eUb levels are increased in the serum and plasma of patients with inflammatory diseases [82,83], suggesting a potential role of eUb during the inflammatory processes. Specifically, intravenous administration of eUb reduces TNF-α plasma levels, and the mortality induced by endotoxin in pigs [84]. In a lung polytrauma pig model, intravenous administration of Ub improves oxygenation and decreases circulating lactate levels as well as inflammatory cytokines (IL-8, IL-10, TNF-α, and CXCL12) in the pulmonary tissue [85]. Similarly, eUb enhances the Th2 cytokine response and improves pulmonary function in post-ischemic lungs when administered before reperfusion in rats [86]. On the contrary, in a model of ischemia-reperfusion heart injury, administration of eUb with an osmotic pump does not provoke any changes in circulating levels of IL-4, IL-10, or IL-13 [87]. Yet, it was able to protect against cardiac injury, which suggested that eUb’s anti-inflammatory properties may be restricted to sites of damaged tissue instead of having a systemic effect.

In human peripheral blood mononuclear cells (PBMCs), eUb prevents the LPS-induced TNF-α and IFN-γ production and promotes an increase in the anti-inflammatory cytokines IL-8 and IL-4 [16,88]. In contrast, in RAW264.7 cells, a murine macrophage cell line, eUb synergizes with LPS to induce TNF-α production [89], indicating that the anti-inflammatory properties of eUb may be cell-type dependent and/or species specific. On the other hand, eUb and a small Ub-derived peptide decreased the number of antigen-forming colonies of murine-spleen cells stimulated with sheep red blood cells (SRBC) [90,91]. In one of these works [91], the authors reported that the Ub50-59 peptide, with a rigid secondary structure, has a stronger immunosuppressive activity than the full-length protein, suggesting that Ub and its short-derived peptide may share a common receptor. Moreover, eUb can decrease the one-way mixed leukocyte in vitro reaction and improve skin graft survival, without any deleterious effect on body weight in mice [92]. Furthermore, an anti-inflammatory Th2 cytokine profile and an M2 polarization are provoked in human PBMCs, and macrophages incubated with eUb, respectively [88,93]. In macrophages, eUb has been shown to promote the anti-inflammatory M2 polarization, an effect that could be blocked by the addition of 100 µM of the CXCR4 inhibitor, AMD3100 [94]. Although this observation indicated that eUb-induced M2 polarization is via CXCR4, the high AMD3100 concentration used in that study makes it difficult to draw a solid conclusion since AMD3100 at doses higher than 10 µM has been shown to activate CXCR7 [95].

Although experimental data pointed out that eUb is a promising candidate to treat inflammatory diseases, the deleterious effects of this protein in cancer must be considered. eUb reduces apoptosis and promotes lung metastasis, as well as tumor progression of B16 melanoma in mice, which is related to increased matrix metalloproteinase-9 (MMP9) and vascular endothelial growth factor (VEGF) production [93,96]. Interestingly, an increase in anti-inflammatory cytokines was reported in this study. eUb does not affect the migration or apoptosis of the HepG2 cells [94], notwithstanding, HepG2 hepatoma cells reduced apoptosis and increased migration when co-incubated with eUb-pretreated macrophages [93,94]. Considering that eUb induces M2 macrophage polarization, it was suggested that eUb promoted tumor progression by inducing the tumor-associated anti-inflammatory process [94]. Therefore, in case of a possible use of Ub as an anti-inflammatory biotherapeutic, it should be important to consider the patient’s general health status before administration.

