Impact of Lysine Succinylation on the Biology of Fungi

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

The review article entitled Impact of Lysine Succinylation on the Biology of Fungi is well-written and I recommend publishing, few suggestions can be revised before acceptance of this review paper.

·        Add introduction of discovery and regulation of Ksucc.

·        Add a detail table of enzymes related to succinylated in these fungi with references.

·        Add brief information of comparison of succinylation sites among various fungal species.

The main question addressed by the research: Clinical contexts in the management of mycotoxin.

Figures look fine, only high resolution images need to add

 

Author Response

Reviewer #1: The review article entitled Impact of Lysine Succinylation on the Biology of Fungi is well-written and I recommend publishing, few suggestions can be revised before acceptance of this review paper.

Reviewer comment: Add introduction of discovery and regulation of Ksucc.

Answer: I agree and have revised as followed

PTMs has undergone a significant transformation following the discovery of lysine succinylation (Ksucc).

This newly recognized PTM has seized the attention of researchers due to its regulatory implications in a wide array of biological processes. Lysine succinylation now stands alongside extensively studied modifications like acetylation and methylation, contributing to the intricate web of cellular regulatory networks[1].

The exploration of lysine succinylation began with advancements in mass spectrometry-based proteomics, providing researchers with the tools to systematically examine protein modifications on a global scale. This technological progress has led to the identification of numerous succinylated proteins across diverse organisms, revealing the ubiquity and significance of this PTM [2]. Notably, in the realm of fungal biology, the complex interplay between lysine succinylation and cellular processes, including metabolism and stress response, has taken center stage[3]. The regulation of Ksuc involves a multifaceted process that includes enzymes responsible for both the addition and removal of succinyl groups. Sirtuins, a class of NAD+-dependent deacylases, assume a pivotal role in dynamically controlling succinylation levels, introducing an additional layer of complexity to the regulatory landscape[4]. A comprehensive understanding of the mechanisms governing Ksucc dynamics holds the promise of unraveling its functional consequences across various cellular contexts.

Reviewer comment:  Add a detail table of enzymes related to succinylated in these fungi with references.

Answer: I agree and have revised as followed

The regulation of succinylation involves two enzyme types, lysine succinyltransferases, and desuccinylases. Currently, there have been no reported enzymes in fungi that perform both succinylation and desuccinylation. The only enzyme identified to date with lysine succinyltransferase activity is the histone acetyltransferase p300 [5]. Due to the structural resemblance of these short-chain acyl-CoAs (such as malonyl-CoA, succinyl-CoA, and glutaryl-CoA) to acetyl-CoA, there was a suggestion that acetyltransferases catalyze promiscuous acyltransferase activity [5]. Sirtuins, alternatively termed Sir2 (Silent information regulator 2) proteins, constitute a category of NAD+-dependent deacetylases. Within mammalian cells, seven sirtuins (SIRT1–7) have been recognized. Existing evidence indicates that SIRT3–5 and SIRT7 possess desuccinylase activity [5] (Table 1).

 

Table 1: Lysine Succinylated Enzymes in the Fungi

Fungus	Enzymes	Function	References
Trichophyton rubrum	There are 18 proteins annotated as acetyltransferases (TERG_00136T0, TERG_00160T0, TERG_00920T0, TERG_00960T0, TERG_02546T0, TERG_03442T0, TERG_03711T0, TERG_04055T0, TERG_04687T0, TERG_04983T0, TERG_05174T0, TERG_05450T0, TERG_05561T0, TERG_06411T0, TERG_06572T0, TERG_07217T0, TERG_07375T0, and TERG_07548T0	Additional experiments are required to investigate whether one or several of these acetyltransferases possess succinyltransferase activities, or if there is another succinyltransferase in T. rubrum.	[6]
Yeast	In yeast, five Sir2 proteins have been acknowledged (Sir2p and Hst1–4)	Can catalyze other forms of lysine acylation.   in yeast, Hst2 exhibits a higher affinity for binding propionyl-lysine and butyryl-lysine compared to acetyl-lysine	[7-9]
Trichophyton rubrum	T. rubrum, five potential deacylases have been designated as Sir2 family histone deacetylases (TERG_03010T0, TERG_03268T0, TERG_05234T0, TERG_06970T0, and TERG_07330T0).	Further investigations are required to explore the activities of these Sir2 proteins. We are curious about whether one of these deacetylases might be involved in desuccinylase activities or if there is another desuccinylase present in these dermatophytes	[6]

 

 

 

Reviewer comment: Add brief information of comparison of succinylation sites among various fungal species.

