bims-minimp Biomed News
on Mitochondria, innate immunity, proteostasis
Issue of 2022–05–15
28 papers selected by
Hanna Salmonowicz, International Institute of Molecular Mechanisms and Machines of the Polish Academy of Sciences



  1. Elife. 2022 May 13. pii: e74552. [Epub ahead of print]11
      Proliferating cells undergo metabolic changes in synchrony with cell cycle progression and cell division. Mitochondria provide fuel, metabolites, and ATP during different phases of the cell cycle, however it is not completely understood how mitochondrial function and the cell cycle are coordinated. CLUH is a post-transcriptional regulator of mRNAs encoding mitochondrial proteins involved in oxidative phosphorylation and several metabolic pathways. Here, we show a role of CLUH in regulating the expression of astrin, which is involved in metaphase to anaphase progression, centrosome integrity, and mTORC1 inhibition. We find that CLUH binds both the SPAG5 mRNA and its product astrin, and controls the synthesis and the stability of the full-length astrin-1 isoform. We show that CLUH interacts with astrin-1 specifically during interphase. Astrin-depleted cells show mTORC1 hyperactivation and enhanced anabolism. On the other hand, cells lacking CLUH show decreased astrin levels and increased mTORC1 signaling, but cannot sustain anaplerotic and anabolic pathways. In absence of CLUH, cells fail to grow during G1, and progress faster through the cell cycle, indicating dysregulated matching of growth, metabolism and cell cycling. Our data reveal a role of CLUH in coupling growth signaling pathways and mitochondrial metabolism with cell cycle progression.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.74552
  2. Biochim Biophys Acta Bioenerg. 2022 May 06. pii: S0005-2728(22)00037-8. [Epub ahead of print] 148568
      The filamentous ascomycete Podospora anserina is a well-established model system to study organismic aging. Its senescence syndrome has been investigated for more than fifty years and turned out to have a strong mitochondrial etiology. Several different mitochondrial pathways were demonstrated to affect aging and lifespan. Here, we present an update of the literature focusing on the cooperative interplay between different processes.
    Keywords:  Aging; Lifespan; Mitochondria; Mitochondrial proteases; Podospora anserina; Respiratory chain
    DOI:  https://doi.org/10.1016/j.bbabio.2022.148568
  3. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo coordinated rounds of fusion and fission that are critical for maintaining the functional integrity of this essential organelle. While a growing number of proteins have been identified as important regulators of mitochondrial dynamics, the direct role of membrane lipid composition during the fusion and fission processes is poorly understood. To address these shortcomings, we devised a protein-engineering platform that allows for the acute remodeling of structural phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, we modified a bacterial phospholipase C (Bacillus cereus (Bc)PI-PLC) to initiate the rapid hydrolysis of phosphatidylinositol (PI) and locally generate diacylglycerol (DAG); an important intracellular signaling molecule and metabolic precursor that is used in diverse lipid biosynthetic pathways. Spatial restriction of enzyme activity was achieved using a chemically inducible system consisting of a rapamycin-dependent dimerization module (FKBP-BcPI-PLC) along with an OMM targeting sequence tagged with the FKBP-rapamycin binding domain (OMM-FRB). Using these unique molecular tools, we show that recruitment of FKBP-BcPI-PLC to the OMM not only causes the expected local accumulation of DAG, but also initiates the rapid and uniform fragmentation of the mitochondrial network. Mitochondrial fission induced by FKBP-BcPI-PLC is accompanied by profound swelling of the mitochondrial matrix along with vesiculation of the inner mitochondrial membrane (IMM) and a general loss of cristae, which all occur within minutes of tethering FKBP-BcPI-PLC to the OMM. Expression of dominant-negative constructs targeting essential GTPases known to regulate OMM fission suggest that both dynamin-related protein 1 (Drp1) and dynamin 2 (Dnm2) work together to drive efficient BcPI-PLC-induced mitochondrial division. However, results using a validated Drp1 knockout cell line show that the loss of Drp1 alone is sufficient to prevent the mitochondrial fragmentation initiated by FKBP-BcPI-PLC recruitment, indicating that Drp1 likely functions upstream or independent of Dnm2 in this context. Interestingly, unlike the induced OMM fission, removal of Drp1 from cells does not prevent the matrix swelling or OMM constrictions observed in response to acute generation of DAG within the OMM. Ongoing experiments are now focused on characterizing new methods to sequentially metabolize the DAG generated within the OMM as well as investigate how local lipid composition influences the binding and oligomerization of membrane-shaping proteins that may function in concert with Drp1 to regulate mitochondrial remodeling. Overall, these studies establish a direct relationship between lipid metabolism within the OMM and clinically relevant morphological changes that are known to manifest in mitochondrial-associated diseases.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3682
