bims-mikwok Biomed News
on Mitochondrial quality control
Issue of 2020‒12‒20
eleven papers selected by
Avinash N. Mukkala
University of Toronto

  1. Sci Rep. 2020 Dec 18. 10(1): 22334
      Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play nevertheless a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. A paradigm for this process is the sequestration and autophagic degradation of dysfunctional mitochondria. Although studies have shown that K63-ubiquitylation of mitochondrial proteins by the ubiquitin ligase Parkin is important in this process, it is presently not clear if this modification also suffices to initiate this cascade of events. To address this question, we have engineered the ubiquitin ligase ProxE3, which in an inducible manner synthesizes K63-linked ubiquitin chains on the surface of mitochondria. We found that the presence of K63-linked ubiquitin chains on mitochondria resulted in the recruitment of the ubiquitin adaptor p62 and induced a dramatic redistribution of mitochondria, which was reminiscent to the Parkin-facilitated sequestration in response to mitochondrial uncoupler. However, ProxE3 did not induce autophagic degradation of mitochondria. Our data show that K63-linked ubiquitin chains at the mitochondrial membrane are sufficient for the induction of mitochondrial sequestration, but not mitophagy, without the need of extrinsically inflicting mitochondrial dysfunction.
  2. Methods Mol Biol. 2021 ;2245 215-224
      Immunofluorescence staining is a widely used and powerful tool for the visualization and colocalization of two or more proteins and/or cellular organelles. For colocalization studies in fixed cells, one target protein/organelle is immunostained and visualized by one fluorophore and the other target protein/organelle is immunostained and visualized by a different fluorophore whose excitation emission spectra does not overlap with the first fluorophore. Parkin (PARK2) is an E3 ubiquitin ligase which performs ubiquitination of surface proteins of dysfunctional mitochondria to mark them for autolysosomal degradation. Here we describe the immunofluorescence staining of parkin protein and immunofluorescence or dye-based methods to visualize mitochondria and study the colocalization of parkin and mitochondria in primary human or mouse chondrocytes or cell lines.
    Keywords:  Autophagy; CCCP; Chondrocytes; Confocal microscopy; IL-1β; Immunofluorescence; Inflammation; MitoTracker Deep Red; Mitochondria; Mitochondrial dysfunction; Mitophagy; Osteoarthritis; Oxidative stress; PINK1; Parkin
  3. Cell Calcium. 2020 Nov 26. pii: S0143-4160(20)30163-9. [Epub ahead of print]93 102321
      Mitochondria are autonomous and dynamic cellular organelles orchestrating a diverse range of cellular activities. Numerous cell-signaling pathways target these organelles and Ca2+ is one of the most significant. Mitochondria are able to rapidly and transiently take up Ca2+, thanks to the mitochondrial Ca2+ uniporter complex, as well as to extrude it through the Na+/Ca2+ and H+/Ca2+ exchangers. The transient accumulation of Ca2+ in the mitochondrial matrix impacts on mitochondrial functions and cell pathophysiology. Here we summarize the role of mitochondrial Ca2+ signaling in both physiological (yang) and pathological (yin) processes and the methods that can be used to investigate mitochondrial Ca2+ homeostasis. As an example of the pivotal role of mitochondria in pathology, we described the state of the art of mitochondrial Ca2+ alterations in different pathological conditions, with a special focus on Alzheimer's disease.
    Keywords:  ATP; Alzheimer’s disease; Autophagy; Calcium signaling; Cell death; Cell metabolism; Chemical biosensors; GECI; MCUC; Mitochondria; Presenilins
  4. Methods Mol Biol. 2021 ;2245 195-213
      Chondrocytes, the only cells in articular cartilage, are metabolically active and responsible for the turnover of extracellular matrix and maintenance of the tissue homeostasis. Changes in chondrocyte function can cause degradation of the matrix and loss of articular cartilage integrity, leading to development and progression of osteoarthritis (OA). These changes are exemplified by accumulated mitochondrial damage and dysfunction. Because mitochondria are the critical organelles to produce energy and play a key role in cellular processes, the approaches to assess mitochondrial function under both physiological and pathological conditions enable us to uncover the mechanisms on how dysfunction of mitochondria in chondrocytes mediates signaling pathways that are involved in disturbance of cartilage homeostasis. In this chapter, we describe the methods to evaluate mitochondrial biogenesis, activity and mitochondrial DNA (mtDNA) integrity in chondrocytes.
