys protein and Hcy-S-S-Cys protein [36]. The genetic or nutritional deficiencies contribute to the defect of Hcy metabolizing to Cys or Met, resulting in the accumulation of Hcy and its protein-related products and increasing the flux of HTL pathway strongly associated with considerable pathological environments in humans. To our knowledge, the research about Hcy has received amazing development in the past decades due to the desire to understand the effect of Hcy and its related metabolites on cardiovascular and neurologic diseases [37-39].

Fig. (1).

Pathways of Hcy Metabolism. ① the generative pathway of homocysteine; ‚ the remethylation pathway; ƒ the transsulfuration pathway; „ the homocysteine-thiolactone pathway; … the reversible pathway of homocysteine generation. Abbreviations: Hcy, homocysteine; Met, methionine; AdoMet, adenosylmethionine; AdoHcy, adenosylhomocysteine; Cys, cysteine; HTL, homocysteine thiolactone; N-Hcy-protein, N-homocysteined protein; S-Hcy-protein, S-homocysteined protein; MS, Met synthase; MAT, Met S-adenosyltransferase; CBS, cystathionine β-synthase; CSE, cystathionine γ–lyase; MetRS, methionyl-tRNA synthetase; PON1, paraoxonase 1; SAHH, S-adenosylhomocysteine hydrolase; VB₆, vitamin B₆; VB_12, vitamin B₁₂.

Moreover, Hcy is also metabolized to excitatory amino acid neurotransmitters, such as homocysteic acid (HCA) and cysteine sulfinic acid [40, 41], which leads to activation of N-methyl-Daspartate (NMDA) glutamate receptor. According to the “glutamatergic hypothesis” of depression, another actor that should be considered in the multifactorial pathogenesis of this disease might be the homocysteic acid (HCA), a neurotoxic compound that derives from Hcy [42]. Increased levels of HCA have been found in the PFC of treatment-resistant depression (TRD) [43]. HCA might contribute to the dysfunction of the glutamatergic system in TRD, and an increase of HCA might be detected in major depressive disorder (MDD) patients with hyperhomocysteinemia (HHcy) [44]. Genetic polymorphisms in MTHFR and CBS genes or a deficiency in essential cofactors in the one-carbon metabolism pathway (vitamin B₆, B₁₂, folate) can lead to HHcy, which is known to decrease the SAM-dependent synthesis of dopamine, noradrenaline [42]. Supplementation with vitamin B₆, B₁₂, and folate is known to reduce HHcy [39]. Almeida et al. [45] have found that B vitamins (0.5 mg of vitamin B₁₂, 2 mg of folic acid, and 25 mg of vitamin B₆) enhance and sustain antidepressant response over 1 year.

Previous researches find elevated levels of Hcy in Alzheimer’s disease (AD) [46, 47] and cognitive impairment [48]. A preliminary study suggests that plasma HCA may be a useful indicator as an early diagnostic marker for mild cognitive impairment (MCI). HCA is possibly upstream from neurodegeneration in the Alzheimer’s disease pathology because it promotes amyloid polymerization and tau phosphorylation in AD [49]. Adequate vitamin B₆, vitamin B₁₂, and folate intakes were significantly associated with better cognitive reserve by affecting methylation levels of specific redox-related genes [50].

2.2. Protein Homocysteinylation

Homocysteinylation is recognized as a chemical non-enzymatic reaction between proteins and Hcy or HTL, including S- or N-homocysteinylation [51]. S-homocystei- nylation involves both the addition of reduced Hcy to available cysteinyl residues in proteins and a disulfide exchange reaction with optional disulfide, for example, S-homocysteinylated transthyretin (Fig. 2) [52-54]. Homocysteine modification affects the function of many proteins, but only a few proteins can be S-homocysteinylated, mainly due to analytical limitations at first. The following study has revealed that in most plasma and tissue, Hcy links to the protein by disulfide bond after treatment with 2-mercaptoethanol [55]. Moreover, exogenous Hcy could link to protein and be detected quantitatively by administration with 2-mercaptoethanol. These investigations have revealed for the first time the phenomenon for protein S-homocysteinylation [56, 57]. S-homocysteinylation alters the original structure of the protein, but the effect of these alterations on health and disease is not well known [5].