  4. Sci Rep. 2022 May 11. 12(1): 7704
      Aging of sensory organs is associated with a decline in mitochondrial function and the accumulation of dysfunctional mitochondria. Impaired mitophagy blocks the turnover of dysfunctional mitochondria and leads to their accumulation. Urolithin A (UA) induces mitophagy in various mammalian cells. This study was aimed at investigating the effect of the mitophagy activator, UA, on premature senescent auditory cells. The levels of cellular senescence-associated p53 and p21 significantly increased in H2O2-induced senescent House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants. However, the levels of mitophagy-related molecules significantly decreased. UA significantly decreased the expression of senescence-associated p53 and p21, and increased the expression of mitophagy-related proteins, in H2O2-induced senescent cells and cochlear explants. The percentage of β-galactosidase-stained senescent cells also reduced in H2O2-treated cells and cochlear explants upon UA pre-treatment. The formation of mitophagosomes and mitophagolysosomes was restored upon UA pre-treatment of H2O2-induced senescent cells. The knockdown of mitophagy-related genes (Parkin and Bnip3) resulted in annulment of UA-induced anti-senescent activity. UA significantly increased the ATP content, mitochondrial DNA (mtDNA) integrity, and mitochondrial membrane potential in senescent HEI-OC1 cells. These findings indicate that UA counteracted mitophagy decline and prevented premature senescence in auditory cells. Hence, UA administration might be a promising strategy for preventing mitochondrial dysfunction in patients with age-related hearing loss.
    DOI:  https://doi.org/10.1038/s41598-022-11894-2
  5. FASEB J. 2022 May;36 Suppl 1
      Phosphorylation has long been appreciated to influence mitochondrial metabolism via the regulation of pyruvate dehydrogenase. However, the extent to which phosphorylation broadly influences mitochondrial function remains unclear, despite the presence of multiple protein phosphatases within the organelle. We recently demonstrated that deletion of the mitochondrial matrix phosphatase Pptc7 unexpectedly caused perinatal lethality in mice, suggesting that the regulation of mitochondrial phosphorylation is essential in mammalian development. Pptc7-/- mice exhibit severe metabolic deficiencies, including hypoglycemia and lactic acidosis, and die within one day of birth. Biochemical and proteomic approaches revealed that Pptc7-/- tissues have decreased mitochondrial function concomitant with a post-transcriptional downregulation of mitochondrial proteins. Multiple elevated mitochondrial protein phosphorylation sites in Pptc7-/- tissues suggest novel functional connections between Pptc7-mediated dephosphorylation and these observed metabolic consequences. Interestingly, these modifications occur on components of the import machinery of the mitochondria and within the mitochondrial targeting sequences of select nuclear-encoded precursor proteins. Collectively, our data reveal an unappreciated role for a matrix-localized phosphatase in the post-translational regulation of the mitochondrial proteome and organismal metabolic homeostasis.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R6264
  6. FASEB J. 2022 May;36 Suppl 1
      Peroxisomes are dynamic and ubiquitous organelles that house many metabolic pathways and interact with other organelles such as the endoplasmic reticulum, lipid droplets, and mitochondria. One mechanism for organelle interaction is through membrane contact sites. While contact sites between multiple organelles have been identified, little is known about the proteins that serve as molecular tethers in such sites. We study organelle dynamics using peroxisome-like organelles called glycosomes in the early diverging organism Trypanosoma brucei and have identified a novel peroxin (protein involved in peroxisome biogenesis) that is essential for mitochondrial morphology. Silencing this protein leads to a significant growth defect and swollen mitochondria. Multiple mitochondrial membrane transport channels have been identified in immunoprecipitation studies. Based on these findings, we hypothesize that this protein that we have named a putative peroxisome-mitochondrial contact protein (PPMCP), localizes to glycosomes and mitochondria at contact points, which facilitate the transfer of metabolites between the two organelles. Disruption of this connection results in "leaky" mitochondria and cell death. Current work is focused on resolving the metabolic defects in PPMCP-deficient cells and identifying additional molecular components of these contact sites. This work forwards our understanding of how contact sites are established and the role they play in interorganelle communication.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2441
  7. Cell Rep. 2022 May 10. pii: S2211-1247(22)00564-2. [Epub ahead of print]39(6): 110797
      The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.