Effects of eUb have been also reported on cell proliferation. Intraperitoneal administration of eUb in mice subjected to chemically-induced inhibition of hematopoiesis resulted in a quadruple increase in bone marrow cell count within 24 h. Notably, this effect contrasted with the reduction of peripheral blood cell count, suggesting that eUb could regulate stem cell activity, normalizing the release of functional cells into the bloodstream [97]. Moreover, in a recent investigation, it was described that eUb can modulate the erythroblast/megakaryocyte ratio and reduce cell size during bone marrow’s proliferative activity, accentuating Ub’s role in modulating hematopoiesis [97]. Studies involving the Ba/F3 cell line (a murine pro B cell line) demonstrated that Ub overexpression results in its secretion in these cells. The same study also established that the addition of eUb inhibits proliferation across multiple blood cell lines (HL-60, KT-3, U937, and Daudi) [48]. Collectively, these findings highlighted the potential of eUb as a key regulator in hematological cell proliferative processes.

In the context of mouse tumor lung cells, eUb treatment failed to exert any significant impact on cellular proliferation. Neither ERK pathway activation nor STAT3 effects were observed, but there was a notable activation of AKT3 [98]. These observations suggest that Ub plays distinct roles across hematopoietic cells and tumor epithelial cells in proliferation.

The neuronal cells are another potential target of Ub. In 1986 and 1987, Meyer et al. [99,100] showed that treatment with a specific anti-Ub antibody decreases sodium-dependent neurotransmitter transport in rat synaptosomes, an effect that is not related to cell polarization or antibody internalization. Although the authors suggested that there may exist a ubiquitination signal in the outer face of the cellular membrane necessary for neurotransmitter uptake, the possibility that free eUb may directly participate in such a mechanism should not be excluded.

The role of eUb in brain repair has been also described. In swine, administration of eUb 30 min after brain injury (BI) reduces cerebral perfusion pressure, due to decreased intracranial but not arterial pressure, and reduces the intravenous fluid administration requirement during resuscitation after BI [101]. Interestingly, the authors [101], also found that eUb concentration in CSF increased after the treatment administration, suggesting that Ub can cross the BBB. Furthermore, it has been shown that eUb reduces contusion volume in rats [102] and promotes microglia/macrophage activation after BI [103]. Altogether, these observations suggested that after BI, eUb promotes the activation of phagocytic cells in the CNS to accelerate the brain healing process.

An interesting use for monomeric eUb has been proposed by Abarca-Castro et al. [104]. They suggested that monomeric Ub could be used as a therapeutic to limit the harmful effects on the neurodevelopment of offspring due to the inflammatory response caused in pregnant mothers by pre-eclampsia. They argue that pre-eclampsia, characterized by hypertension and organ damage during pregnancy, is linked to the offspring’s cognitive deficits, behavioral abnormalities, and neurodevelopmental issues [105], which significantly affect their development and adult life. The evidence indicated that the cholinergic anti-inflammatory pathway (CAP) could significantly impact the fetus’ and the newborn’s development by functioning as a neuroimmunology network facilitating internal monitoring. This pathway connects the CNS with the vagus nerve, regulating inflammation in the body [106].

In addition, Ub can disassemble amyloid-β42 aggregates in vitro and prevent uptake and cytotoxicity of the aggregates in SH-SY5Y cells when Ub is added to the cell culture [80]. Therefore, eUb may be useful to treat Alzheimer’s disease and pre-eclampsia, considering that in both pathologies amyloid-β42 aggregates are involved [107,108].

Ischemic heart disease (IHD) is one of the primary causes of death worldwide. IHD is characterized by acute myocardial infarction due to acute lethal ischemia-reperfusion (I/R) injury, and cardiomyocyte death, which can result in heart failure [109,110]. In coronary heart disease (CHD), augmented levels of eUb showed a positive correlation with worsening CHD, suggesting that eUb could play a role, even as a biomarker, for CHD progression [111].