Answer: I agree and have revised as followed

 

7.1. Comparison of succinylation sites among various fungal species

Histone lysine can also undergo decoration with succinylation and lysine malonylation. These modifications lead to a nearly identical charge shift as phosphorylation in histone residues, necessitating the use of highly precise mass spectrometry systems for their analysis [10]. In Saccharomyces cerevisiae, seven sites of lysine succinylation have been identified: H2AK13, H2AK21, H2BK34, H2BK46, H3K79, H4K31, and H4K77 [11]. The potential impact of these PTMs within the core domains of histones, closely engaged with DNA, suggests a role for succinylation in the modulation of histone–DNA interactions. To comprehend their functions, studies have utilized lysine substitutions, replacing them with arginine (R) or alanine (A) to prevent succinylation, or with glutamic acid (E) or aspartic acid (D) to simulate constant lysine succinylation [144]. The majority of these substitutions exhibited no discernible effect on the phenotype of S. cerevisiae. Notably, only the H4K77E mutant exhibited a loss of gene silencing at telomeres and rDNA, implying a role of H4K77suc in stabilizing nucleosome assembly [146].

Similarly, in a study conducted by Frankovsky et al, using a proteomic mass spectrometry analysis on purified yeast mitochondria, revealed the presence of 314 succinylated mitochondrial proteins and identified 1763 previously undiscovered succinylation sites. Among the structures impacted by succinylation is the mitochondrial nucleoid, a complex formed by mitochondrial DNA (mtDNA) and mitochondrial proteins. Their investigation unveiled that Abf2p, the primary constituent of mt-nucleoids responsible for compacting mtDNA in S. cerevisiae, undergoes succinylation in vivo at a minimum of thirteen lysine residues [12]. In a related study in Aspergillus flavus, investigation into succinylated proteins, comparing strains with varying aflatoxin production capabilities, revealed 1240 lysine succinylation sites in 768 proteins. Among these, 1103 lysine succinylation (Ksuc) sites in 685 proteins were quantified. The A. flavus standard strain NRRL3357, subjected to sodium succinate treatment, was employed to scrutinize aflatoxin biosynthesis[13]. This exploration led to the pioneering discovery of Ksuc's involvement in the secondary metabolic pathway and aflatoxin biosynthesis in A. flavus [14]. Employing tandem mass tag (TMT)-labeled quantitative lysine succinylome, they extensively documented the variations in Ksuc levels between A. flavus strains exhibiting high and low aflatoxin production under natural conditions. The quantitative global proteomics data highlighted that the upregulated proteins in the high aflatoxin yield group were predominantly associated with carbon-related metabolism, while the downregulated proteins were enriched in oxidative phosphorylation. Subsequent functional analysis revealed a direct correlation between the upregulated proteins involved in carbon-related metabolism and aflatoxin production [15] .

Morealso, Wang et al performed a Systematic analysis of the lysine succinylome in the model medicinal mushroom Ganoderma lucidum.  Additionally, succinylated sites K90 and K106 were identified in the conserved Fve region of the immunomodulatory protein LZ8 [16]. Whereas, in pyricularia oryzae, the analysis revealed a total of 2109 lysine succinylation sites within 714 proteins. Ten distinctive succinylation sequence patterns were recognized, with K*******Ksuc and K**Ksuc emerging as the two most favored ones. Lysine succinylation was observed in 10 crucial enzymes associated with the tricarboxylic acid (TCA) cycle. Among these, PGK and GAPDH exhibited the highest succinylation site count (12 sites), while FBP and PFK had the least (1 site) [17]. Likewise, the lysine succinylome from Candida albicans SC5314 was systematically characterized using an integrated proteomic approach. Notably, the comprehensive analysis revealed succinylation on every enzyme involved in the tricarboxylic acid (TCA) cycle.  As aconitrate hydratase (ACO), a pivotal enzyme in the conversion of citric acid to isocitrate, has 19 succinylated sites. Furthermore, three categories of rate-limiting enzymes were enriched in succinylation. Firstly, citrate synthase (CS) exhibited 9 succinylated sites. Secondly, isocitrate dehydrogenase (IDH1) and isocitrate dehydrogenase (NAD+) (IDH3) were succinylated at 2 and 7 sites, respectively. Thirdly, 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase) (DLST) featured 4 succinylated sites, while 2-oxoglutarate dehydrogenase complex E1 component (OGDH) had 2 sites [18].

