bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022‒01‒09
28 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

  1. Blood Adv. 2022 Jan 04. pii: bloodadvances.2021005609. [Epub ahead of print]
      Iron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase Six-transmembrane epithelial antigen of the prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CrispR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and Tfrc1 suggesting cells were iron limited. We show transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89 supporting a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89, led to increased intracellular UTI89 proliferation suggesting that the endolysosomal system is retaining Fe3+ that can be used for intravesicular pathogen proliferation. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.
  2. Adv Biol Regul. 2021 Dec 06. pii: S2212-4926(21)00072-5. [Epub ahead of print] 100854
      Aberrant signaling of mechanistic target of rapamycin (mTOR' aka mammalian target of rapamycin) is shown to be linked to tumorigenesis of numerous malignancies including glioblastoma (GB). Glioblastoma mTOR is a serine threonine kinase that functions by forming two multiprotein complexes. There complexes are named mTORC1 and mTORC2 and downstream activated substrate execute cellular and metabolic functions. This signaling cascade of PI3K/AKT/mTOR is often upregulated due to frequent loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. Since rapamycin and it's analogue are less effective in treatment of GB, we used novel ATP-competitive dual inhibitors of mTORC1 and mTORC2, namely, Torin1, Torin2, and XL388. Torin2 caused a concentration dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6KSer235/236 and 4E-BP1Thr37/46 as well as the mTORC2 substrate AKTSer473 resulting in suppression of tumor cell proliferation and migration. Torin1 showed similar effects only at higher doses. Another small molecule compound, XL388 suppressed cell proliferation at a higher dose but failed to inhibit cell migration. Torin1 suppressed phosphorylation of PRAS40Thr246, however Torin2 completely abolished it. XL388 treatment inhibited the phosphorylation of PRAS40Thr246 at higher doses only. These findings underscore the use of novel compounds in treatment of cancer. In addition, formulation of third generation mTOR inhibitor "Rapalink-1" may provide new aspects to target mTOR pathways. Numerous inhibitors are currently being used in clinical trials that are aimed to target activated mTOR pathways.
    Keywords:  Glioblastoma; mTOR; mTORC1 mTORC2
  3. J Cell Sci. 2022 Jan 04. pii: jcs.258687. [Epub ahead of print]
      The human Apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found in cells stably expressing each APOE isoform that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-golgi compartment is degraded by autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that may contribute to AD pathogenesis.
    Keywords:  APOE; APOE4; Alzheimer's disease; Chaperone-mediated autophagy; LC3-associated endocytosis
  4. Front Cell Dev Biol. 2021 ;9 821719
    Keywords:  FUS; IL6-receptor; PLA2; PX domains; TOM1; endosomes; lysosomes; v-ATPase
  5. Mol Ther Methods Clin Dev. 2022 Mar 10. 24 71-87
      More than 50 lysosomal storage diseases (LSDs) are associated with lysosomal dysfunctions with the frequency of 1:5,000 live births. As a result of missing enzyme activity, the lysosome dysfunction accumulates undegraded or partially degraded molecules, affecting the entire body. Most of them are life-threatening diseases where patients could die within the first or second decade of life. Approximately 20 LSDs have the approved treatments, which do not provide the cure for the disorder. Therefore, the delivery of missing genes through gene therapy is a promising approach for LSDs. Over the years, ex vivo lentiviral-mediated gene therapy for LSDs has been approached using different strategies. Several clinical trials for LSDs are under investigation.Ex vivo lentiviral-mediated gene therapy needs optimization in dose, time of delivery, and promoter-driven expression. Choosing suitable promoters seems to be one of the important factors for the effective expression of the dysfunctional enzyme. This review summarizes the research on therapy for LSDs that has used different lentiviral vectors, emphasizing gene promoters.