    Keywords:  APEX2; CP: Cell biology; CP: Immunology; ER-mitochondria contact site; HIV-1; TRIM5α; ULK1 complex; autophagy; inflammation; mitochondrial metabolism; proteomics; tripartite motif
    DOI:  https://doi.org/10.1016/j.celrep.2022.110797
  8. FASEB J. 2022 May;36 Suppl 1
      Mitochondrial ATPase ATAD3A is essential for cholesterol transport, mitochondrial structure, and cell survival. However, the relationship between ATAD3A and non-alcoholic fatty liver disease (NAFLD) is largely unknown. In this study, we found that ATAD3A was upregulated in the progression of NAFLD in livers from rats with diet-induced non-alcoholic steatohepatitis (NASH) and in human livers with NAFLD. CRISPR-Cas9 was used to delete ATAD3A function in Huh7 human hepatocellular carcinoma cells to assess the influence of ATAD3A deletion on liver cells with free cholesterol (FC) overload induced by treatment with cholesterol plus 58035, an inhibitor for acetyl-CoA acetyltransferase, the enzyme converting FC to cholesterol ester. The results showed that ATAD3A KO exacerbated FC accumulation under FC overload in Huh7 cells. Triglyceride (TG) levels were also significantly increased in ATAD3A KO Huh7 cells under FC overload and control conditions via inhibited lipolysis mediated by upregulation of perilipin 2. Moreover, loss of ATAD3A downregulated mitophagy-associated PTEN-induced kinase 1 expression in Huh7 cells under FC overload and control conditions, suggesting that ATAD3A KO blocks mitophagy. Consistently, mitochondrial mass was increased in ATAD3A KO cells under FC overload as indicated by mitochondrial protein translocase of outer mitochondrial membrane 20 (TOM20). The results also showed that loss of ATAD3A impaired mitochondrial basal respiration and ATP production in Huh7 cells under FC overload, accompanied by downregulation of mitochondrial ATP synthase. In conclusion, loss of ATAD3A promotes the progression of NAFLD through the accumulation of FC, TG, and damaged mitochondria in hepatocytes.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4389
  9. J Cell Sci. 2022 May 01. pii: jcs259596. [Epub ahead of print]135(9):
      The heterotrimeric BAG6 complex coordinates the direct handover of newly synthesised tail-anchored (TA) membrane proteins from an SGTA-bound preloading complex to the endoplasmic reticulum (ER) delivery component TRC40. In contrast, defective precursors, including aberrant TA proteins, form a stable complex with this cytosolic protein quality control factor, enabling such clients to be either productively re-routed or selectively degraded. We identify the mitochondrial antiviral-signalling protein (MAVS) as an endogenous TA client of both SGTA and the BAG6 complex. Our data suggest that the BAG6 complex binds to a cytosolic pool of MAVS before its misinsertion into the ER membrane, from where it can subsequently be removed via ATP13A1-mediated dislocation. This BAG6-associated fraction of MAVS is dynamic and responds to the activation of an innate immune response, suggesting that BAG6 may modulate the pool of MAVS that is available for coordinating the cellular response to viral infection.
    Keywords:  BioID2; ER membrane complex; Protein targeting; SGTA; Tail-anchored proteins
    DOI:  https://doi.org/10.1242/jcs.259596
  10. FASEB J. 2022 May;36 Suppl 1
      Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin-related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome-mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3665
  11. Int J Mol Sci. 2022 Apr 25. pii: 4741. [Epub ahead of print]23(9):
      Function of mitochondria largely depends on a characteristic ultrastructure with typical invaginations, namely the cristae of the inner mitochondrial membrane. The mitochondrial signature phospholipid cardiolipin (CL), the F1Fo-ATP-synthase, and the 'mitochondrial contact site and cristae organizing system' (MICOS) complex are involved in this process. Previous studies with Podospora anserina demonstrated that manipulation of MICOS leads to altered cristae structure and prolongs lifespan. While longevity of Mic10-subcomplex mutants is induced by mitohormesis, the underlying mechanism in the Mic60-subcomplex deletion mutants was unclear. Since several studies indicated a connection between MICOS and phospholipid composition, we now analyzed the impact of MICOS on mitochondrial phospholipid metabolism. Data from lipidomic analysis identified alterations in phospholipid profile and acyl composition of CL in Mic60-subcomplex mutants. These changes appear to have beneficial effects on membrane properties and promote longevity. Impairments of CL remodeling in a PaMIC60 ablated mutant lead to a complete abrogation of longevity. This effect is reversed by supplementation of the growth medium with linoleic acid, a fatty acid which allows the formation of tetra-octadecanoyl CL. In the PaMic60 deletion mutant, this CL species appears to lead to longevity. Overall, our data demonstrate a tight connection between MICOS, the regulation of mitochondrial phospholipid homeostasis, and aging of P. anserina.