Xu et al., carried out the first succinylome profile of Trichophyton rubrum reveals lysine succinylation on proteins involved in various key cellular processes. Among these, are secreted proteases, crucial for the virulence of dermatophytes by breaking down hard keratin tissues during infection, comprised eight succinylated members. These includes aminopeptidase, aspartic endopeptidase Pep2, leucine aminopeptidase 1, leucine aminopeptidase 2, subtilisin-like protease, Peptidase S41 family protein, tripeptidyl-peptidase SED2, and carboxypeptidase S1. Additionally, in the context of dermatophyte infection [19], their study identified succinylation in the mdr2-encoded ABC multidrug transporter (at K361 and K368) and the AcuE-encoded malate synthase (at K161, K319, K483, K486, and K501). Furthermore, two Rho-type GTPases, Rho GTPase Rho1 and Rho-GDP dissociation inhibitor (Rho-GDI), were found to be succinylated in our investigation [6]

 

Reviewer comment: The main question addressed by the research: Clinical contexts in the management of mycotoxin.

Answer: I agree and have revised as followed

The findings reported in this review are expected to contribute to the comprehension of the impact of lysine succinylation on the biology of fungi, thereby enhancing our understanding of these clinically significant pathogenic fungi.

Reviewer comment: Figures look fine, only high resolution images need to add

Answer: I agree and have revised as followed

The images resolution has been improved to 300 dpi to enhance clarity

 

Authors reply: OK! We added to introduction, Lysine Succinylated Enzymes in the Fungi and Comparison of succinylation sites among various fungal species

Reviewer 2 Report

Comments and Suggestions for Authors

The review cimb-2791099 with the title of “Impact of Lysine Succinylation on the Biology of Fungi” investigated the impact of lysine succinylation (Ksuc) on fungal biology, its reversible chemical alterations, evolutionarily conserved dynamics, and diverse role in protein functionality, cellular processes, and secondary metabolites, offering potential insights for innovative approaches in mycotoxin management.

The review is well written and organized, and below are some comments for improving the manuscript:

-The figures caption should include information about the tool used to create the images.

-I recommend to the authors to change the genus and species names to italics throughout the manuscript. In the first part of the review, they are not italicized.

-I recommend to the authors to clearly present the meaning or the full names of abbreviations when used for the first time in the text (e.g., YES media; VBS).

-Check the text; some words lack spaces, for example: 'elucidatethis’.

-Improve the images resolution to a minimum of 300 dpi. Some figures exhibit poor clarity.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Reviewer #2: The review cimb-2791099 with the title of “Impact of Lysine Succinylation on the Biology of Fungi” investigated the impact of lysine succinylation (Ksuc) on fungal biology, its reversible chemical alterations, evolutionarily conserved dynamics, and diverse role in protein functionality, cellular processes, and secondary metabolites, offering potential insights for innovative approaches in mycotoxin management.

The review is well written and organized, and below are some comments for improving the manuscript:

Reviewer comment: -The figures caption should include information about the tool used to create the images.

Answer: I agree and have revised as followed

Figure created with BioRender.com

Reviewer comment: -I recommend to the authors to change the genus and species names to italics throughout the manuscript. In the first part of the review, they are not italicized.

Answer: I agree and have revised as followed

The genus and species names has been italicized such as Aspergillus flavus throughout the manuscript

Reviewer comment: -I recommend to the authors to clearly present the meaning or the full names of abbreviations when used for the first time in the text (e.g., YES media; VBS).

Answer: I agree and have revised as followed

Yeast Extract Supplemented (YES)media; Versicolorin B Synthase (VBS)

Reviewer comment: -Check the text; some words lack spaces, for example: 'elucidatethis’.

Answer: I agree and have revised as followed

 elucidate this

Reviewer comment: -Improve the images resolution to a minimum of 300 dpi. Some figures exhibit poor clarity.

Answer: I agree and have revised as followed

The images resolution has been improved to 300 dpi to enhance clarity

Reviewer comment: Minor editing of English language required

Answer: I agree and have revised as followed

The English have been manuscript checked and edited by a colleague fluent in English writing.

 

Authors reply:  OK! We have made the corrections throughout the whole manuscript and highlighted them in yellow.

Reviewer 3 Report

Comments and Suggestions for Authors

This is a good review article on the molecular mechanisms underlying lysine succinylation (Ksuc) and its diverse functions in fungi, supported by biochemical, physiological, and pathological studies. This is a clear, section-by-section description of a more profound comprehension of Ksuc and its impact on the biology of fungi, and is expected to provide useful insights for further development of related research. The subject matter of this manuscript is within the scope of CIMB, I recommend publication in its current form.

Author Response

Reviewer #3: This is a good review article on the molecular mechanisms underlying lysine succinylation (Ksuc) and its diverse functions in fungi, supported by biochemical, physiological, and pathological studies. This is a clear, section-by-section description of a more profound comprehension of Ksuc and its impact on the biology of fungi, and is expected to provide useful insights for further development of related research. The subject matter of this manuscript is within the scope of CIMB, I recommend publication in its current form.

Authors reply:  OK! Thank you very much Prof.