    Keywords:  gene promoter; gene therapy; lentivirus; lysosomal storage disease; stem cell therapy
  6. Acta Physiol (Oxf). 2022 Jan 03. e13757
      Coordinated action among various organelles maintain cellular functions. For instance, mitochondria and lysosomes are the main organelles contributing to cellular metabolism and provide energy for cardiomyocyte contraction. They also provide essential signaling platforms in the cell that regulate many key processes such as autophagy, apoptosis, oxidative stress, inflammation, and cell death. Often, abnormalities in mitochondrial or lysosomal structures and functions bring about cardiovascular disease (CVD). Although the communication between mitochondria and lysosomes throughout the cardiovascular system is intensely studied, the regulatory mechanisms have not been completely understood. We summarize the most recent studies related to mitochondria and lysosomes' role in CVDs and their potential connections and communications under cardiac pathophysiological conditions. Further we discuss limitations and future perspectives regarding diagnosis, therapeutic strategies, and drug discovery in CVDs.
    Keywords:  autophagy; cardiovascular disease; interaction; lysosome; mechanism; metabolism; mitochondrion
  7. Front Cell Dev Biol. 2021 ;9 798262
      For a long time, lysosomes were considered as mere waste bags for cellular constituents. Thankfully, studies carried out in the past 15 years were brimming with elegant and crucial breakthroughs in lysosome research, uncovering their complex roles as nutrient sensors and characterizing them as crucial multifaceted signaling organelles. This review presents the scientific knowledge on lysosome physiology and functions, starting with their discovery and reviewing up to date ground-breaking discoveries highlighting their heterogeneous functions as well as pending questions that remain to be answered. We also review the roles of lysosomes in anti-cancer drug resistance and how they undergo a series of molecular and functional changes during malignant transformation which lead to tumor aggression, angiogenesis, and metastases. Finally, we discuss the strategy of targeting lysosomes in cancer which could lead to the development of new and effective targeted therapies.
    Keywords:  BORC; TFEB; autophagy; cancer; endocytosis; lysosome; mTOR; reformation
  8. Autophagy. 2022 Jan 05. 1-16
      Barth syndrome (BTHS) is an X-linked genetic disorder caused by mutations in the TAFAZZIN/Taz gene which encodes a transacylase required for cardiolipin remodeling. Cardiolipin is a mitochondrial signature phospholipid that plays a pivotal role in maintaining mitochondrial membrane structure, respiration, mtDNA biogenesis, and mitophagy. Mutations in the TAFAZZIN gene deplete mature cardiolipin, leading to mitochondrial dysfunction, dilated cardiomyopathy, and premature death in BTHS patients. Currently, there is no effective treatment for this debilitating condition. In this study, we showed that TAFAZZIN deficiency caused hyperactivation of MTORC1 signaling and defective mitophagy, leading to accumulation of autophagic vacuoles and dysfunctional mitochondria in the heart of Tafazzin knockdown mice, a rodent model of BTHS. Consequently, treatment of TAFAZZIN knockdown mice with rapamycin, a potent inhibitor of MTORC1, not only restored mitophagy, but also mitigated mitochondrial dysfunction and dilated cardiomyopathy. Taken together, these findings identify MTORC1 as a novel therapeutic target for BTHS, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for BTHS.Abbreviations: BTHS: Barth syndrome; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CL: cardiolipin; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; KD: knockdown; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LV: left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; OCR: oxygen consumption rate; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB/S6K: ribosomal protein S6 kinase beta; SQSTM1/p62: sequestosome 1; TLCL: tetralinoleoyl cardiolipin; WT: wild-type.
    Keywords:  BTHS; MTORC1; TAFAZZIN; cardiolipin; mitophagy; rapamycin
  9. Alzheimers Dement. 2022 Jan 03.
      Increased activation of the contact system protein high molecular weight kininogen (HK) has been shown in plasma and cerebrospinal fluid of Alzheimer's disease (AD) patients, but its potential role in the brain has not been explored. We assessed HK levels in brain tissue from 20 AD patients and controls and modeled the effects of HK on microglia-like cells in culture. We show increased levels of HK in the hippocampus of AD patients, which colocalized with amyloid beta (Aβ) deposits and activated microglia. Treatment of microglia with HK led to cell clustering and elevated levels of phagocytosed Aβ. We demonstrate that microglia internalize HK and traffic it to lysosomes, which is accompanied by reduced activity of lysosomal cathepsins L and S. Our results suggest that HK accumulation in the AD hippocampus may alter microglial uptake and degradation of Aβ fibrils, possibly contributing to microglial dysfunction in AD.
