Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Mitochondrial Ubiquitin Ligase MITOL/MARCH5

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101579

Synonyms

Historical Background

E3 ubiquitin ligases are a large family of proteins that recognize target substrates and mediate the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme to the substrate. These modifications can have diverse effects on the substrate, ranging from proteasome-dependent degradation to modulation of protein function, including activation and/or localization. The activity of most E3 ubiquitin ligases is specified by a RING domain, which is a protein structural domain of the zinc finger type containing a C3HC4 amino acid motif. The mitochondrial ubiquitin ligase MITOL/MARCH5 belongs to the membrane-associated MARCH family of E3 ubiquitin ligases, which were originally discovered as structural homologs to Kaposi’s sarcoma-associated herpesvirus K3 and K5 ubiquitin E3 ligases. Hereafter, the term “MITOL” is used consistently when referring to this protein. MITOL is an integral mitochondrial outer-membrane protein, and its N-terminal RING domain is exposed to the cytoplasm (Yonashiro et al. 2006) (PMID: 16874301) (Fig. 1). It also has auto-ubiquitination activity. Northern blot analysis has shown that MITOL is expressed in all mouse tissues. MITOL is the first E3 ubiquitin ligase found to be localized specifically in the mitochondria, where it plays various roles.
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 1

Structure of MITOL. MITOL is an integral mitochondrial outer-membrane protein, the N-terminal RING domain of which is exposed to the cytoplasm

Regulation of Mitochondrial Morphology by MITOL

Upon the knockdown of MITOL in HeLa cells using RNA interference, the mitochondria rapidly fragmented (Nakamura et al. 2006; Yonashiro et al. 2006) (PMID: 16874301, PMID: 16936636), suggesting that MITOL regulates mitochondrial morphology. Drp1 and Fis1, which are the main mitochondrial fission factors, were initially identified as substrates of MITOL (Nakamura et al. 2006; Yonashiro et al. 2006) (PMID: 16874301, PMID: 16936636). Later, Mid49, which mediates Drp1 recruitment and mitochondrial fission, was also found to be a substrate for it (Xu et al. 2016) (PMID: 26564796). MITOL promotes the degradation of these substrates through the ubiquitin–proteasomal pathway. Therefore, MITOL dysfunction induced mitochondrial fragmentation via the accumulation of these mitochondrial fission proteins. Under normal physiological conditions, MITOL inhibits excess mitochondrial fission by ubiquitinating Drp1/Fis1/MID49 (Fig. 2). In MITOL-knockdown cells, a significant increase in reactive oxygen species (ROS) from mitochondria was observed, leading to cellular senescence through Drp1 and mitofusin 1 (Park et al. 2010) (PMID: 20103533). Since overactivation of mitochondrial fission proteins such as Drp1 has been shown to induce mitochondrial damage and increase mitochondrial ROS production, the accumulation of Drp1/Fis1/MID49 in mitochondria may contribute to the generation of ROS in MITOL-knockdown cells. In cardiomyocytes, MITOL plays a protective role against apoptosis through Drp1 inhibition (Wang et al. 2016) (PMID: 27444773). In conclusion, MITOL regulates mitochondrial dynamics through the control of mitochondrial fission proteins and protects mitochondria against the toxicity induced by excess mitochondrial fission.
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 2

Regulation of mitochondrial morphology by MITOL. MITOL protects mitochondria against the toxicity induced by excess mitochondrial fission

Degradation of Misfolded Proteins by MITOL

Misfolded and aggregated proteins are prone to accumulate in mitochondria and induce mitochondrial dysfunction. This pathological condition is closely associated with the development of degenerative neurological disorders. For instance, mutations of superoxide dismutase 1 (SOD1) are associated with the onset of amyotrophic lateral sclerosis. The polyglutamine diseases are a group of neurodegenerative disorders caused by expanded glutamine chains (polyQ). Similarly, the accumulation of polyQ in mitochondria is considered a possible cause of neurotoxicity. MITOL interacts with and ubiquitinates mutant SOD1 and expanded polyQ, and attenuates their cytotoxicity by degradation through the ubiquitin–proteasome pathway (Yonashiro et al. 2009; Sugiura et al. 2011) (PMID: 19741096, PMID: 20851218). MITOL knockdown resulted in the accumulation of these denatured proteins in mitochondria and neuronal cell death, suggesting that MITOL plays a critical role in the degradation of misfolded proteins in mitochondria and promotes neuronal cell survival. MITOL can directly recognize its misfolded substrates via an intrinsically disordered C-terminal domain. When this disordered domain is deleted, MITOL fails to associate with and ubiquitinate mutant SOD1 and does not induce the degradation of mutant SOD1 and polyQ. Thus, MITOL is involved in mitochondrial quality control (Fig. 3).
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 3