Due to the limited proliferative capacity of myocytes [112], it becomes relevant to prevent its apoptosis during heart damage. Previous studies have established that β-AR stimulation can induce apoptosis in cardiac myocytes through the activation of the glycogen synthase kinase-3 (GSK-3β) and mitochondrial pathways [113]. During in vitro and in vivo experiments, pre-treatment with eUb reduces β-AR-stimulated myocyte apoptosis by inhibiting activation of GSK-3β and the c-Jun N-terminal kinase (JNK), as well as suppressing cytosolic cytochrome c release, effects that are mediated by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway [52,114]. Additionally, eUb promotes the production of matrix metalloproteinase-2 and -9 (MMP-2 and -9) and the tissue inhibitors of MMPs (TIMPs) in cardiac cells exposed to ISO [87,114]. MMPs’ proteolytic activity participates in extracellular matrix remodeling, facilitating cell migration to promote tissue repair [115]. Although MMP2 overexpression is related to increased heart failure [116], the protective roles of this enzyme against cardiac hypertrophy [117] and against Angiotensin II-induced hypertension [118] have also been reported. Thus, induced MMP2 overexpression may be part of the protective mechanisms of eUb against heart failure.

In a mouse model of myocardial I/R injury, eUb treatment reduces apoptosis, oxidative stress, and mitochondrial fission, but increases mitochondrial biogenesis in a CXCR4-dependent manner [115]. The authors also reported that, in isolated hearts, eUb treatment reduces infarct size and restores heart function after I/R injury by preventing myocyte apoptosis [115]. Moreover, in another study, it was found that eUb reduces the area at infarct risk and improves heart function by increasing the percentage of fractional shortening and the ejection fraction of the heart after I/R. Additionally, eUb reduces the inflammatory response in the heart by reducing neutrophils and macrophage infiltration [87].

Cardiac angiogenesis, the process by which new blood vessels are generated, is also important for heart tissue repair after an IHD [119], and VEGF-A is a well-known regulator of this process [120]. It is reported that eUb promotes the expression of VEGF-A in cardiac microvascular endothelial (CMEC) cells via CXCR4 activation, proposing another cardiac-protective mechanism of eUb after IHD [121]. Furthermore, eUb promotes CMEC migration, a necessary process for cell repopulation in newly created blood vessels [121].

After heart tissue damage, cardiac fibroblasts (CFs) proliferate and produce extracellular matrix (EM) components to promote wound healing [122]. However, uncontrolled fibroblast proliferation and excessive EM production may result in a stiff scar which limits muscle contraction. CFs can differentiate into α-smooth muscle actin (α-SMA) or positive myofibroblasts (MFs) [123]; MFs are more contractile than CFs [122,124]. Thus, scar produced by this type of cells affects to a lesser extent heart contraction [123]. In 2018, it was shown that eUb, through its interaction with CXCR4, stimulates the activation of the ERK1/2 pathway in cardiac fibroblasts, which increases the production of VEGF-A and decreases expression of β3 integrin, influencing fibroblast-mediated activities such as angiogenesis [125]. Cell migration into wounds and fibroblast growth promoted by fetal bovine serum are likewise reduced by eUb therapy [125]. Furthermore, eUb increases the production of MFs [125], which in turn results in increased contraction of fibroblast-populated collagen gel pads [125]. In line with the previous study, it was discovered that eUb has no direct pro-proliferative effect on cardiac fibroblasts [126]. Instead, a truncated form of Ub (1-74), but not the full-length protein, inhibits the pro-proliferative effects of CXCL12 [126], a CXCR4 agonist known to enhance cardiac fibroblast proliferation [127]. Interestingly, Ub (1-74) is known to bind, but not activate, CXCR4 [21]. Ub (1-74) is the product of the processing of Ub (1-76) by the IDE [128]. Importantly, IDE inhibition blocks the conversion of Ub (1-76) to Ub (1-74) and restores SDF-1′s pro-proliferative effects in cardiac fibroblasts [126].

The evidence reviewed above indicates that eUb plays a crucial role in cardiac tissue repair and remodeling after ischemic heart disease. It stimulates the formation of new blood vessels, enhances cell migration and tube network formation, and regulates fibroblast behavior in heart tissue. Additionally, eUb has heart-protective effects and improves cardiac function following ischemia/reperfusion injury. Further research is needed to understand the underlying molecular pathways of eUb in the aforementioned processes.