    Keywords:  MICOS; Podospora anserina; aging; cardiolipin; cristae; mitochondria; tafazzin
    DOI:  https://doi.org/10.3390/ijms23094741
  12. FASEB J. 2022 May;36 Suppl 1
      ATP-dependent AAA+ proteases are critical regulators of mitochondrial functions, playing crucial roles in the mitochondrial quality control response system. The past years have provided much structural insight into the molecular mechanisms associated with degradation of substrates by these proteolytic machines. Recent cryo-electron microscopy (cryo-EM) studies have provided critical insights into a conserved, AAA+-mediated hand-over-hand substrate translocation mechanism required to processively engage, unfold, and degrade proteolytic substrates. However, the underlying mechanisms regulating their various activities are not well understood. Numerous prior studies suggest that AAA+ protease have evolved numerous layers of regulation to control or tune proteolytic activity to meet cellular needs. Herein, we present compelling biochemical and structural data that support a long-range allosteric model linking substrate binding to proteolytic activity.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0I120
  13. FASEB J. 2022 May;36 Suppl 1
      Mitochondria produce over 90% of cellular ATP and actively participate in maintaining ion homeostasis, redox status, lipid metabolism, and cell growth. Changes in the matrix volume of mitochondria affect their functional and structural integrity. Modest volume increases associated with modulation of the inner mitochondrial membrane can activate electron transfer chain and oxidative phosphorylation, whereas excessive swelling impairs structural organization of the membrane and initiate mitochondria-mediated cell death mechanisms. Therefore, clarifying the precise mechanisms of excessive mitochondrial swelling is important for regulation of mitochondria-mediated cell death pathways in response to energy and oxidative stresses. Opening of non-selective mitochondrial permeability transition pores (mPTP) is the primary cause of excessive matrix swelling. The molecular identity remains unknown and recent studies suggest the existence of two or more types of mPTP that can be composed of different protein(s). The adenine nucleotide translocator (ANT) and FO F1 -ATP synthase were proposed to be potential mPTP core components that can act together or independently each other. Here, we elucidated the role of ANT in mPTP opening by applying both experimental and computational approaches. mPTP opening was evaluated in cardiac mitochondria that were exposed to moderate and high Ca2+ concentrations in the absence and presence of respiratory substrates and ADP. We developed a detailed model of the ANT transport mechanism including the matrix (ANTM ), cytosolic (ANTC ), and pore (ANTP ) states of the transporter that was able to simulate our experimental data. In addition, we corroborated and simulated our ANT model based on previous ANT kinetics data. The model was successful not only in simulating ANT pore state transition, but also explained the potential role of ANT in mPTP opening in cardiac mitochondria.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5305
  14. FASEB J. 2022 May;36 Suppl 1
      Mitochondria-early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE-mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti-import) and high ferroportin (FPN) (pro-export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple-negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome-mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE-mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re-expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro-Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE-mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE-mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1-mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin-dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re-expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re-expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE-mitochondria interactions to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cell migration. Thus, we propose DMT1 as a key player associated with aggressive phenotypes in breast cancer.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R5276
  15. FASEB J. 2022 May;36 Suppl 1
      Mitochondrial oxidative phosphorylation plays a significant role in cellular functions such as nutrient metabolism, ATP synthesis, and respiratory capacity. Mitochondria's branching network, the reticulum, is active through the fusion (connection) and fission (fragmentation) dynamics. As humans age, however, there is a loss of mitochondrial fusion which increases fragmentation and this results in the loss of mitochondrial function. Therefore, the purpose of this study is to investigate mitochondrial dysfunction in human skeletal muscle derived cells (SkM). The discrepancies of the oxygen consumption rate (OCR) of the mitochondria between the young versus old human cells may reflect the driving factor behind aging. In order to determine mitochondrial dysfunction, OCR was measured between a primary human SkM from an 18 year-old-male (18M) and 66 year old male (66M) were purchased from Cook MyoSite Inc. (Pittsburgh, PA). OCR was measured using the Cell Mito Stress Test by SeaHorse Analytics XFp Analyzer (Agilent Technologies; Santa Clara, CA). Data generated by Seahorse Report Generator (Mean±SEM). Basal OCR and Maximal OCR were higher in 18M compared to 66M (Basal: 28.51 ± 1.61 and 20.43 ± 