Paolo Boretto e Davide de Vito
DRP1 is the human-referred name of Dynamine-1-like protein (DNM1L). It's an 80 kDa mechano-chemical GTPase of the dynamin superfamily that regulates mitochondrial fission in human.
The protein is recruited from the cytosol to the mitochondrial outer membrane where it oligomerizes at discrete foci. Some of these foci develop into mitochondrial scission sites.
Recent studies provided evidence about DRP1 role in pathological state in humans.
(Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein, 2013)
DRP1 is encode by the DNM1L gene, located on cromosome 12, in th band 12p11.21
CHEMICAL STRUCTURE AND IMAGES
DRP1 is formed by three distinct domain: the GTP-binding domain, the bundle signalling element (BSE) and the stalk, which is fundamental for a stable dimerization and dynamic oligomerization of dynamin. Both the BSE and stalk contain sequences of the middle domain and the GTPase effector domain (GED).
The amino (N-) terminal G domain of DNM1L is composed of a central b-sheet of eight b-strands surrounded by eight ahelices.
The BSE is composed of a three helix bundle. The BSE’s central localization within the DNM1L molecule and its architecture involving elements from widely dispersed regions in the DNM1L sequence suggest a function as transmitter of conformational changes from the G domain to the stalk.
The stalk constitutes an elongated, antiparallel four-helix bundle.
(Structural insights into oligomerization and mitochondrial remodelling of dynamin 1-like protein, 2013)
Protein Aminoacids Percentage
SYNTHESIS AND TURNOVER
DRP1 is ubiquitously expressed, with highest levels found in skeletal muscles, heart, kidney and brain. There are 6 isoforms of this protein and the isoform 1 is brain-specific. DRP1 is primarily a cytosolic protein that traslocates to the outher mitochondrial membrane after activation by interaction with some non-GTPase receptor protein as Fis1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor). (Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis, 2004)
- Phosphorylation/dephosphorylation on two sites near the GED domain regulate DRP1 activity: phosphorylation on Ser-637 by PKA inhibits DRP1 and mitochondrial fission, while phosphorylation on Ser-616 by cyclin B1-CDK1 or CaMK (calcium-calmodulin-dependent kinase) activates it. Dephosphorylation of Ser-637 by calcineurin also enhances DRP1 activity.
- S-nitrosylation by nitric oxyde increases DNM1L dimerization and mitochondrial fission.
- SENP5 (Nucleolar small ubiquitin-like) modifier protease moves to the mitochondria during mitosis and desumoylates DRP1, which leads to the activation of DRP1
- MARCH5 (Membrane-associated RING-CH protein 5) and parkin are ubiquitin ligases that can regulate DRP1 activation and degradation.
- Mdivi-1 is a mitochondrial division inhibitor 1 that inhibits DRP1
(Perturbations in mitochondrial dynamics induced by human mutant PINK1 can be rescued by the mitochondrial division inhibitor mdivi-1, 2010)
DRP1 is involved in the regulation of mitochondrial fission-fusion process. Mitochondria are mobile organelles that exist in dynamic networks and continuously join by the process of fusion and divide by the process of fission.
Fission and fusion fine-tune fundamental cellular processes such as calcium homeostasis and the generation of ATP and reactive oxygen species and consequently have important roles in cell-cycle progression, apoptosis, mitophagy, and oxygen sensing. In particular there are five noncanonical mitochondrial capabilities which are involved in the dynamic cycle of the mitochondria:
1) Mitochondria are linked to the endoplasmic reticulum to facilitate calcium flux into the mitochondria. This connection is regulated by Mitofusin-2 (Mfn-2).
2) Mitochondria number is regulated by mitochondrial biogenesis to meet the energy demands of the cell and compensate for cell damage. PGC-1α (Peroxisomeproliferator–activated receptor γ coactivator 1α) play a key role in the regulation because of its transcriptional coactivator effect on Mitofusin-2. (Regulation of mitofusin-2 expression in skeletal muscle, 2009)
3) Mitochondria have a quality-control program called mitophagy which maintains cellular health by selectively enclosing damaged and depolarized mitochondria in autophagic vacuoles for elimination by lysosomes. Fission process facilitates mitophagy.