Indiscriminate use of antibiotics has promoted drug-resistant microorganisms, which nowadays have become a severe health problem worldwide [129]. Thus, finding new antimicrobial agents is an urgent need. Antimicrobial peptides (AMPs) are important components of the host defense against pathogens [130]. In 2003, Kieffer et al. described nicotine-stimulated chromaffin-secreted granules as a potential source of eUb in bovines [131]. Interestingly, the authors also found that Ub inhibits the growth of M. luteus, B. megaterium, and N. crassa with a minimal inhibitory concentration (MIC) of 60 µM [131]. Furthermore, synthetic peptides derived from the positively charged hydrophobic Ub c-term (Ub65-76) show higher growth-inhibitory capabilities than full Ub against bacteria, yeast, and fungi. In addition, Ub65-76 induced membrane destabilization in A. fumigatus and inhibits calcineurin phosphatase activity, a crucial enzyme in the regulation of hyphal growth and morphology in some filamentous fungi [131]. Like these observations, Alonso et al. [132] found that Ub incubated with lysosomal cathepsins but not full Ub or cathepsins alone induces a bactericidal effect against Mycobacterium, reinforcing the fact that Ub-derived peptides have higher antimicrobial activity than full-length Ub. In 2007, Jin-Young Kim et al. [133] obtained a small 4 kDa peptide from human amniotic fluid, identified as part of the N-term of Ub1-18 and named AFP-1. This peptide has antimicrobial activity at the µM range against a broad spectrum of bacteria, fungi, and yeast [133]. Interestingly, another N-term Ub peptide (Ub1-34) synergizes with the Ub65-76 synthetic peptide to suppress the growth of fungi and yeast [131]. Moreover, a truncated Ub form lacking the two c-term glycine residues (named by the authors cgUb) from the oyster Crassotrea gigas showed bacteriostatic activity against Gram-negative and -positive bacteria at the low µM range, but no hemolytic activity when exposed to human red blood cells [134]. On the other hand, antimicrobial eUb-derived peptides are also produced by the V8 endoprotease of Staphylococcus aureus or by cathepsins secreted by activated leukocytes in the extracellular space [135]. Considering all the previous findings, it is clear that the full-length Ub has poor antimicrobial activity. However, Ub peptides produced in lysosomes and exosomes of macrophages and chromaffin cells [131,136] may be an important source of these AMPs.

It has been suggested that Ub is secreted to the extracellular space by the yeast Pichia pastoris as a response to cellular stress [137]. Although the role of eUb in yeast remains to be elucidated it has been shown that eUb reduces cell growth by promoting G2 arrest in Schizoacharomyces pombe, which can be abrogated by the addition of the proteasome inhibitor Lactacystin [138]. In this case, the authors hypothesized that eUb is internalized to cells, which results in the unprogrammed degradation of cell cycle proteins. A similar effect has been observed in the KT-33 human cell line, where eUb promoted STAT3 ubiquitination and degradation; these effects are also diminished by the addition of proteasome inhibitors [48]. These observations reinforce the reprogramming of the ubiquitin/proteasome pathway induced by eUb as another mechanism of action.

In the marine invertebrate Halocynthia roretzi, extracellular ubiquitination of the 70-kDa main VC component (HrVC70) by a sperm extracellular ubiquitinating enzyme is relevant for egg fertilization (reviewed in [139]). This phenomenon can be promoted by the addition of Ub and ATP to the media and can be blocked by adding an anti-Ub antibody or proteasome inhibitors [140]. These observations indicate that degradation of HrVC70 by the extracellular ubiquitination/proteasome system is important during egg fertilization in this invertebrate.