    Keywords:  Chondrocyte; Mitochondrial DNA; Mitochondrial biogenesis; Mitochondrial transcription factor A; Osteoarthritis; Peroxisome proliferator-activated receptor γ coactivator 1α
  5. Front Cell Dev Biol. 2020 ;8 598112
      The emergence of endosymbiosis between aerobic alpha-proteobacterium and anaerobic eukaryotic cell precursors opened the chapter of eukaryotic evolution. Multiple functions of mitochondria originated from the ancient precursors of mitochondria and underwent remodeling in eukaryotic cells. Due to the dependence on mitochondrial functions, eukaryotic cells need to constantly adjust mitochondrial output based on energy demand and cellular stress. Meanwhile, eukaryotes conduct the metabolic cooperation between different cells through the involvement of mitochondria. Under some conditions, mitochondria might also be transferred to nearby cells to provide a protective mechanism. However, the endosymbiont relationship determines the existence of various types of mitochondrial injury, such as proteotoxic stress, mutational meltdown, oxidative injure, and immune activation caused by released mitochondrial contents. Eukaryotes have a repertoire of mitochondrial optimization processes, including various mitochondrial quality-control proteins, regulation of mitochondrial dynamics and activation of mitochondrial autophagy. When these quality-control processes fail, eukaryotic cells can activate apoptosis to intercept uncontrolled cell death, thereby minimizing the damage to extracellular tissue. In this review, we describe the intracellular and extracellular context-based regulation of mitochondrial output in eukaryotic cells, and introduce new findings on multifaceted quality-control processes to deal with mitochondrial defects.
    Keywords:  apoptosis; eukaryotes; metabolism; mitochondria; mitochondrial dynamics; mitochondrial transfer; mitophagy
  6. Front Physiol. 2020 ;11 543962
      Tumor cells without mitochondrial (mt) DNA (ρ0 cells) are auxotrophic for uridine, and their growth is supported by pyruvate. While ATP synthesis in ρ0 cells relies on glycolysis, they fail to form tumors unless they acquire mitochondria from stromal cells. Mitochondrial acquisition restores respiration that is essential for de novo pyrimidine biosynthesis and for mitochondrial ATP production. The physiological processes that underpin intercellular mitochondrial transfer to tumor cells lacking mtDNA and the metabolic remodeling and restored tumorigenic properties of cells that acquire mitochondria are not well understood. Here, we investigated the changes in mitochondrial and nuclear gene expression that accompany mtDNA deletion and acquisition in metastatic murine 4T1 breast cancer cells. Loss of mitochondrial gene expression in 4T1ρ0 cells was restored in cells recovered from subcutaneous tumors that grew from 4T1ρ0 cells following acquisition of mtDNA from host cells. In contrast, the expression of most nuclear genes that encode respiratory complex subunits and mitochondrial ribosomal subunits was not greatly affected by loss of mtDNA, indicating ineffective mitochondria-to-nucleus communication systems for these nuclear genes. Further, analysis of nuclear genes whose expression was compromised in 4T1ρ0 cells showed that immune- and stress-related genes were the most highly differentially expressed, representing over 70% of those with greater than 16-fold higher expression in 4T1 compared with 4T1ρ0 cells. The monocyte recruiting chemokine, Ccl2, and Psmb8, a subunit of the immunoproteasome that generates MHCI-binding peptides, were the most highly differentially expressed. Early monocyte/macrophage recruitment into the tumor mass was compromised in 4T1ρ0 cells but recovered before mtDNA could be detected. Taken together, our results show that mitochondrial acquisition by tumor cells without mtDNA results in bioenergetic remodeling and re-expression of genes involved in immune function and stress adaptation.
    Keywords:  4T1 model; breast cancer; gene expression; mitochondrial DNA; tumor macrophages
  7. Redox Biol. 2020 Dec 01. pii: S2213-2317(20)31029-6. [Epub ahead of print]38 101824
      Sestrin2 (Sesn2) is a stress-inducible protein that declines with aging in the heart. We reported that rescue Sesn2 levels in aged mouse hearts through gene therapy improves the resistance of aged hearts to ischemia and reperfusion (I/R) insults. We hypothesize that Sesn2 as a scaffold protein maintains mitochondrial integrity to protect heart from ischemic injury during I/R. Young C57BL/6 J (3-6 months), aged C57BL/6 J (24-26 months), and young Sesn2 KO (3-6 months, C57BL/6 J background) mice were subjected to in vivo regional ischemia and reperfusion. The left ventricle was collected for transcriptomics, proteomics and metabolomics analysis. The results demonstrated that Sesn2 deficiency leads to aging-like cardiac diastolic dysfunction and intolerance to ischemia reperfusion stress. Seahorse analysis demonstrated that Sesn2 deficiency in aged and young Sesn2 KO versus young hearts lead to impaired mitochondrial respiration rate with defects in Complex I and Complex II activity. The Sesn2 targeted proteomics analysis revealed that Sesn2 plays a critical role in maintaining mitochondrial functional integrity through modulating mitochondria biosynthesis and assembling of oxidative phosphorylation (OXPHOS) complexes. The RNA-Seq data showed that alterations in the expression of mitochondrial compositional and functional genes and substrate metabolism related genes in young Sesn2 KO and aged versus young hearts. Further immunofluorescence and immunoprecipitation analysis demonstrated that Sesn2 is translocated into mitochondria and interacts with OXPHOS components to maintain mitochondrial integrity in response to I/R stress. Biochemical analysis revealed that Sesn2 is associated with citrate cycle components to modulate pyruvate dehydrogenase and isocitrate dehydrogenase activities during I/R stress. Thus, Sesn2 serves as a scaffold protein interacting with OXPHOS components to maintain mitochondrial integrity under I/R stress. Age-related downregulation of cardiac Sesn2 fragilizes mitochondrial functional integrity in response to ischemic stress.