    Keywords:  Alzheimer's disease; cathepsin; high molecular weight kininogen; microglia; phagocytosis
  10. Proc Natl Acad Sci U S A. 2022 Jan 11. pii: e2115261119. [Epub ahead of print]119(2):
      The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin-Benson-Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.
    Keywords:  CO2; Chlamydomonas; TOR kinase; amino acids
  11. Exp Cell Res. 2022 Jan 03. pii: S0014-4827(21)00563-2. [Epub ahead of print] 113007
      Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare, lysosomal disorder caused by mutations in a gene encoding iduronate-2-sulfatase (IDS). IDS deficiency results in an accumulation of glycosaminoglycans (GAGs) and secondary accumulations of other lipids in lysosomes. Symptoms of MPS II include a variety of soft and hard tissue problems, developmental delay, and deterioration of multiple organs. Enzyme replacement therapy is an approved treatment for MPS II, but fails to improve neuronal symptoms. Cell-based neuronal models of MPS II disease are needed for compound screening and drug development for the treatment of the neuronal symptoms in MPS II. In this study, three induced pluripotent stem cell (iPSC) lines were generated from three MPS II patient-derived dermal fibroblast cell lines that were differentiated into neural stem cells and neurons. The disease phenotypes were measured using immunofluorescence staining and Nile red dye staining. In addition, the therapeutic effects of recombinant human IDS enzyme, delta-tocopherol (DT), and hydroxypropyl-beta-cyclodextrin (HPBCD) were determined in the MPS II disease cells. Finally, the neural stem cells from two of the MPS II iPSC lines exhibited typical disease features including a deficiency of IDS activity, abnormal glycosaminoglycan storage, and secondary lipid accumulation. Enzyme replacement therapy partially rescued the disease phenotypes in these cells. DT showed a significant effect in reducing the secondary accumulation of lipids in the MPS II neural stem cells. In contrast, HPBCD displayed limited or no effect in these cells. Our data indicate that these MPS II cells can be used as a cell-based disease model to study disease pathogenesis, evaluate drug efficacy, and screen compounds for drug development.
    Keywords:  Delta-tocopherol; Hunter syndrome; Hydroxypropyl-beta-cyclodextrin; Induced pluripotent stem cells; MPS II; Mucopolysaccharidosis type II; Neural stem cells
  12. ACS Nano. 2022 Jan 03.
      Rapid and efficient isolation of intact lysosomes is necessary to study their functions and metabolites by proteomic analysis. We developed a swift and robust nanoparticle-based magnetic separation method in which magnetic-plasmonic hybrid nanoparticles (MPNPs) conjugated with amino dextran (aDxt) were targeted to the lumen of lysosomes via the endocytosis pathway. For well-directed magnetic separation of the lysosomes, it is important to trace the intracellular trafficking of the aDxt-conjugated MPNPs (aDxt-MPNPs) in the endocytosis pathway. Therefore, we analyzed the intracellular transport process of the aDxt-MPNPs by investigating the time-dependent colocalization of plasmonic scattering of aDxt-MPNPs and immunostained marker proteins of organelles using the threshold Manders' colocalization coefficient (Rt). Detailed analysis of time variations of Rt for early and late endosomes and lysosomes allowed us to derive the transport kinetics of aDxt-MPNPs in a cell. After confirming the incubation time required for sufficient accumulation of aDxt-MPNPs in lysosomes, the lysosomes were magnetically isolated as intact as possible. By varying the elapsed time from homogenization to complete isolation of lysosomes (tdelay) and temperature (T), the influences of tdelay and T on the protein composition of the lysosomes were investigated by polyacrylamide gel electrophoresis and amino acid analysis. We found that the intactness of lysosomes could become impaired quite quickly, and to isolate lysosomes as intact as possible with high purity, tdelay = 30 min and T = 4 °C were optimal settings.
