Among the post-translational modifications, ubiquitination is one of the most extensively studied. Ubiquitination consists of the attachment of ubiquitin, a small protein of 76 amino acids in length, on specific lysine (K) residues of the targeted protein [1]. The attached ubiquitin can undergo further ubiquitination, and depending on the specific lysine residue on the ubiquitin sequence (K6, K11, K27, K29, K33, K48 and K63), different poly-ubiquitin chains can be formed and are named after the specific lysine residue [2].

The different ubiquitination types lead to different cell fates: among the most important, K48 ubiquitination brings the tagged protein to the proteasome for degradation, K63 ubiquitination is related to various cellular processes such as endocytic trafficking, inflammation and DNA repair, K11 is implicated in mitotic regulation and endoplasmic-reticulum-associated degradation (ERAD), K6 is involved in DNA repairs and DNA modifications such as methylation, and K27 is involved in mitochondrial DNA repair [3].

The process of ubiquitination is a multi-reaction cascade and requires three different classes of enzymes, each one catalyzing a different step of the reaction. The enzymes are classified into E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzymes) and E3 (ubiquitin ligases). The E1 activating enzyme activates ubiquitin at its C-terminus in an ATP-dependent manner: this enables ubiquitin to be transferred by the E2 conjugating enzymes to the E3 ligases that are capable of binding both the E2 and the specific substrate [4,5].

The third step, the most important of the cascade, is called “ligation” and is mediated by an E3 ubiquitin ligase that transfers ubiquitin from the E2 to the specific substrate. E3 ligases mediate the specificity of the ubiquitination reaction by recognizing specific structures and motifs on the targeted protein. Outside of the catalytic core, the rest of the structure of the E3 ligases is responsible for recognizing the specific substrate. Paired with the specific localization of those enzymes, which allows ubiquitination of the co-localized substrates, this creates a vast network of post-translational regulation, resulting in protein quality control, degradation of overproduced proteins and their activation and localization [5].

To allow ubiquitination and control of so many different substrates, in the human genome, there are more than a thousand different E3 enzymes, divided into four classes based on the structure of their catalytic domain: HECT (homologous to the E6AP carboxyl terminus), RING (Really Interesting New Gene), U-Box and RBR (RING-IBR-RING, a hybrid between HECT and RING). The four classes of E3 enzymes are responsible for different cellular functions [5].

Each E3 ligase can recognize and interact with several substrates. Depending on their substrates, as well as the different lysine residues that are ubiquitinated, E3 ligases can then be involved in a plethora of cellular and physiological processes. Moreover, alteration in the function of the E3 ligases can lead to several pathological scenarios [6]. In fact, the loss of activity of an E3 ligase would result in the accumulation of its targets, which may lead to pathology. On the other hand, excessive activity of one or more E3 ligases would mean complete degradation of their targets and ultimately their loss of function [6].

Alterations and mutations of a wide number of E3 ligases have been linked to different neurological diseases, such as encephalopathy, ataxia or Parkinson’s disease [7,8].

Furthermore, dysregulation of E3 ligases, both in the form of hyperactivation and downregulation, has been associated with cancer progression, as well as with evasion from cell death, and metastasis [9].

Another common trait in many tumors is the alteration of mitochondria: this leads not only to metabolic changes in the neoplastic cells but also to an alteration in the production of reactive oxygen species, as well as in their signaling, increased proliferation and changes in the interactions with the tumor microenvironment [10].

When merging these two concepts, it becomes clear that understanding the alterations in the mitochondrial E3 ubiquitin ligases may be relevant for finding new strategies for cancer therapy. There are only three E3 ligases specifically localized in the mitochondria: MARCH5, RNF185 and MUL1, also called collectively “mito E3 ligases”. These enzymes are all RING finger-type ligases and are capable of ubiquitinating also cytosolic targets.

The Membrane-Associated RING-CH Protein V protein (MARCH5) is located in the outer mitochondrial membrane (OMM). Also known as MITOL and RNF153, it was first identified in 2006 and associated with mitochondrial dynamics: in fact, MARCH5 targets for K-48 ubiquitination the mitochondrial proteins Mitofusin 2 (MFN2) and Dynamin Related Protein 1 (DRP1), thus contributing to the regulation of mitochondrial fission and fusion [11]. Other well-established targets of MARCH5 include the DRP1 receptor MiD49 [11], the mitochondrial quality control protein FUN14-Domain-Containing Protein 1 (FUNDC1) [12], the retinoic acid-inducible gene-I (RIG-I) receptor [13], the ER-associated endoribonuclease IRE1α [14] and the mitochondrial antiviral signaling (MAVS) protein [15]. MARCH5 has thus been canonically involved in mitochondrial protein quality control, mitophagy, ER stress and unfolded protein response (UPR), innate immune response and prevention of cell senescence. Via ubiquitination of MFN2, MARCH5 mediates the physical interaction between mitochondria and the ER, allowing the exchange of lipids and calcium ions at the membrane contact site. Depletion of MARCH5 has been related to the disruption of the membrane contact site and the aggravation of derangements caused by progressive diseases [16]. Due to its role in mitochondrial quality control, MARCH5 has been associated with neurodegenerative diseases, where damaged proteins may translocate to the mitochondria. Consequently, this connects MARCH5 also to senescence and aging. This suggests the use of MARCH5 activators, such as berberine, as anti-aging drugs [17].

MARCH5 is regulated by auto-ubiquitination [16], targeting itself for proteasomal degradation to maintain correct mitochondrial homeostasis. In addition, it can be negatively regulated by the mir-30a microRNA [17].

In the context of cancer, MARCH5 tends to act as an oncogene, with different mechanisms that depend on the cellular context and are characterized by the specific targets of the ligase.

In fact, MARCH5 can be found upregulated in breast cancer (BC): high levels of the ligase have been found in several BC cell lines, as well as in cultures derived from BC patients [18], mainly due to a downregulation of mir-30a. MARCH5 expression in the context of BC is related to poor prognosis [19]. Increased MARCH5 expression results in lower levels of its targets such as DRP1 and MFN2 and altered autophagy, ultimately leading to dysfunctional mitochondria, increased reactive oxygen species (ROS) production and increased aggressiveness and metastatic potential. This was proven by knocking down MARCH5 in a model of BC cells that led to reduced cell growth and metastatic ability. This was also shown in vivo in an MDB-MB xenograft on MARCH5 KO mice where it caused slower tumor growth and a smaller number of metastases [19].

3. RNF185