Mitochondrial quality control of MITOL. MITOL recognizes and ubiquitinates misfolded proteins such as mutant SOD1 or expanded polyQ through the intrinsic disordered domain at its C-terminus and promotes their degradation

MITOL Protects Mitochondria Against Nitrosative Stress

In a yeast two-hybrid screen, microtubule-associated protein 1B-light chain 1 (MAP1B-LC1) was identified as a substrate of MITOL. MAP1B is a protein complex that consists of a heavy chain and a light chain. It plays an important role not only in the stability of the cytoskeleton but also in the inhibition of retrograde transport of mitochondria. In addition, MAP1B and its homolog are reported to induce mitochondrial dysfunction and cell death via a specific domain named MAGD (mitochondrial aggregation and genome destruction). Furthermore, MAP1B-LC1 is reported to be activated by S-nitrosylation on cysteine residue 257 via its conformational change (Stroissnigg et al. 2007) (PMID: 17704770). Thus, S-nitrosylated MAP1B-LC1 is a negative regulator of mitochondrial dynamics and neuronal cell survival. MITOL specifically ubiquitinates S-nitrosylated MAP1B-LC1 and promotes its proteasomal degradation, thereby inhibiting mitochondrial damage and neuronal cell death (Yonashiro et al. 2012) (PMID: 22308378). The balance between MAP1B-LC1 activation by S-nitrosylation and downregulation by MITOL is critical for neuronal cell survival. MITOL plays a protective role against nitrosative stress-mediated disruption of mitochondrial dynamics (Fig. 4). However, excessive nitric oxide (NO) production inactivates MITOL by direct S-nitrosylation. MITOL inactivation by excess NO may be associated with neurological diseases caused by nitrosative stress-mediated mitochondrial dysfunction. From another perspective, MITOL promotes MAP1B-LC1 degradation to prevent the excessive stabilization of microtubules.
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 4

Role of MITOL in nitrosative signaling. MITOL protects neuronal cells against the cytotoxicity of S-nitrosylated MAP1B-LC1. However, when excess NO inactivates MITOL by direct S-nitrosylation, it causes neuronal cell death

Role of MITOL in Interaction of Mitochondria and Endoplasmic Reticulum

Mitochondria play an important role in exchanging Ca2+ and metabolizing lipids by positioning at sites close to the endoplasmic reticulum (ER), which is called mitochondria-associated ER membrane (MAM). Mitofusin 2 (Mfn2) is a mitochondrial fusion factor that is enriched at the MAM. Mfn2 tethers the ER to mitochondria by forming homotypic and heterotypic complexes with Mfn2 and/or Mfn1 on the mitochondrial surface (Merkwirth and Langer 2008) (PMID: 19109886). MITOL interacts with the Mfn2 HR1 domain and ubiquitinates mitochondrial Mfn2, which does not then undergo proteasomal degradation. MITOL mediates the addition of a lysine-63-linked polyubiquitin chain to lysine 192 in Mfn2, and promotes Mfn2 binding to GTP, resulting in the formation of an Mfn2 oligomer (Sugiura et al. 2013) (PMID: 23727017). Therefore, MITOL knockdown was shown to inhibit Mfn2 oligomer formation and cause Mfn2 mislocalization and MAM reduction. In addition, in MITOL-knockdown cells stimulated with histamine, the mitochondrial Ca2+ concentration was significantly lower than that in control cells. MITOL thus regulates ER tethering to mitochondria by activating Mfn2 via lysine 192 ubiquitination (Fig. 5). Since mutations in Mfn2 are the cause of Charcot–Marie–Tooth type 2A hereditary neuropathy, MAM regulation by MITOL may be involved in the mechanisms underlying this disease.
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 5