2.18; Maximal: 54.98 ± 6.74 and 28.68 ± 3.91 pmol/min). In addition, the results reflected that ATP production as well as Spare Respiratory Capacity (SRC) were higher in 18M compared to 66M (ATP Production: 23.88 ± 1.37 and 16.84 ± 2.04; SRC: 26.47 ± 5.13 and 8.25 ± 1.73 pmol/min). Basal OCR, Maximal OCR, ATP Production, and SRC of the 18M cells were higher than those of the 66M cells, revealing a greater mitochondrial function in the primary skeletal muscle-derived cells derived from the young compared to the old. This could be accounted for by the fact that the young cells have a higher stress adaptability compared to the old cells. The discrepancies in the OCR between the young versus the old human cells, therefore, augment our understanding of how mitochondrial dysfunction may serve as a driving force behind aging in humans. Combining data from additional samples to be obtained, this study will elucidate the various mechanisms that propel age-related mitochondrial dysfunction and provide crucial information to prevent skeletal muscle pathologies. Therefore, this project will unveil the effects of aging on skeletal muscle mitochondrial dysfunction and mitigate age-related muscle pathologies.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2665
  16. FASEB J. 2022 May;36 Suppl 1
      Mitochondria undergo permeability transition (PT) resulting in the opening of the non-selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass <1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+ -sensitive fluorescence probe Calcium Green-5N. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin-permeabilized cells was higher than the CRC of digitonin-permeabilized cells. In addition, the mitochondrial CRC of saponin-permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin-permeabilized cells has more advantages than isolated mitochondria.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4561
  17. FASEB J. 2022 May;36 Suppl 1
       BACKGROUND: Sarcopenic obesity is a highly prevalent disease with poor survival and ineffective medical interventions. Mitochondrial dysfunction is purported to be central in the pathogenesis of sarcopenic obesity by impairing both organelle biogenesis and quality control. We have previously identified an orally available mitochondrial-targeted furazano[3,4-b]pyrazine named BAM15 that selectively lowers respiratory coupling efficiency and protects against diet-induced obesity in mice. Here, we tested the hypothesis that mitochondrial uncoupling simultaneously attenuates loss of muscle function and weight gain in a mouse model of sarcopenic obesity.
    METHODS: 80-week-old male C57BL/6J mice with obesity were randomized to 10 weeks of high fat diet (CTRL) or BAM15 (BAM15; 0.1% w/w in high fat diet) treatment. Body composition, muscle function, energy expenditure, and locomotor activity were determined after treatment. Skeletal muscle was harvested and evaluated for histology, gene expression, protein signaling, and mitochondrial structure and function.
    RESULTS: BAM15 decreased body weight (~25% reduction, P<0.001) which was attributable to increased energy expenditure (~20% increment, P<0.001). BAM15 increased muscle mass (~13% increment, P<0.001), strength (~37% increment, P<0.0001), and locomotor activity (~25% increment, P<0.001). Improvements in physical function were mediated in part by reductions in skeletal muscle inflammation (IL-6 and gp130, both P<0.05), enhanced mitochondrial function, and improved endoplasmic reticulum homeostasis and reduced inflammation. Specifically, BAM15 activated mitochondrial quality control through AMPK (PINK1-ubiquitin binding and LC3II, P<0.01), increased electron transport chain activity (citrate synthase and complex II activity, all P<0.05), restricted endoplasmic reticulum (ER) misfolding (decreased oligomer A11 insoluble/soluble ratio, P<0.0001) while limiting ER stress (decreased PERK signaling, P<0.0001), apoptotic signaling (decreased cytochrome C release and Caspase-3/9 activation, all P<0.001), and muscle protein degradation (decreased 14-kDa actin fragment insoluble/soluble ratio, P<0.001).
    CONCLUSIONS: Mitochondrial uncoupling agents such as BAM15 may mitigate age-related decline in muscle mass and function by molecular and cellular bioenergetic adaptations that confer protection against sarcopenic obesity through activation of mitochondrial quality control and attenuation of ER stress.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L7532
  18. FASEB J. 2022 May;36 Suppl 1
      The asymmetric distribution of phospholipids in membranes is a fundamental principle of cellular compartmentalization and organization and is a crucial factor regulating organelle shape and biogenesis. Phosphatidylethanolamine (PE), a nonbilayer phospholipid that generates negative membrane curvature, has many cellular roles, including regulating mitochondrial architecture and lipidation of the autophagy factor LC3. Previously, we demonstrated in budding yeast that the PE synthase Psd1, which canonically operates on the mitochondrial inner membrane, unexpectedly also localizes to the ER. However, it has been unclear what the role of ER-localized Psd1 is. We now resolve this mystery and demonstrate that ER-localized Psd1 is a critical regulator of lipid droplet (LD) biogenesis at ER subdomains. While it is constitutively targeted to the ER membrane, Psd1 transiently concentrates on the ER to LD attachment sites specifically during stimulated LD biogenesis. Using an inducible LD biogenesis assay, we demonstrate that ER-localized Psd1 is required for normal LD formation. Further, we identify a LD binding motif on Psd1 and show that this motif is required for Psd1 to influence LD morphogenesis. We also find that the role of phosphatidylserine decarboxylase (PSD) enzymes in LD formation is conserved in fission yeast, though occurs through a distinct targeting mechanism than in budding yeast. Thus, we have identified PSD enzymes as novel regulators of LDs and demonstrate that both mitochondria and LDs are organized and shaped by the spatial positioning of a single PE synthesis enzyme.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R850