Fig. (2).

Pathways of protein homocysteinylation. Abbreviations: Hcy, homocysteine; MetRS, methionyl-tRNA synthetase; HTL, homocysteine thiolactone.

N-homocysteinylation is an irreversible process of non-enzymatic acylation between the carbonyl group and Lys residues of proteins, for instance, albumin [58], collagen [59], cytochrome C [60], and fibrinogen [61]; and the level of N-Hcy-protein is dominant directly in plasma ‘total Hcy’ [62, 63]. N-Hcy-protein was first established in human endothelial cells in the last century [64, 65], and subsequent pieces of research have confirmed the activity of N-Hcy-protein in humans [66] and mice [67]. There are two steps about the mechanism underlying N-Hcy-protein generation (Fig. 2). The first step is the metabolic conversion of Hcy to HTL catalyzed by MetRS, and the second step is the reaction with HTL and Lys residues of protein to afford N-Hcy-protein. Additionally, homocysteine modification could be induced by two other uncommon ways in humans (Fig. 2). The one way is the production of S-nitroso-Hcy through nitric oxide-mediated mechanism [68], and another is demethylation of protein Met residues to Hcy catalyzed by cuprum and ferrum [69]. Like other protein modifications, it is well established that protein homocysteinylation leads to alterations in some biological protein structures and functions, for example, protein cross-linking [70], protein aggregation [71], and the generation of autoantibodies directed against homocysteinylated proteins after activation of the immune response [72]. Thus, the role of homocysteinylation in protein damage has gained increasing importance.

N-homocysteinylation results in loss of protein functions due to changes in the protein structure and becomes susceptible to further oxidative damage. It has been observed that the structural and functional consequences resulting from N-homocysteinylation depend on the pI of the proteins. Basic proteins are resistant to structural and functional alterations due to N-homocysteinylation, whereas acidic proteins are sensitive to N-homocysteinylation and cause denaturation of proteins. Therefore, it is a very definite possibility that cells or tissues abundant with acidic proteins might be the major targets for Hcy-induced cytotoxicity [73]. Furthermore, large bodies of evidence support the fact that homocysteinylation results in the accumulation of damaged and dysfunctional products and even diseases [74, 75]. Increased N-homocysteinylation of tau and microtubule-associated protein 1 (MAP1) is a mechanism of brain aging in AD and vascular dementia, which depends on the concentration of Hcy and expression of methionine tRNAsynthetase (MARS) enzyme [76]. It has also been reported that homocysteinylated-proteins affect expressions of vascular endothelial cell-related genes in human beings, associated with cardiovascular and central nervous system diseases [77]. Some blood coagulation genes (CD9, ANXA8, SCUBE1) and lipid metabolism gene (SULT1E1) are affected by N- homocysteinylated proteins and Hcy. Most interestingly, epigenetic mechanisms involving chromatin/histone modifications might lead to HTL-induced endothelial dysfunction [78]. Additionally, in human fetal brains, down-regulation in the expressions of neural tube closure-related genes (Cecr2, Smarca4, and Dnmt3b), along with high levels of Hcy and H3K79-Hcy, contributes to the onset of neural tube defects [79]. These pieces of evidence suggest that protein homocysteinylation is an important intermediate process in the pathological consequences of various diseases.