    Keywords:  intracellular trafficking; lysosome; magnetic separation; nanoparticle; organelle; plasmonic imaging
  13. J Cardiovasc Pharmacol. 2021 Dec 27.
      ABSTRACT: The abnormal proliferation of vascular smooth muscle cells (VSMCs) is a key pathological characteristic of vascular proliferative diseases. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays an important role in regulating cell growth, motility, proliferation, and survival, as well as gene expression in response to hypoxia, growth factors, and nutrients. Increasing evidence shows that mTOR also regulates VSMC proliferation in vascular proliferative diseases and that mTOR inhibitors, such as rapamycin, effectively restrain VSMC proliferation. However, the molecular mechanisms linking mTOR to vascular proliferative diseases remain elusive. In our review, we summarize the key roles of the mTOR and the recent discoveries in vascular proliferative diseases, focusing on the therapeutic potential of mTOR inhibitors to target mTOR signaling pathway for the treatment of vascular proliferative diseases. Herein, we discuss mTOR inhibitors as promising candidates to prevent VSMC-associated vascular proliferative diseases.
  14. Cell Rep. 2022 Jan 04. pii: S2211-1247(21)01687-9. [Epub ahead of print]38(1): 110187
      Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.
    Keywords:  ALG-2; Candida albicans; ESCRT; calcium; candidalysin; epithelia; lysosome; membrane damage; plasma membrane; repair
  15. Front Med (Lausanne). 2021 ;8 743150
      Autosomal recessive polycystic kidney disease (ARPKD) is a severe renal cystic disease caused mainly by the polycystic kidney and hepatic disease 1 (PKHD1). However, the genetic cause, pathologic features, and mechanism of action of ARPKD are not well known. Here, we identified a family with ARPKD. Two siblings harbored biallelic variants in PKHD1 (c.7205G>A, c.7973T>A). We determined that the "de novo" variant, c.7205G>A, arose from the mosaicism of the father and had a 7.4% level. Pathologic characterization, using biopsy analysis, was evidenced with predominant cystic dilation in proximal tubules, slight ectasia of collecting ducts, defective ciliogenesis, and impaired cell-cell junctions in renal tubules and collecting ducts. Exosome proteomics in the urine from patients with ARPKD were markedly different from those of controls, with the most significant alterations occurring in mitochondrial and lysosomal proteins. Expression of the proteins of OXPHOS was downregulated sharply, in parallel with upregulated expression of the proteins involved in glycolysis in patients with ARPKD. Several lysosomal proteins associated with renal lesions were more abundant in the exosome of the patient than in controls. Moreover, the lysosomal enzyme sulfamidase, which is produced by the SGSH gene, was abrupt uniquely in the exosome of the patient. Consistently, swollen mitochondria and abundant lysosomes were visualized in the mutant tubular epithelial cells of patients with mutant PKHD1. Collectively, these findings provide new insights on the pathophysiology of the polycystic kidney due to PKHD1 deficiency. PKHD1 mosaicism should be considered in genetic testing of ARPKD patients.
    Keywords:  ARPKD; PKHD1; exosome; lysosome; mitochondria; mosaicism
  16. Biochim Biophys Acta Mol Cell Biol Lipids. 2022 Jan 04. pii: S1388-1981(21)00235-3. [Epub ahead of print] 159107
      Phosphoinositides are a family of signaling lipids that play a profound role in regulating protein function at the membrane-cytosol interface of all cellular membranes. Underscoring their importance, mutations or alterations in phosphoinositide metabolizing enzymes lead to host of developmental, neurodegenerative, or and metabolic disorders that are devastating for human health. In addition to lipid enzymes, phosphoinositide metabolism is regulated and controlled at membrane contact sites (MCS). Regions of close opposition typically between the ER and other cellular membranes, MCS are non-vesicular lipid transport portals that engage in extensive communication to influence organelle homeostasis. This review focuses on lipid transport, specifically phosphoinositide lipid transport and metabolism at MCS.