  19. FASEB J. 2022 May;36 Suppl 1
      A gradual breakdown in the cellular networks that assure protein stability, termed proteostasis, contributes significantly to the accumulation of misfolded and aggregated proteins that severely compromises cellular function later in life. A progressive decline in the efficiency of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) causes cells to become particularly susceptible to disturbances affecting the biogenesis, folding, stability, and turnover of membrane proteins. While the dynamics of ER stress response mechanisms during aging have been well-characterized, less is understood about how the function of other organelles important for membrane protein biogenesis and turnover might change over time. We hypothesized that age-dependent changes in the function of the Golgi and endosomes could contribute to the collapse in proteostasis during adulthood. Using a reverse genetic approach in Caenorhabditis elegans, we probed the function of these two compartments by knocking down the expression of cogc-2, a member of the Conserved Oligomeric Golgi (COG) complex and of rme-8, encoding an endosomal protein important for the retrieval of Golgi residents and the recycling of plasma membrane proteins. A key component of the C. elegans innate immune response is the secretion of immune effector proteins into the intestinal lumen, which can itself trigger the ER stress response due to the increased flux through the secretory pathway. Therefore, as a proxy for the integrity of vesicular transport pathways we measured the survival of juvenile and adult animals infected with the bacterial pathogen Pseudomonas aeruginosa following RNAi treatments targeting cogc-2 or rme-8. While there was a negligible effect on late larval stage animals, reduced expression of cogc-2 and rme-8 enhanced the sensitivity of post-reproductive adult animals to infection. This suggests that components of vesicular transport pathways downstream of the ER could also contribute to proteostasis in an age-dependent manner through their roles in ensuring the proper localization of membrane proteins. Follow-up studies will use in vivo fluorescent reporters of protein stability to determine whether interfering with protein transport through the Golgi and endosomes accelerates the decline in proteostasis in older animals. Our studies imply that organelles involved in late steps of membrane protein biogenesis and stability play increasingly important roles in maintaining the proteome during aging.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L7998
  20. FASEB J. 2022 May;36 Suppl 1
      Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax ) in isolated human airway smooth muscle (hASM) cells using 1-methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax , the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil-immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP-mediated disruption of the mitochondrial proton gradient does not affect SDHmax , while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3283
  21. FASEB J. 2022 May;36 Suppl 1
       BACKGROUND: The accumulation of senescent cells is a hallmark of aging in the skeletal muscle which causes reactive oxygen species (ROS) and mitochondrial dysfunction. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that clearance of p21high cells enhances muscle function including grip strength, hanging endurance, and maximal walking speed in mice. However, it is still unclear how the muscle performance enhances through the clearance of p21high cells. One of the potential mechanisms is mitochondrial function and/or mitochondria-derived ROS. Therefore, this study investigated the linkage between mitochondria and p21high cells in aging or high fat diet-induced muscle dysfunction.
    METHODS: Five p21-Cre/+; +/+ (P) and p21-Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Mitochondrial respiration was measured, by high-resolution respirometry, in permeabilized muscle fibers from the soleus muscle. Mitochondria-derive ROS production was determined using Amplex Red assays. One-way ANOVAs with Bonferroni post-hoc were used to determine differences between groups (P < 0.05).
    RESULTS: Reductions in complex I + II state 3 respiration were observed in p21-Cre mice with high fat diet but the clearance of p21high cells using tamoxifen enhanced complex I + II state 3 respiration (Lean P: 35.2 ± 3.13 pmol·s-1 ·mg-1 ; Obese P: 17.45 ± 2.85 pmol·s-1 ·mg-1 ; Obese PD: 30.04 ± 3.19 pmol·s-1 ·mg-1 ; P < 0.05). State 4 respiration did not differ between groups (P > 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased in high fat diet group but clearing p21high cells restored respiratory function (Lean P: 4.20 ± 1.18; Obese P: 2.21 ± 1.11; Obese PD: 3.85 ± 1.60; P<0.05). Also, high fat diet increased mitochondrial ROS production, but tamoxifen treatment attenuated ROS production (Lean P: 10.16 ± 0.41 pmol·s-1 ·mg-1 ; Obese P: 26.83 ± 0.54 pmol·s-1 ·mg-1 ; Obese PD: 12.23 ± 0.54 pmol·s-1 ·mg-1 ; P<0.05).