In boars, Petelak et al. (2019) [141] found an indirect correlation between the degree of ubiquitinated membrane proteins in the extracellular space of sperm and its capability to induce blastocyst formation in fertilized oocytes, effects that were improved by treatment of sperm with a Ub-blocking antibody [141]. On the other hand, embryo implantation can be reduced by administrating Ub-neutralizing antibodies in mice [142]. Interestingly, Ub has been found as a biomarker in the secretome during blastocyst formation and development in mice and in humans [143]. Together, these observations suggest a direct effect of the amount of eUb on sperm quality, related to its capability to promote blastocyst formation, as well as in blastocyst development in different mammalian species.

One of the first steps in angiosperm pollination requires the adhesion of pollen to the stigma. Then, the growth of pollen tubes through the pistil allows sperm cells to be discharged to the ovule. In 2006, Kim et al. [144], found that Ub was co-purified with the stigma/stylar cys-rich adhesin (SCA), from Lilium longiflorum pistils. The authors also reported Ub as an important protein to induce pollen adhesion to the stigma [144] and described that exogenously added Ub promotes the SCA-induced adhesion of pollen tubes. This suggests that eUb may have an important role during the early stages of pollination in Lilium longiflorum.

Biotechnological drug products are biological medicinal molecules obtained from live sources using genetic engineering. This group includes cytokines, growth factors, hormones, interferons, and regulatory peptides and proteins [145]. In addition to therapeutic proteins, specifically monoclonal antibodies, peptides are one of the biotherapeutics that have gained relevance in recent years for the development of biopharmaceutical products.

The difference between a peptide and a protein can be established based on their size, with proteins being those structures with 50 or more amino acid residues [146]. However, there are cases in which this division is not clear, such as insulin (5.7 kDa) or eUb (8.6 kDa), which can be considered as a long peptide or a small protein.

Recently, the number of naturally occurring peptides that are known to regulate physiological functions and that could serve as models for the development of biotherapeutics has increased [146]. Besides peptide-derived hormones (e.g., insulin, vasopressin, and gonadotropin-releasing hormone), there are neuropeptides (e.g., enkephalin, substance P, oxytocin) and antimicrobial peptides (e.g., human neutrophil peptides and human beta-defensins) [147,148]. Interestingly, several neuropeptides and antimicrobial peptides are produced by immune cells under inflammatory conditions or after antigenic stimulation and bind to GPCRs, which are expressed in different immune cells, including T cells, macrophages, monocytes, DCs, and neutrophils [149].

Peptides have the characteristics to overcome the main disadvantages of the two most relevant groups of current drugs, being more specific (and less toxic) than drugs with a small structure and having better bioavailability than therapeutic proteins (>100 kDa) [150]. Nevertheless, peptide-derived drugs must overcome some main challenges: (i) they should be as close as possible to endogenous human proteins to reduce the risk of immunogenicity; (ii) they should be parenterally administered to avoid extended proteolysis and acid degradation in the stomach, and (iii) their structural properties need to be improved because they have low half-lives and high conformational freedom [146,150].

The physicochemical and biological characteristics of eUb make it an attractive molecule for biotherapeutic development. First, Ub is a highly conserved protein in eukaryote cells [151], which reduces the probability of generating adverse events and/or therapeutic failure due to the induction of anti-drug antibodies (ADAs). Second, Ub is not glycosylated, thus avoiding low microheterogeneity owing to the reduction of glycosylated isoforms in the final product, which allows better quality control. Third, Ub is highly stable, with a T_m of 95 °C at pH 7.0 [152], which could be a desirable property for storing Ub at room temperature as a lyophilized powder and/or possibly in solution.