4) Mitochondria actively traverse the citosol on dynein and kinesine tracks. Dynein also regulates fission. (PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility, 2011)
5) Mitochondria are oxygen sensors in cells within the homeostatic oxygen-sensing system, such as pulmonary arterial and ductus arteriosus smooth-muscle cells. These specialized mitochondria vary production of diffusible reactive oxygen species by the electron transport chain in proportion to cellular oxygen levels, permitting redox regulation of ion channels, enzymes, and transcription factors. Mitochondrial dynamics are an early step in this redox signaling mechanism. (Acute oxygen-sensing mechanisms, 2005)
In synthesis, fission increases reacting oxygen species production, facilitates mitophagy and accelerates cell proliferation. In contrast, fusion enhances comunication with the endoplasmic reticulum, increases ATP production and exercises a quality control on the mitochondria by diluiting DNA mutations and oxidized proteins.
Fusion and Fission Mechanism
Fusion: this process is mediated by the coordinated activities of Mfn-2 in the outer mitochondrial membrane and OPA1 (optic atrophy-1- proteine) in the inner one. Mitofusin-2 is also located in the endoplasmic reticulum, where it alters morphologic features and promotes endoplasmic reticulum–mitochondrial tethering. During the fusion, two mitofusin isoforms form homodimers or heterodimers that join two adjacent mitochondria, with a mechanism similar to a Velcro fastener.
Fission: during the process DRP1 is activated, moves to the outher mitochondrial membrane and form the fission apparatus guided by some proteins as MFF and Fis1. Activation usually reflect post-transcriptional modification by a kinase, a phosphatase, or ubiquitin ligase or by sumoylation. after the assembly of the fission apparatus, mitochondria fragmented into two parts. (Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer, 2012)
Relation between Fission and Fusion Dynamics and Mitochondrial Metabolism
Pyruvate generated in the cytosol by glycolysis either enters the mitochondria and fuels oxidative metabolism or remains in the cytosol and is converted to lactate by lactate dehydrogenase (LDH) if mitochondrial metabolism is inhibited. Once inside the mitochondria, pyruvate is converted into acetyl coenzyme A (CoA) by pyruvate dehydrogenase (PDH), the major regulator of oxidative metabolism.
There are two main mechanisms that rule both fission-fusion and mitochondrial metabolism:
1) Hipoxia-inducible factor 1α (HIF-1α) activates pyruvate dehydrogenase kinase (PDK), which inhibits PDH, and promotes the activation of DRP1.
2) Mitochondrial calcium levels regulate PDH activity and apoptosis: an increase of mitochondrial calcium activates PDH but at higher levels promotes apoptosis. In contrast, the rise of cytsolic calcium level activates CaMK and then DRP1.
DRP1 – RELATED DISEASES
Disorders of mitochondrial dynamics are implicated in proliferative, apoposis-resistant diseases.
Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. Excessive proliferation and resistance to apoptosis are hallmarks of cancer cells and structural mitochondrial abnormalities contribute to the imbalance between proliferation and apoptosis in cancer (Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer,2012).
Coordination between mitochondrial division and mitosis (so-called mitotic fission) ensures equitable distribution of mitochondria to daughter cells: at the transition from the G1 phase to the S phase, mitochondria fuse and increase ATP production, while DRP1 inhibition induces mitochondrial hyperfusion and triggers DNA replication and cyclin E accumulation (A hyperfused mitochondrial state achieved at G1-S regulates cyclin E buildup and entry into S phase,2009). The coordination of fission and mitosis is substantially regulated by cyclin B1–CDK1, which simultaneously initiates mitosis and activates DRP1 by phosphorylating serine 616 (Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer,2012). Another mitotic kinase, aurora A, phosphorylates the Raslike GTPase (RalA), leading to mitotic, mitochondrial accumulation of RalA and its effector, ralA binding protein 1 (RalBP1). RalBP1 serves as a scaffold for recruiting DRP1 and cyclin-CDK to mitochondria and inducing fission (RALA and RALBP1 regulate mitochondrial fission at mitosis,2011).
Human lung cancer cell lines exhibit an imbalance of DRP-1/Mfn-2 expression, which promotes a state of mitochondrial fission: these cells had lower levels of the fusion mediator Mfn-2 and markedly higher levels of the mitochondrial fission mediator DRP-1 than normal cells. This finding suggested that the observed mitochondrial fragmentation was related to both impaired fusion and enhanced fission. Fragmentation may accelerate mitotic fission (Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer,2012) and also interrupts intramitochondrial calcium waves, preventing calcium-mediated apoptosis (Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca2+ waves and protects against Ca2+-mediated apoptosis,2004).