2.3. Hydrogen Sulfide and its Metabolism

Hydrogen sulfide (H₂S) is a kind of colorless, flammable, and rotten egg odor-like gas. It easily crosses the plasma membrane because it is soluble in both water and lipid. In the past, H₂S was known as the environmental toxic gas until first identified as the endogenously generated signaling neuromodulator (Fig. 3) [80]. Endogenous H₂S is catalyzed synthetization by CBS and CSE, two vitamin B₆-dependent enzymes [81]. Meanwhile, CBS and CSE are associated with the transsulfuration pathway from Hcy to Cys. In particular, the coexistence of Hcy and Cys contributes to H₂S synthesis by CBS [82]. CBS catalyzes Hcy and serine to form cystathionine and H₂O, and serine is replaced by Cys, leading to the formation of H₂S. The CSE-catalyzed reaction to form H₂S is the β-elimination of Cys to ammonia, H₂S, and pyruvate, or the γ-elimination of Hcy to α-ketobutyrate [81]. As the Cys level is higher than Hcy in our body, the desulfhydration of Cys is the main mechanism underlying H₂S generation by CSE under normal conditions [83]. The third mechanism of H₂S production is Cys converting to 3-mercaptopytuvate (3-MP) by cysteine aminotransferase (CAT) and 3-MP converting to H₂S by 3-mercaptopyruvate sulfurtransferase (3-MST) [84, 85]. H₂S is also possibly synthesized from Cys by catalysis of D-amino acid oxidase (D-AAO) and 3-MST [86]. H₂S is enzymatically oxidized by sulfide: quinone oxidoreductase (SQR) in the cellular mitochondria at very low concentrations [87], which transfers electrons to ubiquinone (coenzyme Q) to involve in aerobic respiration metabolism [88, 89]. Based on current knowledge, H₂S takes part in many physiological processes, ranging from the regulation of vascular tone to neuroprotective effects [90, 91]. Available pieces of evidence suggest that one of the mechanisms underlying H₂S signaling is probably protein sulfhydration [8].

Fig. (3).

Pathways of H₂S production. Abbreviations: Hcy, homocysteine; Cys, cysteine; SQR, sulfide: quinone oxidoreductase; 3-MP, 3-mercaptopytuvate; CBS, cystathionine β-synthase; D-AAO, D-amino acid oxidase; 3-MST, 3-mercaptopyruvate sulfurtransferase; CAT, cysteine aminotransferase; CSE, cystathionine γ–lyase; VB₆, vitamin B₆.

2.4. Protein Sulfhydration

Sulfhydration (also named persulfidation or perthiolation) is believed to be a modification of the –SH group of Cys with H₂S to produce persulfide or –SSH group, functioning like nitrosylation [92]. In some sense, persulfidation is more specialized to describe the process in comparison to other terms. H₂S modifies Cys residues after the oxidative forms of Cys, rather than directly reacting. It is noteworthy that the main form of H₂S is its negative ion (HS^-) in soluble liquid, which is the major form of H₂S activity. The reactivity of sulfhydration is mainly dependent on the dissolvent and functional group of Cys residues [93]; for example, Cys of the low acid dissociation constant are more sensitive to sulfhydration [94].

Nevertheless, they are also more susceptible to oxidants (e.g., H₂O₂), resulting in the formation of sulfenic acid (SOH), sulfinic acid (SO₂H), or other sulfonic acid derivatives [95-97]. The formation of SOH is a reversible process, whereas modification of the thiol group of Cys is irreversible. Therefore, protein sulfhydration has an important role in preventing Cys residues of protein against irreversible modification [98]. Sulfhydration occurs by several pathways as mentioned below, which are the reaction of sulfide with oxidized Cys residues, for instance, cysteine sulfenic acids (Cys-SOH) or cysteine disulfides (-S-S-) [99], the reaction between oxidized sulfide species [100], and the reaction of H₂S₂ with cysteine thiol [101]. Previous studies strongly suggest that sulfhydration of target protein contributes to variations in the structure and function of the modified protein [102, 103].

