**2. Post-Translational Modifications (PTMs) by Ubiquitin-Like (Ubl) Proteins**

Post-translational modifications (PTMs) of proteins play a relevant role in the functional diversity of the proteome. In most eukaryotes, PTMs refer to the covalent and reversible addition of small chemical entities into target proteins following protein biosynthesis in order to exert a dynamic control over protein function in diverse cell biological contexts. The recent advances in the fields of systems biology and proteomics, have pushed forward the interest in deciphering protein modifications and their impact on the cellular microenvironment and disease pathophysiology. The most common PTMs include phosphorylation, acetylation, glycosylation, ubiquitination, acetylation, and hydroxylation, among others.

Ubiquitination is implicated in the pathogenesis of certain human diseases, including liver fibrosis. Ubiquitin has shown to be a marker of non-alcoholic liver fibrosis and it is frequently detected at the border or within the fibrous matrix [33,34]. Under these circumstances, overall changes in the ubiquitinated proteome may reflect either modifications in the ubiquitination cascade or in the proteasomal activity. For example, Cai et al. detected, in a rat model of liver fibrosis, a reduced SMAD specific E3 ubiquitin protein ligase 2 (Smurf-2) mRNA expression, which is a HECT domain E3 Ub ligase that ubiquitinates nuclear Smads and targets them for proteasomal degradation, resulting in an increased Smad2 expression [35]. Gp78, an endoplasmic reticulum (ER)-associated E3 Ub ligase, is also a key player in the ER-associated degradation (ERAD) and responsible for ubiquitination of lipid metabolism mediators, among others. Loss of Gp78 in aged mice caused NASH with fibrosis as a result of spontaneous and random ER stress [36]. On the other hand, Wilson and colleagues have shown that the Ub C-terminal hydrolase L1 (UCHL1) is an absent DUB in quiescent HSCs but its

expression is increased and positively correlates with HSC transdifferentiation, in pre-clinical mouse models and in human livers from NASH and ALD patients. Pharmacological inhibition of UCHL1 in CCl4 and bile-duct ligated (BDL) mice or ablation of UCHL1 in vitro in cultured HSC cells reduces liver fibrogenesis [37]. Likewise, an increase in mRNA expression and immunoreactivity of synoviolin which is an E3 Ub ligase has been observed in myofibroblasts. Fibrotic human livers also showed co-localization of synoviolin and the main fibrotic marker, α-SMA [38]. This compelling evidence implicating ubiquitination in liver fibrosis led several authors to evaluate the impact of ubiquitin-like proteins (Ubls)-mediated PTMs in liver fibrosis, the topic of this Review.

Ubiquitin-like proteins (Ubls) are a family of small proteins involved in PTMs, whose name is derived from ubiquitin, the first discovered member of the family. Besides ubiquitin, the human genome encodes at least eight families of Ubls, that are considered type I Ubls: (SUMO) small ubiquitin-related modifier, NEDD8 (neural precursor cell expressed developmentally downregulated protein 8), ATG8 (autophagy-related protein 8), ATG12 (autophagy-related protein 8), URM1 (ubiquitin-related modifier 1), UFM1 (ubiquitin-fold modifier 1), FAT10 (human leukocyte antigen-F adjacent transcript 10 or ubiquitin D), and ISG15 (interferon-stimulated gene 15) [39]. Even though, sparse studies have shown alteration of the levels of some Ubls in liver fibrosis, namely ATG12 related to autophagy [40], Fat10 and UFM1 [41], and ISG15 specifically in hepatitis C [42], in this Review, we will specially focus on the relevance of NEDD8 and SUMO proteins in liver fibrosis, whose therapeutic role has been addressed in liver fibrosis. The main characteristics of these proteins in comparison to ubiquitin can be found in Table 1.