    Keywords:  Aging; Ischemia reperfusion injury; Metabolism; Mitochondria; Sestrin2
  8. Mitochondrion. 2020 Dec 11. pii: S1567-7249(20)30224-5. [Epub ahead of print]
      Mitochondria play vital role in regulating the cellular energetics and metabolism. Further, it is a signaling hub for cell survival and apoptotic pathways. One of the key determinants that calibrate both cellular energetics and survival functions is mitochondrial calcium (Ca2+) dynamics. Mitochondrial Ca2+ regulates three Ca2+-sensitive dehydrogenase enzymes involved in tricarboxylic acid cycle (TCA) cycle thereby directly controlling ATP synthesis. On the other hand, excessive Ca2+ concentration within the mitochondrial matrix elevates mitochondrial reactive oxygen species (mROS) levels and causes mitochondrial membrane depolarization. This leads to opening of the mitochondrial permeability transition pore (mPTP) and release of cytochrome c into cytosol eventually triggering apoptosis. Therefore, it is critical for cell to maintain mitochondrial Ca2+ concentration. Since cells can neither synthesize nor metabolize Ca2+, it is the dynamic interplay of Ca2+ handling proteins involved in mitochondrial Ca2+ influx and efflux that take the center stage. In this review we would discuss the key molecular machinery regulating mitochondrial Ca2+ concentration. We would focus on the channel complex involved in bringing Ca2+ into mitochondrial matrix i.e. Mitochondrial Ca2+ Uniporter (MCU) and its key regulators Mitochondrial Ca2+ Uptake proteins (MICU1, 2 and 3), MCU regulatory subunit b (MCUb), Essential MCU Regulator (EMRE) and Mitochondrial Ca2+ Uniporter Regulator 1 (MCUR1). Further, we would deliberate on major mitochondrial Ca2+ efflux proteins i.e. Mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) and Leucine zipper EF hand-containing transmembrane1 (Letm1). Moreover, we would highlight the physiological functions of these proteins and discuss their relevance in human pathophysiology. Finally, we would highlight key outstanding questions in the field.
    Keywords:  EMRE; Letm1; MCU complex; MCUR1; MICU 1/2/3; Mitochondrial calcium dynamics; NCLX
  9. EMBO Rep. 2020 Dec 13. e50827
      Many cancer cells maintain enhanced aerobic glycolysis due to irreversible defective mitochondrial oxidative phosphorylation (OXPHOS). This phenomenon, known as the Warburg effect, is recently challenged because most cancer cells maintain OXPHOS. However, how cancer cells coordinate glycolysis and OXPHOS remains largely unknown. Here, we demonstrate that OMA1, a stress-activated mitochondrial protease, promotes colorectal cancer development by driving metabolic reprogramming. OMA1 knockout suppresses colorectal cancer development in AOM/DSS and xenograft mice models of colorectal cancer. OMA1-OPA1 axis is activated by hypoxia, increasing mitochondrial ROS to stabilize HIF-1α, thereby promoting glycolysis in colorectal cancer cells. On the other hand, under hypoxia, OMA1 depletion promotes accumulation of NDUFB5, NDUFB6, NDUFA4, and COX4L1, supporting that OMA1 suppresses OXPHOS in colorectal cancer. Therefore, our findings support a role for OMA1 in coordination of glycolysis and OXPHOS to promote colorectal cancer development and highlight OMA1 as a potential target for colorectal cancer therapy.
    Keywords:  OMA1; colorectal cancer; glycolysis; hypoxia; oxidative phosphorylation
  10. Front Immunol. 2020 ;11 573646
      During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.
    Keywords:  (macroautophagy); autophagy; dendritic cell; hypoxia; hypoxia-inducible factor (HIF)-1α; lipopolysaccharide (LPS)