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Cell Death & Disease Mar 2024Dynamin related protein 1 (DRP1), a pivotal mitochondrial fission protein, is post-translationally modified by multiple mechanisms. Here we identify a new...
Dynamin related protein 1 (DRP1), a pivotal mitochondrial fission protein, is post-translationally modified by multiple mechanisms. Here we identify a new post-translational modification of DRP1 by the ubiquitin-like protein, interferon-stimulated gene 15 (ISG15). DRP1 ISGylation is mediated by ISG15 E3 ligase, HERC5; this promotes mitochondrial fission. DeISGylation of DRP1 however leads to hyperfusion. Heterologous expression of SARS-CoV2 PLpro, a deISGylating enzyme, results in similar mitochondrial filamentation, significant decrease in total DRP1 protein levels and efflux of mtDNA. We report that deISGylated DRP1 gets ubiquitylated and degraded by TRIM25, instead of PARKIN and MITOL. While the cytosolic pool of DRP1 is primarily ISGylated, both mitochondrial and cytosolic fractions may be ubiquitylated. It is known that phosphorylation of DRP1 at S616 residue regulates its mitochondrial localisation; we show that ISGylation of phospho-DRP1 (S616) renders fission competence at mitochondria. This is significant because DRP1 ISGylation affects its functionality and mitochondrial dynamics in Alzheimer's disease pathophysiology.
Topics: Mitochondrial Dynamics; RNA, Viral; Dynamins; Protein Processing, Post-Translational; Mitochondria; Mitochondrial Proteins
PubMed: 38431611
DOI: 10.1038/s41419-024-06543-7 -
Research Square Sep 2023invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors - TarP and TmeA. They collaborate to promote robust actin...
invasion of epithelial cells is a pathogen-driven process involving two functionally distinct effectors - TarP and TmeA. They collaborate to promote robust actin dynamics at sites of entry. Here, we extend studies on the molecular mechanism of invasion by implicating the host GTPase dynamin 2 (Dyn2) in the completion of pathogen uptake. Importantly, Dyn2 function is modulated by TarP and TmeA at the levels of recruitment and activation through oligomerization, respectively. TarP-dependent recruitment requires phosphatidylinositol 3-kinase and the small GTPase Rac1, while TmeA has a post-recruitment role related to Dyn2 oligomerization. This is based on the rescue of invasion duration and efficiency in the absence of TmeA by the Dyn2 oligomer-stabilizing small molecule activator Ryngo 1-23. Notably, Dyn2 also regulated turnover of TarP- and TmeA-associated actin networks, with disrupted Dyn2 function resulting in aberrant turnover dynamics, thus establishing the interdependent functional relationship between Dyn2 and the effectors TarP and TmeA.
PubMed: 37841835
DOI: 10.21203/rs.3.rs-3376558/v1 -
Molecular Neurodegeneration Mar 2024Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological...
BACKGROUND
Dynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson's disease and Alzheimer's disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed.
METHODS
We characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1 mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used. RESULTS IN VITRO: data demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1 mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology.
CONCLUSIONS
This study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.
Topics: Animals; Humans; Mice; Rats; alpha-Synuclein; Autophagy; Dynamins; HeLa Cells; Mitochondria; Mitochondrial Dynamics; Parkinson Disease
PubMed: 38504290
DOI: 10.1186/s13024-024-00708-w -
Autophagy Reports Aug 2023Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal...
Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies.
PubMed: 38107506
DOI: 10.1080/27694127.2023.2247309 -
Cell Death & Disease Jan 2024Mitochondria are highly dynamic organelles capable of altering their sizes and shapes to maintain metabolic balance through coordinated fission and fusion processes. In...
Mitochondria are highly dynamic organelles capable of altering their sizes and shapes to maintain metabolic balance through coordinated fission and fusion processes. In various cancer types, mitochondrial hyperfragmentation has been frequently observed, contributing to the progression of cancer toward metastasis. Inverted formin 2 (INF2), which resides in the endoplasmic reticulum (ER), has been found to accelerate actin polymerization and drive mitochondrial fission. In this study, we demonstrate that INF2 expression is significantly upregulated in endometrial cancer (EC) and is associated with a poor prognosis in EC patients. INF2 promotes anchorage-dependent and independent EC cell growth in part by facilitating mitochondrial fission. Furthermore, in conditions of energy stress, AMP-activated protein kinase (AMPK) phosphorylates INF2 at Ser1077, leading to increased localization of INF2 to the ER and enhanced recruitment of the dynamin-related protein 1 (DRP1) to mitochondria. This AMPK-mediated phosphorylation of INF2 at Ser1077 facilitates mitochondrial division and promotes EC cell growth. Pathological examination using immunohistochemical analyses revealed a positive correlation between AMPK activity and phosphorylated INF2 (Ser1077) in EC specimens. Collectively, our findings uncover novel molecular mechanisms involving the AMPK-INF2 axis, which regulates mitochondrial dynamics and malignant cell growth in EC.