Mechanistically, sulfhydration of the target protein is regarded as a fundamental mechanism underlying H₂S-regulated signaling pathways. Sulfhydration of Cys is an important paradigm of post-translational protein modification in the process of H₂S signaling and a critical redox mechanism to regulate H₂S-mediated neuroprotection and neurogenesis [104]. Several studies have shown that the sulfhydration of Cathepsin S (Cat S) at Cys25 [105] and Akt at Cys77 [106] are required for H₂S-mediated effects and might exert a vital role in the pathological functions of AD. H₂S keeps vascular health via the upregulation of vascular endothelial growth factor receptor 2 and neuropilin-1, which mainly results from sulfhydration of a transcription factor specificity protein 1 (SP1) at Cys68 and Cys755 residues [107]. Moreover, SP1, as a potent regulator of CSE, is sequestered and inactivated by mutant huntingtin in early Huntington’s disease (HD), leading to cell redox imbalance resulting from depleted Cys biosynthesis [108]. Additionally, sulfhydration of the apoptotic effector protein caspase-3 at Cys163 loses its activity and reduces apoptotic death in primary cortical neurons, suggesting the inhibitory role of protein sulfhydration in enzymatic activities [109]. The sulfhydration of target proteins has a prominent role in the prevention of age-related hippocampal long-term potentiation (LTP) deficit by serine racemase [110] and inhibition of Parkinson’s disease-related lesions by Parkin [111] or protein p66Shc (a modulator in mitochondrial redox signaling) [112], suggesting that this modification is also necessary for physiological states and pathological conditions, possibly including cardiovascular and neuronal systems.

2.5. Homocysteinylation and Sulfhydration in Diseases

According to the fact that H₂S protects humans from improper protein modification in contrast to the impairment of Hcy for protein, in a sense, we assume that H₂S may affect the destructive role of homocysteinylated target proteins by sulfhydration in pathological states.

2.5.1. Protein Homocysteinylation and Sulfhydration in Cardiovascular Diseases

Previous research has investigated that vasorelaxation is one of the essential functions of H₂S in human beings (Fig. 4) [113]. Specifically, H₂S may exert a vasorelaxative effect via the reduction of Hcy buildup, ultimately resulting in falling blood pressure [114]. It has been reported that the level of H₂S is significantly reduced in Hcy-treated rats, and exogenous H₂S inhibits Hcy-exposed myocardial injury [115]. High plasma albumin cysteinylation (CysAlb) and Hcy levels are observed in the intestinal ischemia-reperfusion model of rats [116]. Additionally, in the ischemia reperfusion-induced myocardial injury, administration with H₂S obviously decreases infarct size to improve the contractile function of cardiac muscle. Recent findings have shown a high level of sulfhydration during this process (Fig. 4) [117]. Normal cellular redox homeostasis is an important factor for heart disease. Homocysteinylation of metallothionein (MT) at the Cys residues obstructs zinc linking to the protein and abolishes superoxide dismutase (SOD) activity [118]. However, SOD reacts with H₂S by sulfhydryl modification at Cys111 residue, leading to slower protein unfolding and resistance to oxidation-induced protein aggregation (Fig. 4) [119, 120]. Sulfhydration of SOD may facilitate endothelial protection and reduce the risk of atherosclerosis (AS). AS is a prevalent and frequent cardiovascular disease among old people that begins with a series of inflammatory reactions induced by HHcy [121], generating HTL to modify Lys residue of the target protein [5]. The subsequent study found that H₂S attenuates aortic atherosclerotic plaque formation, in which the underlying mechanism may be associated with Kelch-like ECH-associated protein 1 (Keap1) sulfhydration at Cys151 to activate nuclear factor-like (Nrf) 2 signaling [122]. Acute myocardial infarction (AMI) is positively related to the increasing Hcy in several case-control and cohort studies [123]. Furthermore, existing investigation evaluated that homocysteinylation of low-density lipoprotein (LDL) is increased in AMI patients [124]. Protective mechanisms of H₂S in AMI involve several physiological and pathological processes, including the direct interaction between H₂S and ATP-sensitive potassium channel (K_ATP) channel proteins via sulfhydryl modification (Fig. 4) [125].

