**3. Concluding Remarks**

In the last years, a big effort has been made on the study of the role of PTMs mediated by Ubl in liver fibrosis (Figure 1). In spite of the improved knowledge obtained on this highly dynamic and pan-cellular process of liver fibrosis and its regulation by Ubl PTMs, it is clear that novel tools need to be developed. As an example, in the last years, both tandem ubiquitin-binding entities (TUBEs) and SUMO-binding entities (SUBEs), were developed [90,91]. Briefly, TUBEs and SUBEs are recombinant proteins that comprise tandem repeats of either ubiquitin-associated (UBA) domains or SUMO-interacting motifs (SIMs) thereby recognizing with high affinity ubiquitin and SUMO molecules on modified proteins, respectively. In the liver context, the use of SUBEs has been used very recently to demonstrate the relevance of Liver Kinase B1 (LKB1) SUMOylation during the progression to Hepatocellular Carcinoma (HCC) highlighting its potential for the assessment of ubiquitinated and SUMOylated proteins in liver fibrosis [92]. Other option is to combine the use of transgenic mice with tagged Ubl PTMs where fibrosis is experimentally induced followed by isolation of the different hepatic populations playing a role on the progression of liver fibrosis. For instance, transgenic mouse models, specially dedicated to the study of the ubiquitin-proteasome system have been developed. This is the case of the mouse strains transgenic for a green fluorescent protein (GFP) reporter carrying a constitutively active degradation signal [93]. Moreover, Mayor and colleagues have developed a transgenic mouse expressing biotinylated ubiquitin and demonstrated its use for the isolation of ubiquitinated proteins from the liver by taking advantage of the specificity and strength of the biotin-avidin interaction [94]. Even though similar approaches for other Ubl modifications, such as NEDD8 and SUMO, have been used in cultured cells [95], novel in vivo approaches should be investigated. Importantly, studies to analyze the intermediates on the multiple types of hepatic cells participating in liver fibrosis and not only on HSC, as occurs in the majority of the studies found in literature, should be performed. And the reason for that is that to cure fibrosis is important not only to promote the apoptosis and the reversal of the activation of HSCs, but also to take out the injury insult mainly acting on liver hepatocytes, that is in fact driving the liver fibrosis cascade. Finally, regarding potential therapeutic approaches targeting Ubl PTMs, compelling evidence indicates that whereas NEDDylation inhibition provides a global mechanism for reversing liver fibrosis, with respect to ubiquitination and SUMOylation, we believe that potential therapeutic approaches in liver fibrosis should be more specific aiming at specific ligases with targets playing an important role in the fibrosis pathogenic processes.

**Figure 1.** Schematic representation of the post-translational modifications (PTMs) described to date occurring in the main hepatic cell types involved during liver fibrosis, hepatocytes, Kupffer cells (KCs), and hepatic stellate cells (HSCs). Damage causing the transition from a normal healthy liver to a fibrotic liver are also referred, as well as the small-molecule inhibitors of PTMs that have resulted effective in the reversion of liver fibrosis.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/12/1575/s1, Table S1: UNIPROT entry of the different species where homology of the Ubls was compared.

**Author Contributions:** Writing—original draft preparation, All; writing—review and editing, All.

**Funding:** This research was funded by Gobierno Vasco-Departamento de Salud 2013111114 (to M.L.M.-C.), ELKARTEK 2016, Departamento de Industria del Gobierno Vasco (to M.L.M.-C.), Ministerio de Ciencia, Innovación y Universidades MICINN: SAF2017-87301-R and RTI2018-096759-1-100 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER (to M.L.M.-C. and T.C.D. respectively), BIOEF (Basque Foundation for Innovation and Health Research): EITB Maratoia BIO15/CA/014; Asociación Española contra el Cáncer (M.S.-M., T.C.D. and M.L.M.-C.), Daniel Alagille Award from EASL (to T.C.D.), Fundación Científica de la Asociación Española Contra el Cancer (AECC Scientific Foundation) Rare Tumor Calls 2017 (to M.L.M.-C.), La Caixa Foundation Program (to M.L.M.-C.). Gilead Sciences International Research Scholars Program in Liver Disease (to M.V.-R.). Ciberehd\_ISCIII\_MINECO is funded by the Instituto de Salud Carlos III. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644).

**Conflicts of Interest:** Martínez-Chantar advises for Mitotherapeutix LLC.