Topics: Female; Humans; AMP-Activated Protein Kinases; Dynamins; Endometrial Neoplasms; Formins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Phosphorylation
PubMed: 38233384
DOI: 10.1038/s41419-024-06431-0 -
Redox Biology Apr 2024Hyperglycemia increases the heart sensitivity to ischemia-reperfusion (IR), but the underlying cellular mechanisms remain unclear. Mitochondrial dynamics (the processes...
Hyperglycemia increases the heart sensitivity to ischemia-reperfusion (IR), but the underlying cellular mechanisms remain unclear. Mitochondrial dynamics (the processes that govern mitochondrial morphology and their interactions with other organelles, such as the reticulum), has emerged as a key factor in the heart vulnerability to IR. However, it is unknown whether mitochondrial dynamics contributes to hyperglycemia deleterious effect during IR. We hypothesized that (i) the higher heart vulnerability to IR in hyperglycemic conditions could be explained by hyperglycemia effect on the complex interplay between mitochondrial dynamics, Ca homeostasis, and reactive oxygen species (ROS) production; and (ii) the activation of DRP1, a key regulator of mitochondrial dynamics, could play a central role. Using transmission electron microscopy and proteomic analysis, we showed that the interactions between sarcoplasmic reticulum and mitochondria and mitochondrial fission were increased during IR in isolated rat hearts perfused with a hyperglycemic buffer compared with hearts perfused with a normoglycemic buffer. In isolated mitochondria and cardiomyocytes, hyperglycemia increased mitochondrial ROS production and Ca uptake. This was associated with higher RyR2 instability. These results could contribute to explain the early mPTP activation in mitochondria from isolated hearts perfused with a hyperglycemic buffer and in hearts from streptozotocin-treated rats (to increase the blood glucose). DRP1 inhibition by Mdivi-1 during the hyperglycemic phase and before IR induction, normalized Ca homeostasis, ROS production, mPTP activation, and reduced the heart sensitivity to IR in streptozotocin-treated rats. In conclusion, hyperglycemia-dependent DRP1 activation results in higher reticulum-mitochondria calcium exchange that contribute to the higher heart vulnerability to IR.
Topics: Animals; Rats; Calcium; Coronary Artery Disease; Hyperglycemia; Mitochondria, Heart; Mitochondrial Dynamics; Myocardial Reperfusion Injury; Proteomics; Reactive Oxygen Species; Reperfusion; Ryanodine Receptor Calcium Release Channel; Streptozocin; Dynamins
PubMed: 38266577
DOI: 10.1016/j.redox.2024.103044 -
Research Square Jul 2023The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial fission, but the regulatory mechanisms remain ambiguous. Here we found that a...
The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial fission, but the regulatory mechanisms remain ambiguous. Here we found that a conserved, intrinsically disordered, six-residue hort near otif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function and . Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, leading to the fission of model membranes . , the availability of the native CT-SLiM is a requirement for productive mitochondrial fission, as both non-native extension and deletion of the CT-SLiM severely impair its progression. Thus, contrary to prevailing models, Drp1-catalyzed mitochondrial fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.
PubMed: 37503116
DOI: 10.21203/rs.3.rs-3161608/v1 -
Nutrition & Diabetes Apr 2024The number of patients with type 1 diabetes rises rapidly around the world in recent years. Maternal diabetes has a detrimental effect on reproductive outcomes due to...
BACKGROUND
The number of patients with type 1 diabetes rises rapidly around the world in recent years. Maternal diabetes has a detrimental effect on reproductive outcomes due to decreased oocyte quality. However, the strategies to improve the oocyte quality and artificial reproductive technology (ART) efficiency of infertile females suffering from diabetes have not been fully studied. In this study, we aimed to examine the effects of nicotinamide mononucleotide (NMN) on oocyte maturation of mouse with type 1 diabetes mouse and explore the underlying mechanisms of NMN's effect.
METHODS
Streptozotocin (STZ) was used to establish the mouse models with type 1 diabetes. The successful establishment of the models was confirmed by the results of body weight test, fasting blood glucose test and haematoxylin and eosin (H&E) staining. The in vitro maturation (IVM) rate of oocytes from diabetic mice was examined. Immunofluorescence staining (IF) was performed to examine the reactive oxygen species (ROS) level, spindle/chromosome structure, mitochondrial function, actin dynamics, DNA damage and histone modification of oocytes, which are potential factors affecting the oocyte quality. The quantitative reverse transcription PCR (RT-qPCR) was used to detect the mRNA levels of Sod1, Opa1, Mfn2, Drp1, Sirt1 and Sirt3 in oocytes.