    CONCLUSION: These results demonstrated that p21high cells may play a causal role in mitochondrial dysfunction and ROS emission in the skeletal muscle with obesity and other chronic diseases. Therefore, targeting p21high cells may enhance muscle performance through decreasing mitochondria-derived ROS and enhancing mitochondrial function.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L7969
  22. Nat Commun. 2022 May 13. 13(1): 2673
      The folded mitochondria inner membrane-cristae is the structural foundation for oxidative phosphorylation (OXPHOS) and energy production. By mechanically simulating mitochondria morphogenesis, we speculate that efficient sculpting of the cristae is organelle non-autonomous. It has long been inferred that folding requires buckling in living systems. However, the tethering force for cristae formation and regulation has not been identified. Combining electron tomography, proteomics strategies, super resolution live cell imaging and mathematical modeling, we reveal that the mitochondria localized actin motor-myosin 19 (Myo19) is critical for maintaining cristae structure, by associating with the SAM-MICOS super complex. We discover that depletion of Myo19 or disruption of its motor activity leads to altered mitochondria membrane potential and decreased OXPHOS. We propose that Myo19 may act as a mechanical tether for effective ridging of the mitochondria cristae, thus sustaining the energy homeostasis essential for various cellular functions.
    DOI:  https://doi.org/10.1038/s41467-022-30431-3
  23. Cancer Res. 2022 May 11. pii: canres.3910.2021. [Epub ahead of print]
      Micropeptides are a recently discovered class of molecules that play vital roles in various cellular processes, including differentiation, proliferation, and apoptosis. Here, we sought to identify cancer-associated micropeptides and to uncover their mechanistic functions. A micropeptide named short trans-membrane protein 1 (STMP1) that localizes at the inner mitochondrial membrane was identified to be upregulated in various cancer types and associated with metastasis and recurrence of hepatocellular carcinoma. Both gain- and loss-of-function studies revealed that STMP1 increased dynamin-related protein 1 (DRP1) activation to promote mitochondrial fission and enhanced migration of tumor cells. STMP1 silencing inhibited in vivo tumor metastasis in xenograft mouse models. Overexpression of STMP1 led to redistribution of mitochondria to the leading edge of cells and enhanced lamellipodia formation. Treatment with a DRP1 inhibitor abrogated the promotive effect of STMP1 on mitochondrial fission, lamellipodia formation, and tumor cell migration in vitro and metastasis in vivo. Furthermore, STMP1 interacted with myosin heavy chain 9 (MYH9), the subunit of non-muscle myosin II, and silencing MYH9 abrogated STMP1-induced DRP1 activation, mitochondrial fission, and cell migration. Collectively, this study identifies STMP1 as a critical regulator of metastasis and a novel unit of the mitochondrial fission protein machinery, providing a potential therapeutic target for treating metastases.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-3910
  24. FASEB J. 2022 May;36 Suppl 1
      Ribosome biogenesis occurs in the nucleolus and relies upon RNA Polymerase I (Pol I) transcription of the rDNA into rRNA. This process is critical for normal cells but is dysregulated in diseases such as Alzheimer's disease and cancer. To date, however, we have not yet explored the potential relationship between nucleoli and mitochondria. Mitochondria are critical for cellular energy and mitochondrial disorders exhibit dysregulation of nutrient and energy homeostasis. Like nucleoli, mitochondria are also dysregulated in Alzheimer's disease and cancer, suggesting we may have overlooked a regulatory relationship between these two organelles. In pursuing novel regulators of nucleoli number and function, we have identified a mitochondrial protein that when individually depleted by siRNA in breast epithelial cells (MCF10As) causes a change in nucleolar number - sulfite oxidase (SUOX). Upon SUOX depletion, the nucleolar number decreases from the typical 2-3 nucleoli to 1 nucleolus. SiRNA-mediated SUOX depletion reduces nucleolar rRNA levels in a high-throughput 5-EU assay (Bryant et al. 2021) and reduces global protein translation by a puromycin incorporation assay, indicating defective nucleolar function. Furthermore, combined metabolomic and transcriptomic analyses following siRNA-mediated SUOX depletion reveal disruption in the activated methyl cycle, consistent with aberrant ribosome biogenesis. Experiments are underway to validate defective methylation experimentally at specific steps in ribosome biogenesis. The glutamate cycle is also disrupted, congruent with the presentation of the recessive nervous system disorder Isolated Sulfite Oxidase Deficiency (ISOD). Thus, we have defined a novel mitochondrial regulator of nucleolar number and function, nucleolar rRNA biogenesis, and protein translation with implications in human disease. Our work will open up a new avenue of nucleolus-mitochondria relationships and regulation, and potentially connect mitochondrial disorders with ribosome biogenesis as well as identify how mitochondrial homeostasis is critical for nucleolar function.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4250
  25. Exp Biol Med (Maywood). 2022 May 10. 15353702221094235
      Mitochondria need to interact with the nucleus under homeostasis and stress to maintain cellular demands and nuclear transcriptional programs. Disrupted mitonuclear interaction is involved in many disease processes. However, the role of mitonuclear signaling regulators in endotoxin-induced acute lung injury (ALI) remains unknown. Nicotinamide adenine dinucleotide (NAD+) is closely related to mitonuclear interaction with its central role in mitochondrial metabolism. In the current study, C57BL/6J mice were administrated with lipopolysaccharide 15 mg/kg to induce endotoxin-induced ALI and investigated whether the NAD+ precursor nicotinamide mononucleotide (NMN) could preserve mitonuclear interaction and alleviate ALI. After pretreatment with NMN for 7 days, NAD+ levels in the mitochondrial, nucleus, and total intracellular were significantly increased in endotoxemia mice. Moreover, supplementation of NMN alleviated lung pathologic injury, reduced ROS levels, increased MnSOD activities, mitigated mitochondrial dysfunction, ameliorated the defects in the nucleus morphology, and these cytoprotective effects were accompanied by preserving mitonuclear interaction (including mitonuclear protein imbalance and the mitochondrial unfolded protein response, UPRmt). Furthermore, NAD+-mediated mitonuclear protein imbalance and UPRmt are probably regulated by deacetylase Sirtuin1 (SIRT1). Taken together, our results indicated that NMN pretreatment ameliorated ALI by inducing mitonuclear protein imbalance and activating the UPRmt in an SIRT1-dependent manner.