The state of the art points out that Ub is an endogenous protein involved in regulating immune, nervous, and cardiovascular systems, which are related to illnesses with profound relevance; thus, it could represent an underestimated protein as a potential therapeutic agent. Therefore, we searched for ubiquitin-based biotherapeutics on DrugBank [153], Clinical Trials [154], and Cortellis Drug Discovery Intelligence [155]. No marketed biopharmaceutical products based on Ub were found in DrugBank at the moment of writing this review. On the other hand, 97 clinical studies using the search entry “Ubiquitin” were found on ClinicalTrials.gov. Twenty-three trials are focused on molecules to which MoA is indirectly related to Ub. Out of these 23 trials, 9 analyze the Ub levels (mRNA or protein concentration) as a biomarker, and 65 studies are related to proteins of the proteasome system except Ub, such as Ub carboxy-hydrolase L1 (UCH-L1), Ub-protein ligase E3A (UBE3A), and levels of ubiquitinated proteins in general, among others. Additionally, using the same searching criteria, 1293 entries were found in Clarivate Drug Discovery Intelligence with similar results: all the information found is related to biomarker development or to therapeutic usages of the proteins related to the ubiquitination system.

Interestingly, Ub is not currently being developed as a biotherapeutic despite the preclinical evidence shown in this review. One possibility is that Ub could have pleiotropic effects that make it difficult to study its mechanism of action and its biological effects. On the other hand, cases of the indirect use of Ub as a biotherapeutic are described below.

DLEs are complex mixtures of low-molecular-weight peptides (<10 kDa) obtained from the lysis and dialysis of buffy coats from healthy donors [156]. DLEs have been used as co-adjuvants in the treatment of viral, parasitic, fungal, and mycobacterial infections, as well as primary immunodeficiencies, and allergies [157,158]. Transferon Oral^®, a human DLE (hDLE) manufactured under good manufacturing compliance, regulates the production of the inflammatory cytokines TNF-α, IL-6 e, and IFN-γ and increases the percent of survival when orally administrated in a murine model of cutaneous herpes simplex virus-1 (HSV-1) infection [159]. In addition, this hDLE increases the percent of survival of puppies infected with canine parvovirus when subcutaneously administrated by decreasing circulating levels of cortisol and catecholamines and increasing plasma levels of norepinephrine and serotonin [160,161].

Since their discovery in 1950–1970, the mechanism of action of DLEs has been partially understood owing to their complex compositions. Vallejo-Castillo et al. [162] performed a peptidome analysis by mass spectrometry and found that Ub1-76 and Ub1-74 are two of the main components of Transferon Oral^®. Additionally, Polonini et al. [163] identified the ubiquitin-40S ribosomal protein (also known as the 40S ribosomal protein S27a), and the ubiquitin-ribosomal protein L40 among the main components of Imuno TF^®, a dialyzable extract obtained from pig spleen. Furthermore, Vallejo-Castillo et al. [162] performed a proof-of-concept murine HSV-1 infection assay and observed an increment of the percent of survival of HSV-1-infected mice orally administrated with eUb (0.750 µg/48 h during 10 post-infection days) with respect to the infected/not treated control. They hypothesize that orally administered Ub might stimulate the intragastric vagus nerve endings, favoring the activation of the anti-inflammatory vagal arch [162].

Although vast research is needed to clarify the in vitro and in vivo MoAs of Ub, the above information points out that Ub is an interesting biomolecule for the development of biotherapeutics.

Ub has been suggested for use as a scaffold protein with the capacity to bind to different targets (Affilin^®). Several binding proteins have been proposed as an alternative to the use of antibodies; Affilin^TM was initially described using γ-B-crystallin as a scaffold [164,165], and more recently, a ubiquitin dimer has been proposed as an ideal scaffold for binding multiple targets by introducing mutations in surface-exposed amino acid residues and creating a phage display library [45,166,167]. The use of ubiquitin as a scaffold lies in its physicochemical and biochemical properties as mentioned above. Because Ub does not have post-translational modifications, it may be easily obtained in a recombinant form in bacteria [167]; its poor half-life in circulation, however, might be one of its drawbacks—this could need linking to other proteins, like Fc or BSA [167]. To overcome these restrictions, Affilin^® has also been employed in combination with antibodies and fab in various forms as a bispecific molecule and pair to adenoviral vectors [168,169]. Multiple targets focused on cancer therapy have been evaluated; however, their in vivo evaluation is still necessary.