The submitted review by Mousa, Shkolnik, Alalouf, and Brik summarizes how chemical approaches are being applied to study ubiquitin-like proteins (UBLs). It is significant for leaders in synthetic tools for biochemical studies, such as Ashraf Brik, to periodically author reviews like this, as they can be invaluable to researchers, many of whom primarily have biological backgrounds and often purchase tools without knowing the underlying chemistry, potential pitfalls, or the collaborative opportunities to develop new tools based on existing ones. This review, therefore, holds substantial value for the field.
The purpose of my review is to help enhance the manuscript's quality so it will benefit the field and improve researchers' accessibility to these tools. However, I believe the manuscript requires substantial revisions to meet these goals. The authors seem to have followed a typical UBLs review format without fully articulating the rationale behind writing this manuscript. This approach led to errors in biological details and numerous missing points. Shifting the focus from UBLs biology to the chemical protein synthesis strategies would enable them to produce an innovative review that would more effectively serve the UBL research community. My comments below elaborate on these points, with a specific focus on enhancing clarity and direction.
Abstract
The abstract is well-written, but the focus should be shifted. The opening sentence could be rephrased to set a more relevant context: "Chemical protein synthesis has emerged as a powerful toolkit for producing modified proteins. Ubiquitin and UBLs, which consist of 70–150 amino acids, are well-suited to chemical synthesis..."
The authors can then continue with the rest of the abstract, concluding with the sentence: "In this review, we highlight..."
Introduction
1. To better align the focus with chemical synthesis, it may be beneficial to shift the introduction's emphasis from ubiquitination to synthetic methods. Consider beginning the introduction with the current third paragraph.
2. Change "Including 5 Sumo paralogs" to "Five SUMO paralogs."
3. The sentence "Given the similarity with Ub, these studies…" lacks clarity and relevance to the rest of the introduction. Please revise this sentence for better cohesion.
Figures and Tables:
1. Figure 1: This figure lacks clarity and rationale for the UBLs chosen. If the selection is based on those used in chemical synthesis, the reasoning should be specified. The structures shown are also not particularly informative. Consider highlighting key features relevant to synthesis, such as lysine residues, Gly-Gly motifs, E1 binding motifs, and hydrophobic patches, rather than simply showing amorphic sequences and fold structures.
2. Table 1: This table contains multiple inaccuracies, particularly regarding NEDD8 and its complex biological roles. For instance, mono-NEDD8 is conjugated to cullins via an isopeptide bond, a crucial point that is not even mentioned, and it is not involved in polyNEDD8 or hybrid chains with Ub/SUMO as inaccurately listed under "Chain Type." Additionally, the E1 ligase for NEDD8, NAE1, is composed of a dimer of proteins, similar to SUMO’s E1, and unlike the monomeric Ubiquitin E1, Uba1. Most of the biological processes mentioned, such as O₂ regulation and cell cycle regulation, relate to cullins rather than the chains themselves. To enhance the table’s clarity and relevance, it may be beneficial to replace the current sporadic biological data with structured information on chemical synthesis of UBLs. An alternative structure could include columns such as: (i) synthesis method, (ii) UBLs synthesized, (iii) benefits and applications, and (iv) references.
Brief Overview:
1. Figure 2 lacks a figure legend, which should be added for clarity.
2. The order of the topics (UBLs) in the brief overview is unclear. Are they ordered by available tools, similarity to Ub, or prevalence of study?
3. Rather than Figure 1 and Table 1, consider including schematic figures for each UBL similar to the one provided for SUMO (Figure 2), showing known functional roles, or aligning each UBL with ubiquitin to illustrate their similarities and distinctions, or mentioning the methods used for chemical synthesis.
4. References are missing for certain statements, e.g., the UFMylation of Rpn1, Rpl26, and Cyb5R3. Additionally, more recent references should be used to replace outdated ones.
5. The Atg12 UBL is only briefly mentioned (together with Atg8) in the text; however, it is notably absent in both Figure 1 and Table 1. This omission raises the question of why Atg12, which is a significant UBL, was not included in these key sections. Its absence may limit the comprehensiveness of the review and its utility for researchers interested in the full spectrum of UBLs.
6. The NEDD8 section contains inaccuracies similar to those noted above. Specifically, NEDD8's selection by E1 ligases involves residue 72 (differentiating it from ubiquitin), allowing NEDD8 to enter the ubiquitinome, unlike the vice versa. This critical detail should be included in place of the current information on "chains."
Synthesis of UBLs and Conjugates:
In my opinion, making the field more accessible to UBL researchers should be the authors' primary objective, particularly for biochemists who will apply these tools. This section, therefore, should be the focal point of the article. It would be helpful to replace Table 1 with a table or scheme that summarizes the various synthesis methods, improving accessibility for researchers in the UBL field.
Figure 3 has several issues:
1. The legend is minimal and lacks adequate description.
2. Figure 3A. The use of cullin as an example in the NCL (native chemical ligation) method illustration is inappropriate, as cullin was not previously discussed in this context. Additionally, the only enzyme that cleaves the NEDD8-cullin isopeptide bond is the COP9 signalosome (CSN). While NEDP1/DEN1/SENP8 and UCHL3 are involved in NEDD8 processing, they do not cleave isopeptide bonds; rather, they process peptide bonds. This distinction is important, as these enzymes cannot act on the isopeptide bonds required for cullins.

I am pleased with the significant improvements made to the manuscript, and I find the addition of Figure 1 particularly impactful. This addition transforms the review into a valuable resource for the community. With a few minor revisions outlined below, I believe the manuscript will be ready for acceptance:
1. Introduction: Please specify in the introduction that the review focuses exclusively on UBLs that have been studied through chemical or semi-synthetic methods, with the exception of FAT10.
2. Figure 2: The updated figure is much clearer and more informative. However, I noticed that the Gly-Gly motif is only depicted in the PDB structures for Ub, SUMO, and Urm1, but not for the others. What is the rationale behind this? If there is no strong justification, I suggest removing it for consistency.
3. Table 1: The table has improved significantly. While I have not verified every detail (which you should double-check), I noticed an inconsistency: on page 4, you refer to APPBP1, whereas in the table, the same protein is listed as Ula1. Please ensure consistent terminology throughout the manuscript.
4. NEDD8 Hydrolyzing Enzymes: There are still inaccuracies regarding the enzymes that hydrolyze NEDD8. While UCHL3 is indeed used for chemical synthesis purposes, it is not the endogenous enzyme responsible for cleaving NEDD8 from cullins. This distinction should be explicitly stated in the review. The only endogenous enzyme that hydrolyzes cullin-NEDD8 is CSN5/Jab1, the catalytic subunit of the COP9 signalosome. Additionally, UCHL3 and NEDP1 cannot naturally cleave isopeptide bonds.
5. USP21: The authors cannot rely solely on reviews to justify their claims. If such citations are used, they must be properly referenced. Regarding USP21, it is not a NEDD8 hydrolyzing enzyme. Even in the review cited (manuscript 48, https://pmc.ncbi.nlm.nih.gov/articles/PMC9279671/), the text clearly states:
“In addition, it is noteworthy that the hydrolysis activity of USP21 against neural precursor cell expressed developmentally downregulated 8 (NEDD8) conjugates appears to be inconsistent…”
Please correct this in the manuscript.

Thank you very much for the excellent review!! A very minor comment due to a misunderstanding: NAE1 is a dimer consisting of APPBP1 (also known as Ula1) and Uba3. The authors indicated NAE1 and Uba3 as a dimer, which should be corrected in the text and table.