RESULTS
The NMN supplementation increased the oocyte maturation rate of the mice with diabetes. Furthermore, NMN supplementation improved the oocyte quality by rescuing the actin dynamics, reversing meiotic defects, improving the mitochondrial function, reducing ROS level, suppressing DNA damage and restoring changes in histone modifications of oocytes collected from the mice with diabetes.
CONCLUSION
NMN could improve the maturation rate and quality of oocytes in STZ-induced diabetic mice, which provides a significant clue for the treatment of infertility of the patients with diabetes.
Topics: Animals; Mice; Female; Oocytes; Diabetes Mellitus, Type 1; Diabetes Mellitus, Experimental; Reactive Oxygen Species; Nicotinamide Mononucleotide; Mitochondria; Sirtuin 1; Sirtuin 3; In Vitro Oocyte Maturation Techniques; Superoxide Dismutase-1; DNA Damage; Streptozocin; Oogenesis; Dynamins
PubMed: 38653987
DOI: 10.1038/s41387-024-00280-8 -
RSC Medicinal Chemistry Aug 2023We show that dansylcadaverine (1) a known in-cell inhibitor of clathrin mediated endocytosis (CME), moderately inhibits dynamin I (dynI) GTPase activity (IC 45 μM) and...
We show that dansylcadaverine (1) a known in-cell inhibitor of clathrin mediated endocytosis (CME), moderately inhibits dynamin I (dynI) GTPase activity (IC 45 μM) and transferrin (Tfn) endocytosis in U2OS cells (IC 205 μM). Synthesis gave a new class of GTP-competitive dynamin inhibitors, the Sulfonadyns™. The introduction of a terminal cinnamyl moiety greatly enhanced dynI inhibition. Rigid diamine or amide links between the dansyl and cinnamyl moieties were detrimental to dynI inhibition. Compounds with inhibition of dynI activity <10 μM were tested in-cell for inhibition of CME. These data unveiled a number of compounds, analogues 33 (()--(6-{[(3-(4-bromophenyl)-2-propen-1-yl]amino}hexyl)-5-isoquinolinesulfonamide)) and 47 (()--(3-{[3-(4-bromophenyl)-2-propen-1-yl]amino}propyl)-1-naphthalenesulfonamide)isomers that showed dyn IC <4 μM, IC <30 μM and IC from 12-265 μM. Both analogues (33 and 47) are at least 10 times more potent that the initial lead, dansylcadaverine (1). Enzyme kinetics revealed these sulfonamide analogues as being GTP competitive inhibitors of dynI. Sulfonadyn-47, the most potent SVE inhibitor observed (IC = 12.3 μM), significantly increased seizure threshold in a 6 Hz mouse psychomotor seizure test at 30 ( = 0.003) and 100 mg kg ip ( < 0.0001), with similar anti-seizure efficacy to the established anti-seizure medication, sodium valproate (400 mg kg). The Sulfonadyn™ class of drugs target dynamin and show promise as novel leads for future anti-seizure medications.
PubMed: 37593570
DOI: 10.1039/d2md00371f -
BioRxiv : the Preprint Server For... Apr 2024Mitochondrial health relies on the membrane fission mediated by dynamin-related protein 1 (Drp1). Previous structural studies of Drp1 on remodeled membranes were...
Mitochondrial health relies on the membrane fission mediated by dynamin-related protein 1 (Drp1). Previous structural studies of Drp1 on remodeled membranes were hampered by heterogeneity, leaving a critical gap in the understanding of the mitochondrial fission mechanism. Here we present a cryo-electron microscopy structure of full-length human Drp1 decorated on membrane tubules. Using the reconstruction of average subtracted tubular regions (RASTR) technique, we report that Drp1 forms a locally ordered lattice along the tubule without global helical symmetry. The filaments in the lattice are similar to dynamin rungs with conserved stalk interactions. Adjacent filaments are connected by GTPase domain interactions in a novel stacked conformation. Additionally, we observed contact between Drp1 and membrane that can be assigned to variable domain sequence. We identified two states of the Drp1 lattice representing conformational changes related to membrane curvature differences. Together these structures revealed a putative mechanism by which Drp1 constricts mitochondria membranes in a stepwise, "ratchet" manner.
PubMed: 38617273
DOI: 10.1101/2024.04.04.588123