    Keywords:  Nicotinamide mononucleotide; acute lung injury; lipopolysaccharide; mitochondria; mitochondrial unfolded protein response
    DOI:  https://doi.org/10.1177/15353702221094235
  26. FASEB J. 2022 May;36 Suppl 1
      Eral1 is a GTPase and ribosomal assembly factor of the mitochondrial small subunit (SSU). Eral1 binds 12SrRNA at a critical juncture, and its association and dissociation are necessary for mitoribosome maturation. Previous work in the literature indicated the proteolytic complex mitochondrial ClpXP (mtClpXP) as a regulator of dissociation: mtClpXP physically associates with Eral1 in vivo and knockout of ClpP results in Eral1 stabilization, accumulation of Eral1-bound small subunit precursors, and reduced levels of mature mitoribosomes. In order for mtClpXP to both permit Eral1 association and enact its dissociation from the assembling small subunit, it must either recognize a specific Eral1 conformation that indicates completion of its assembly factor function or precisely tune Eral1 levels to achieve a metastable state. We aim to describe the recognition strategy that mtClpXP utilizes to unfold and degrade Eral1 and enable ribosome assembly. We have demonstrated that mtClpXP directly unfolds and degrades Eral1 in vitro. Evolutionary coupling analysis between human Eral1 and mtClpX has pointed to select residues that may mediate the interaction between these proteins. We have shown that mutation of at least one such Eral1 residue blocks its unfolding and degradation. We are utilizing a peptide SPOT array to characterize additional Eral1 motifs that assist in recognition and unfolding by mtClpX and hypothesize that the exposure of these sequences aligns with the Eral1 state that is recognized by mtClpX.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.L7724
  27. Int J Mol Sci. 2022 May 01. pii: 5039. [Epub ahead of print]23(9):
      Mitochondrial Complex II is composed of four core subunits and mutations to any of the subunits result in lowered Complex II activity. Surprisingly, although mutations in any of the subunits can yield similar clinical outcomes, there are distinct differences in the patterns of clinical disease most commonly associated with mutations in different subunits. Thus, mutations to the SdhA subunit most often result in mitochondrial disease phenotypes, whilst mutations to the other subunits SdhB-D more commonly result in tumour formation. The reason the clinical outcomes are so different is unknown. Here, we individually antisense-inhibited three of the Complex II subunits, SdhA, SdhB or SdhC, in the simple model organism Dictyostelium discoideum. Whilst SdhB and SdhC knockdown resulted in growth defects on bacterial lawns, antisense inhibition of SdhA expression resulted in a different pattern of phenotypic defects, including impairments of growth in liquid medium, enhanced intracellular proliferation of the bacterial pathogen Legionella pneumophila and phagocytosis. Knockdown of the individual subunits also produced different abnormalities in mitochondrial function with only SdhA knockdown resulting in broad mitochondrial dysfunction. Furthermore, these defects were shown to be mediated by the chronic activation of the cellular energy sensor AMP-activated protein kinase. Our results are in agreement with a role for loss of function of SdhA but not the other Complex II subunits in impairing mitochondrial oxidative phosphorylation and they suggest a role for AMP-activated protein kinase in mediating the cytopathological outcomes.
    Keywords:  Complex II; Dictyostelium discoideum; mitochondria; succinate dehydrogenase
    DOI:  https://doi.org/10.3390/ijms23095039
  28. Commun Biol. 2022 May 12. 5(1): 453
      Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1-8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson's disease.
    DOI:  https://doi.org/10.1038/s42003-022-03389-7