    Keywords:  Cholesterol; Extended-synaptotagmins; K(V)2; Lipid transfer; Membrane contact sites; OSBP; PI; PI(4)P; PI(4,5)P(2); Phosphoinositides; TMEM24
  17. Front Cell Infect Microbiol. 2021 ;11 794152
      Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.
    Keywords:  Arf GTPase; Entamoeba histolytica; liver abscess; membrane traffic; stress resistance
  18. Autophagy. 2022 Jan 06. 1-18
      Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D3 and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/β-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1β (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D3 or carbamazepine suppressed pro-inflammatory cytokine expression in vitro. In mice, gavage with TcdB-hyperproducing C. difficile or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D3 and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D3 and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice.
    Keywords:  Autophagic flux; Clostridium difficile; MITF; macrophages; toxin B
  19. Front Pharmacol. 2021 ;12 801234
      Background: The mechanistic target of rapamycin complex 1 (mTORC1) signaling has served as a promising target for therapeutic intervention of major depressive disorder (MDD), but the mTORC1 signaling underlying MDD has not been well elucidated. In the present study, we investigated whether mTORC1 signaling pathway mediates synapse loss induced by chronic stress in the hippocampus. Methods: Chronic restraint stress-induced depression-like behaviors were tested by behavior tests (sucrose preference test, forced swim test and tail suspension test). Synaptic proteins and alternations of phosphorylation levels of mTORC1 signaling-associated molecules were measured using Western blotting. In addition, mRNA changes of immediate early genes (IEGs) and glutamate receptors were measured by RT-PCR. Rapamycin was used to explore the role of mTORC1 signaling in the antidepressant effects of fluoxetine. Results: After successfully establishing the chronic restraint stress paradigm, we observed that the mRNA levels of some IEGs were significantly changed, indicating the activation of neurons and protein synthesis alterations. Then, there was a significant downregulation of glutamate receptors and postsynaptic density protein 95 at protein and mRNA levels. Additionally, synaptic fractionation assay revealed that chronic stress induced synapse loss in the dorsal and ventral hippocampus. Furthermore, these effects were associated with the mTORC1 signaling pathway-mediated protein synthesis, and subsequently the phosphorylation of associated downstream signaling targets was reduced after chronic stress. Finally, we found that intracerebroventricular infusion of rapamycin simulated depression-like behavior and also blocked the antidepressant effects of fluoxetine. Conclusion: Overall, our study suggests that mTORC1 signaling pathway plays a critical role in mediating synapse loss induced by chronic stress, and has part in the behavioral effects of antidepressant treatment.
    Keywords:  chronic restraint stress; depression; fluoxetine; mammalian target of rapamycin; postsynaptic density protein 95
  20. Biochim Biophys Acta Rev Cancer. 2021 Dec 31. pii: S0304-419X(21)00173-6. [Epub ahead of print]1877(1): 188675
      β-galactosylceramidase (GALC) is a lysosomal enzyme that removes β-galactose from β-galactosylceramide, leading to the formation of the oncosuppressor metabolite ceramide. Recent observations have shown that GALC may exert opposite effects on tumor growth by acting as an oncosuppressive or oncogenic enzyme depending on the different experimental approaches, in vitro versus in vivo observations, preclinical versus clinical findings, and tumor type investigated. This review will recapitulate and discuss the contrasting experimental evidence related to the impact of GALC on the biological behavior of cancer and stromal cells and its contribution to tumor progression.