Structural mitochondrial fragmentation in cancer cells could be reversed by either inhibiting DRP1 with the highly specific small molecule DRP1 inhibitor mdivi-1 or with the small-interfering RNA (siRNA) or by overexpressing Mfn-2. In vivo (using a mice xenograft model), all three interventions markedly reduced the mitochondrial fragmentation and decreased tumor mass size (Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer,2012). Moreover, persistent mitochondrial hyperfusion due to Drp1 depletion induces cytochrome c release from mitochondria and also causes mitochondrial dysfunction including accumulation of mtDNA mutation and generation of reactive oxygen species (ROS). This combined effect of genome instability and mitochondrial dysfunction may eventually result in the activation of mitochondrial apoptotic pathway (The role of dynamin-related protein 1 in cancer growth: a promising therapeutic target?,2013).
Figure. Variation of DRP1 levels in cell cycle. APC-Cdh1 (anaphase-promoting complex and coactivator cadherin 1) marks DRP1 for proteosomal degradation, allowing G1-phase reassembly of mitochondrial networks (Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability,2011)
Pulmonary Arterial Hypertension
Pulmonary hypertension (PH) is an increase of blood pressure in the pulmonary artery which exceeds 25 mm Hg at rest or 30 mm Hg with exercise.
Although vasoconstriction, inflammation, and thrombosis contribute to the pathogenesis, an “oncologic view” is emerging, which holds that excessive proliferation and impaired apoptosis mediate disease progression (Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies,2010). Both oxygen-sensing and mitochondrial dynamics are disordered in pulmonary-artery smooth muscle cells (PASMCs), as indicated by normoxic activation of hypoxia-inducible factor 1α (HIF-1α) and mitochondrial fragmentation (An abnormal mitochondrial-hypoxia inducible factor-1alpha-Kv channel pathway disrupts oxygen sensing and triggers pulmonary arterial hypertension in fawn hooded rats: similarities to human pulmonary arterial hypertension,2006), the latter resulting from reduced mitofusin-2–mediated fusion and excessive DRP1-mediated fission (Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension,2012).
HIF-1α activation can result from the redox changes initiated by epigenetic silencing of SOD2 in PAH. Activated HIF-1α suppresses mitochondrial oxidative metabolism (by increasing the expression of pyruvate dehydrogenase kinases, PDKs, thereby blocking pyruvate uptake into the Krebs cycle) while simultaneously upregulating enzymes and transporters that favor glycolysis (i.e. hexokinase-2 and the glucose transporter-1, GLUT1). Moreover, HIF-1α activation in PASMCs is a proliferative stimulus sufficient to cause DRP1-mediated mitochondrial fission (Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension,2012).
Increased fission in PAH PASMC results from DRP1 activation by increased cyclin B1/CDK1 activity and accompanies cell cycle progression from G2 to mitosis. Mdivi-1 (a DRP1-inhibitor) slows proliferation by locking mitochondria in fusion and inhibiting cell-cycle progression, causing G2/M arrest (Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension,2012).
Also, Mitofusin-2 levels are reduced in pulmonary arterial hypertension, contributing to both mitochondrial fragmentation and the proliferative diathesis. Mitofusin-2 down regulation in PAH is associated with low levels of PGC-1α (PGC1α-mediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension,2013), the transcriptional coactivator of mitofusin-2 (Peroxisome proliferator-activated receptor-gamma co-activator 1alpha-mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipid-induced mitochondrial inefficiency,2005). Preclinical testing showed that augmenting mitofusin-2 decreases proliferation and increases apoptosis of pulmonary-artery smooth-muscle cells and leads to partial regression of experimentally induced pulmonary arterial hypertension in vivo in rats (PGC1α-mediated mitofusin-2 deficiency in female rats and humans with pulmonary arterial hypertension,2013).
Figure. Confront control vs PAH PASMCs proliferation. PAH cells have higher levels of DRP1 and cyclin B1 (Dynamin-related protein 1-mediated mitochondrial mitotic fission permits hyperproliferation of vascular smooth muscle cells and offers a novel therapeutic target in pulmonary hypertension,2012)
Mitochondrial dynamics are involved in mechanisms of human diseases and may offer new therapeutic targets. Recently has been developed a small peptide inhibitor P110 that prevents DRP1 activation and fission by interfering with the protein-protein interaction between DRP1 and its mitochondrial adaptor target protein (A novel Drp1 inhibitor diminishes aberrant mitochondrial fission and neurotoxicity,2013). Additional pharmacologic modulator of fission and further studies are needed.
Mitochondrial Dynamics — Mitochondrial Fission and Fusion in Human Diseases,2013