Role of MITOL in endoplasmic reticulum (ER)–mitochondrion tethering. MITOL induces K63-linked ubiquitylation of mitochondrial Mfn2 on K192, resulting in the oligomerization of Mfn2 and thereby the interaction of mitochondria with the ER

Role of MITOL in the Immune System

Mitochondria serve as a platform for antiviral signal transduction. Toll-like receptors are a family of transmembrane receptors that play a key role in innate immunity. TANK (also called I-TRAF) is a negative regulator of Toll-like receptor-mediated induction of proinflammatory cytokines. MITOL mediates the addition of lysine-63-linked polyubiquitin chains to several lysine residues in TANK and impairs the ability of TANK to inhibit TRAF6. Thus, MITOL positively regulates TLR7 signaling via TANK inhibition (Shi et al. 2011) (PMID: 21625535).

Mitochondrial antiviral signaling protein (MAVS) is localized at the outer membrane of mitochondria and regulates downstream antiviral signaling. Upon viral infection, MAVS forms aggregates and transduces antiviral signaling, such as type I interferon induction. To avoid an excess immune response leading to host immunopathology, MAVS aggregates must be downregulated. To achieve this, MITOL ubiquitinates and promotes the proteasomal degradation of MAVS, resulting in the reduction of such aggregates. The RING domain of MITOL recognizes the CARD domain of MAVS. Indeed, MITOL hetero-knockout mice and MITOL-deleted immune cells showed low viral replication and elevated type I interferon responses to RNA viruses. Thus, MITOL functions as a negative regulator of MAVS aggregates by preventing excessive immune reactions (Fig. 6) (Yoo et al. 2015) (PMID: 26246171).
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 6

Role of MITOL in the immune system. MITOL positively regulates TLR7 signaling via TANK inhibition. On the other hand, MITOL functions as a negative regulator of MAVS aggregates by preventing excessive immune reactions

Role of MITOL in Maintaining the Stemness of Embryonic Stem Cells

Embryonic stem cells (ESCs) are pluripotent stem cells that can differentiate into all lineages of the mature organism. MITOL is involved in the maintenance of ESC pluripotency. Knockdown of MITOL in mouse ESCs led to differentiation from naive pluripotency. ESC pluripotency is regulated by a combination of extrinsic and intrinsic factors. Kruppel-like factor 4 (KLF4) is a member of the KLF family of transcription factors, which maintains ESC stemness and prevents ESC differentiation. Knockdown of Klf4 decreased both mRNA and protein levels of MITOL, whereas its overexpression upregulated MITOL expression, indicating that MITOL expression is regulated by Klf4. MITOL mediates K63-linked polyubiquitination of Prkar1a, a negative regulatory subunit of PKA, resulting in inhibition of the Raf/MEK/ERK pathway by PKA activation. Indeed, MITOL can partially replace the use of Klf4 for somatic cell reprogramming. Thus, MITOL plays an important role in maintaining the stemness of mouse ESCs through the Klf4–March5–PKA–ERK pathway (Fig. 7) (Gu et al. 2015) (PMID: 26033541).
Mitochondrial Ubiquitin Ligase MITOL/MARCH5, Fig. 7

Role of MITOL in embryonic stem cell (ESC) pluripotency. MITOL regulates mouse ESC pluripotency via the Klf4–March5–Prkar1a/PKA–ERK pathway

Summary

Mitochondrial morphology is strictly regulated by a balance between fission and fusion. Disruption in this balance induces mitochondrial impairment, resulting in a variety of diseases including neurodegenerative disorders, heart failure, and aging-associated diseases. The mitochondrial ubiquitin ligase MITOL plays an important role in the regulation of mitochondrial dynamics via Drp1 and Mfn2 (Nagashima et al. 2014), suggesting that MITOL dysfunction causes various diseases. Furthermore, MITOL has multiple functions in mitochondria, including in mitochondrial quality control via misfolded protein degradation, nitrosative stress response, signaling in innate immunity, and embryonic development. Further studies on MITOL should deepen our understanding of various diseases including aging-associated diseases.

References

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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Laboratory of Molecular Biochemistry, School of Life SciencesTokyo University of Pharmacy and Life SciencesHachioji, TokyoJapan