    Keywords:  Ceramide; Galactosylceramidase; Galactosylceramide; Lipidome; Melanoma; Sphingolipids
  21. ACS Chem Neurosci. 2022 Jan 05.
      Neuropeptides, functioning as peptide neurotransmitters and hormones, are generated from proneuropeptide precursors by proteolytic processing at dibasic residue sites (i.e., KR, RK, KK, RR). The cysteine proteases cathepsin L and cathepsin V, combined with the serine proteases proprotein convertases 1 and 2 (PC1/3 and PC2), participate in proneuropeptide processing to generate active neuropeptides. To compare the dibasic cleavage properties of these proteases, this study conducted global, unbiased substrate profiling of these processing proteases using a diverse peptide library in multiplex substrate profiling by mass spectrometry (MSP-MS) assays. MSP-MS utilizes a library of 228 14-mer peptides designed to contain all possible protease cleavage sites, including the dibasic residue sites of KR, RK, KK, and RR. The comprehensive MSP-MS analyses demonstrated that cathepsin L and cathepsin V cleave at the N-terminal side and between the dibasic residues (e.g., ↓K↓R, ↓R↓K, and K↓K), with a preference for hydrophobic residues at the P2 position of the cleavage site. In contrast, the serine proteases PC1/3 and PC2 displayed cleavage at the C-terminal side of dibasic residues of a few peptide substrates. Further analyses with a series of dipeptide-AMC and tripeptide-AMC substrates containing variant dibasic sites with hydrophobic P2 residues indicated the preferences of cathepsin L and cathepsin V to cleave between dibasic residue sites with preferences for flanking hydrophobic residues at the P2 position consisting of Leu, Trp, Phe, and Tyr. Such hydrophobic amino acids reside in numerous proneuropeptides such as pro-NPY and proenkephalin that are known to be processed by cathepsin L. Notably, cathepsin L displayed the highest specific activity that was 10-, 64-, and 1268-fold greater than cathepsin V, PC1/3, and PC2, respectively. Peptide-AMC substrates with dibasic residues confirmed that PC1/3 and P2 cleaved almost exclusively at the C-terminal side of dibasic residues. These data demonstrate distinct dibasic cleavage site properties and a broad range of proteolytic activities of cathepsin L and cathepsin V, compared to PC1/3 and PC2, which participate in producing neuropeptides for cell-cell communication.
    Keywords:  cathepsin; mass spectrometry; neuropeptide; peptidomics; proprotein convertase; protease
  22. Mol Cell Proteomics. 2021 Dec 31. pii: S1535-9476(21)00164-X. [Epub ahead of print] 100192
      The amount of any given protein in the brain is determined by the rates of its synthesis and destruction, which are regulated by different cellular mechanisms. Here, we combine metabolic labelling in live mice with global proteomic profiling to simultaneously quantify both the flux and amount of proteins in mouse models of neurodegeneration. In multiple models, protein turnover increases were associated with increasing pathology. This method distinguishes changes in protein expression mediated by synthesis from those mediated by degradation. In the AppNL-F knockin mouse model of Alzheimer's disease increased turnover resulted from imbalances in both synthesis and degradation, converging on proteins associated with synaptic vesicle recycling (Dnm1, Cltc, Rims1) and mitochondria (Fis1, Ndufv1). In contrast to disease models, aging in wildtype mice caused a widespread decrease in protein recycling associated with a decrease in autophagic flux. Overall, this simple multidimensional approach enables the comprehensive mapping of proteome dynamics and identifies affected proteins in mouse models of disease and other live animal test settings.