Fig. (4).

Effects of H₂S or sulfhydration on diseases. H₂S exerts protective effects on vasorelaxation, myocardial injury, redox homeostasis, endothelial injury, and fibrin lysis.

2.5.2. Protein Homocysteinylation and Sulfhydration in Neurologic Diseases

Vascular dysfunction induced by HHcy has an important effect on AD [126]. One aspect of vascular dysfunction is the formation of fibrin clots, which are not easy to dissolve and are produced from Hcy-fibrinogen [61]. It has already been shown that protein homocysteinylation promotes the interaction of fibrinogen with Aβ-peptide, which facilitates the development of fibrin clots and difficult fibrinolysis, enhances Aβ deposition in the cerebral vessel, and deteriorates cognitive defect in the AD model [127, 128]. Moreover, the homocysteinylation of Lys residues increases the neurotoxicity of the Aβ peptide by stabilizing soluble oligomeric intermediates [129]. Homocysteinylation of tau at critical amino acids decreases the binding ability of tau to microtubule protein (MTP), leading to the instability of MTP and disability to promote MTP assembly, which could inhibit the function of tau [130]. Nevertheless, H₂S reduces the polymerization of fibrinogen and increases fibrin lysis in the plasma (Fig. 4) [131]. In AD, owing to diminished protein sulfhydration, exogenous H₂S prevents hyperphosphorylation of tau by sulfhydrating its kinase and glycogen synthase kinase 3β (GSK3β), further ameliorating cognitive deficits [132]. We also find the following pieces of research: the bioactivity of MTHFR is positively associated with its S-sulfhydration level, and H₂S deficiency results in the decrease of MTHFR sulfhydration level and bioactivity in HHcy. However, H₂S donors reverse the decreased bioactivity of MTHFR in HHcy, thus reducing excessive Hcy levels. These studies suggest that H₂S could improve MTHFR bioactivity by sulfhydration, which might provide a candidate therapeutic strategy for HHcy [133]. In the Hcy-treated mice, supplementation of H₂S donors ameliorates Hcy-induced cerebrovascular pathology, cognitive deficits, and toxicity [134]. Based on these observations, we have speculated that appropriate regulation of sulfhydration by H₂S is essential for the inhibition of homocysteinylated protein, which may merit further investigation.

In addition to the above-mentioned effects of sulfhydration on homocysteinylation, some findings seem to provide completely different perspectives for their relationship. A recent study has demonstrated the inhibitory effect of protein homocysteinylation on enzymatic activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) by HTL [135]. However, H₂S causes sulfhydration of GAPDH in neuron dendrites to promote GAPDH binding to ubiquitin-protein ligase E3 (Siah protein). Then, Siah protein links to PSD95, which induces PSD95 degradation and memory dysfunction [136]. The contrary effect of sulfhydration on homocysteinylation may be attributed to the alterations of their substrate and concentration in different pathologic environments.

CONCLUSION

Elevated levels of Hcy and homocysteinylation have been found in many medical diseases. The role of protein homocysteinylation is currently attracting considerable research interest, simply because early intervention to normalize the Hcy-proteins may defer the development of these disorders and protect them from suffering Hcy-proteins-caused cell dysfunction. As discussed in this review, H₂S signaling via sulfhydration regulates a wide variety of cellular functions, and the sulfhydration of the target protein is necessary for organismal physiological health and human diseases. Thus, we speculate that to some extent, sulfhydration of H₂S exerts a beneficial effect on protein homocysteinylation under the condition of physiology or pathology. Nevertheless, some findings have deduced inconsistent results. Apparently, further research on these issues is needed with the help of a better understanding of the association of protein modification. Although contradiction exists, this review on homocysteinylation, sulfhydration, and their relationship in various diseases may help toward a better understanding of the current knowledge for further clinical and basic research. There is little doubt that the field of homocysteinylation and sulfhydration will be a topic of significant research in the future.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.