    Keywords:  Alzheimer's disease; Protein turnover; Proteomics; SILAM; neurodegeneration
  23. Cell Mol Biol Lett. 2022 Jan 03. 27(1): 2
      Sestrins (Sesns), highly conserved stress-inducible metabolic proteins, are known to protect organisms against various noxious stimuli including DNA damage, oxidative stress, starvation, endoplasmic reticulum (ER) stress, and hypoxia. Sesns regulate metabolism mainly through activation of the key energy sensor AMP-dependent protein kinase (AMPK) and inhibition of mammalian target of rapamycin complex 1 (mTORC1). Sesns also play pivotal roles in autophagy activation and apoptosis inhibition in normal cells, while conversely promoting apoptosis in cancer cells. The functions of Sesns in diseases such as metabolic disorders, neurodegenerative diseases, cardiovascular diseases, and cancer have been broadly investigated in the past decades. However, there is a limited number of reviews that have summarized the functions of Sesns in the pathophysiological processes of human diseases, especially musculoskeletal system diseases. One aim of this review is to discuss the biological functions of Sesns in the pathophysiological process and phenotype of diseases. More significantly, we include some new evidence about the musculoskeletal system. Another purpose is to explore whether Sesns could be potential biomarkers or targets in the future diagnostic and therapeutic process.
    Keywords:  Biological functions; Biomarker; Human diseases; Musculoskeletal system disease; Sestrins; Therapeutic target
  24. iScience. 2022 Jan 21. 25(1): 103574
      Heart disease is the leading cause of death with no method to repair damaged myocardium due to the limited proliferative capacity of adult cardiomyocytes. Curiously, mouse neonates and zebrafish can regenerate their hearts via cardiomyocyte de-differentiation and proliferation. However, a molecular mechanism of why these cardiomyocytes can re-enter cell cycle is poorly understood. Here, we identify a unique metabolic state that primes adult zebrafish and neonatal mouse ventricular cardiomyocytes to proliferate. Zebrafish and neonatal mouse hearts display elevated glutamine levels, predisposing them to amino-acid-driven activation of TOR, and that TOR activation is required for zebrafish cardiomyocyte regeneration in vivo. Through a multi-omics approach with cellular validation we identify metabolic and mitochondrial changes during the first week of regeneration. These data suggest that regeneration of zebrafish myocardium is driven by metabolic remodeling and reveals a unique metabolic regulator, TOR-primed state, in which zebrafish and mammalian cardiomyocytes are regeneration competent.
    Keywords:  Biological sciences; Cell biology; Tissue Engineering
  25. Mol Cell. 2022 Jan 06. pii: S1097-2765(21)01077-7. [Epub ahead of print]82(1): 60-74.e5
      Acetyl-CoA is a key intermediate situated at the intersection of many metabolic pathways. The reliance of histone acetylation on acetyl-CoA enables the coordination of gene expression with metabolic state. Abundant acetyl-CoA has been linked to the activation of genes involved in cell growth or tumorigenesis through histone acetylation. However, the role of histone acetylation in transcription under low levels of acetyl-CoA remains poorly understood. Here, we use a yeast starvation model to observe the dramatic alteration in the global occupancy of histone acetylation following carbon starvation; the location of histone acetylation marks shifts from growth-promoting genes to gluconeogenic and fat metabolism genes. This reallocation is mediated by both the histone deacetylase Rpd3p and the acetyltransferase Gcn5p, a component of the SAGA transcriptional coactivator. Our findings reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting.
    Keywords:  Gcn5p; Rpd3p; SAGA; acetyl-CoA; environmental stress response; fat metabolism; gluconeogenesis; glucose starvation; histone acetylation; transcription
  26. iScience. 2021 Dec 17. 24(12): 103446
      KLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1/3 transcriptionally regulates Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis combined with the genome-wide transcription-factor-screening method "TFEL scan", using our original Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased via FoxOs by attenuating insulin signaling. Furthermore, FoxOs elevate the expression levels of amino acid catabolic enzymes and suppress SREBP-1c via KLF15, resulting in accelerated amino acid breakdown and suppressed lipogenesis during fasting. Thus, the FoxO-KLF15 pathway contributes to switching the macronutrient flow in the liver under the control of insulin.
    Keywords:  Diabetology; Molecular biology; Molecular network
  27. Biochem J. 2022 Jan 14. 479(1): 1-22
      Cellular function is based on protein-protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation.
    Keywords:  SLiM; kinase; modular domain; phosphatase; protein–protein interactions