RESUMEN
Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.
RESUMEN
Multiple processes control quiescence of muscle stem cells (MuSCs), which is instrumental to guarantee long-term replenishment of the stem cell pool. Here, we describe that the G-proteins G12-G13 integrate signals from different G-protein-coupled receptors (GPCRs) to control MuSC quiescence via activation of RhoA. Comprehensive screening of GPCR ligands identified two MuSC-niche-derived factors, endothelin-3 (ET-3) and neurotensin (NT), which activate G12-G13 signaling in MuSCs. Stimulation with ET-3 or NT prevented MuSC activation, whereas pharmacological inhibition of ET-3 or NT attenuated MuSC quiescence. Inactivation of Gna12-Gna13 or Rhoa but not of Gnaq-Gna11 completely abrogated MuSC quiescence, which depleted the MuSC pool and was associated with accelerated sarcopenia during aging. Expression of constitutively active RhoA prevented exit from quiescence in Gna12-Gna13 mutant MuSCs, inhibiting cell cycle entry and differentiation via Rock and formins without affecting Rac1-dependent MuSC projections, a hallmark of quiescent MuSCs. The study uncovers a critical role of G12-G13 and RhoA signaling for active regulation of MuSC quiescence.
RESUMEN
All mitochondria import >95% of their proteins from the cytosol. This process is mediated by protein translocases in the mitochondrial membranes, whose subunits are generally highly conserved. Most eukaryotes have two inner membrane protein translocases (TIMs) that are specialized to import either presequence-containing or mitochondrial carrier proteins. In contrast, the parasitic protozoan Trypanosoma brucei has a single TIM complex consisting of one conserved and five unique subunits. Here, we identify candidates for new subunits of the TIM or the presequence translocase-associated motor (PAM) using a protein-protein interaction network of previously characterized TIM and PAM subunits. This analysis reveals that the trypanosomal TIM complex contains an additional trypanosomatid-specific subunit, designated TbTim15. TbTim15 is associated with the TIM complex, lacks transmembrane domains, and localizes to the intermembrane space. TbTim15 is essential for procyclic and bloodstream forms of trypanosomes. It contains two twin CX9C motifs and mediates import of both presequence-containing and mitochondrial carrier proteins. While the precise function of TbTim15 in mitochondrial protein import is unknown, our results are consistent with the notion that it may function as an import receptor for the non-canonical trypanosomal TIM complex.
Asunto(s)
Mitocondrias , Proteínas de Transporte de Membrana Mitocondrial , Membranas Mitocondriales , Transporte de Proteínas , Proteínas Protozoarias , Trypanosoma brucei brucei , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/enzimología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Subunidades de Proteína/metabolismoRESUMEN
The tripartite attachment complex (TAC) of the single mitochondrion of trypanosomes allows precise segregation of its single nucleoid mitochondrial genome during cytokinesis. It couples the segregation of the duplicated mitochondrial genome to the segregation of the basal bodies of the flagella. Here, we provide a model of the molecular architecture of the TAC that explains how its eight essential subunits connect the basal body, across the mitochondrial membranes, with the mitochondrial genome. We also discuss how the TAC subunits are imported into the mitochondrion and how they assemble to form a new TAC. Finally, we present a comparative analysis of the trypanosomal TAC with open and closed mitotic spindles, which reveals conserved concepts between these diverse DNA segregation systems.
Asunto(s)
Trypanosoma brucei brucei , Trypanosoma , Trypanosoma brucei brucei/genética , Mitocondrias , Trypanosoma/genética , ADN Mitocondrial/genética , Membranas Mitocondriales/metabolismoRESUMEN
The protozoan parasite Trypanosoma brucei and its disease-causing relatives are among the few organisms that barely regulate the transcription of protein-coding genes. Yet, alterations in its gene expression are essential to survive in different host environments. Recently, tRNA-derived RNAs have been implicated as regulators of many cellular processes within and beyond translation. Previously, we identified the tRNAThr-3'-half (AGU) as a ribosome-associated non-coding RNA able to enhance global translation. Here we report that the tRNAThr-3'-half is generated upon starvation inside the mitochondria. The tRNAThr-3'-half associates with mitochondrial ribosomes and stimulates translation during stress recovery, positively affecting mitochondrial activity and, consequently, cellular energy production capacity. Our results describe an organelle ribosome-associated ncRNA involved in translation regulation to boost the central hub of energy metabolism as an immediate stress recovery response.
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Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , ARN de Transferencia de Treonina/metabolismo , Ribosomas/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/genética , ARN de Transferencia/metabolismoRESUMEN
The AAA-ATPase Msp1 extracts mislocalised outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pulldown experiments, we show that trypanosomal Msp1 localises to both glycosomes and the mitochondrial outer membrane, where it forms a complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double, and triple RNAi cell lines combined with proteomic analyses, we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with Msp1. pATOM36 is a functional analog of the yeast mitochondrial import complex complex and possibly of human mitochondrial animal-specific carrier homolog 2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.
Asunto(s)
Proteómica , Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/genética , Membranas Mitocondriales , Proteínas de la Membrana , Complejo de la Endopetidasa Proteasomal , Subunidades de Proteína , ATPasas Asociadas con Actividades Celulares Diversas/genéticaRESUMEN
Background and objectives: In recent years, spinal cord stimulation (SCS) has emerged as a promising management option for chronic pain of multiple etiologies. While its effectiveness has been strongly suggested in many patients, the exact mechanism of action of SCS is incompletely understood. This article reviews the leading mechanisms underlying the analgesic and cardiovascular effects of SCS and reports its novel benefits in a case of recurrent pericarditis. Literature review: Throughout history, the analgesic properties of SCS were thought to arise via stimulation of the spinothalamic tract. Although this mechanism has been thoroughly reported, new research and patient outcomes from SCS have revealed various additional properties that cannot be fully explained by this mechanism alone. Evidence suggests that SCS enhances calcitonin gene-related peptide release and modulates inflammatory cytokine secretion, sympathetic tone, and inhibitory neurotransmitter secretion. These distinct mechanisms likely collectively contribute to the therapeutic effects of SCS on the cardiovascular system and pain management. Case report: We report the case of a 48-year-old male patient with recurrent pericarditis, characterized by refractory angina-like pain and reduced left ventricular ejection fraction (LVEF). After 1 year of having a spinal cord stimulator implanted, the patient is free from pain and narcotics, with a reduction of 428â mg equivalent dose of morphine. The patient's LVEF increased from 40% to 45% without changes to his previous medical treatment. This is the first reported case of refractory pericarditis managed with spinal cord stimulation. Conclusion: Recognizing the improved pain management, reduced narcotic usage, and improved LVEF in our patient following SCS is critical to paving the way toward a complete understanding of the mechanism of action of SCS. This case reveals the therapeutic potential of SCS for cardiovascular pathologies other than refractory angina pectoris.
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Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.
Asunto(s)
Interleucina-6 , ARN , Células Endoteliales/metabolismo , Receptor gp130 de Citocinas , Endotelio/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismoRESUMEN
Consistent with other eukaryotes, the Trypanosoma brucei mitochondrial genome encodes mainly hydrophobic core subunits of the oxidative phosphorylation system. These proteins must be co-translationally inserted into the inner mitochondrial membrane and are synthesized by the highly unique trypanosomal mitoribosomes, which have a much higher protein to RNA ratio than any other ribosome. Here, we show that the trypanosomal orthologue of the mitoribosome receptor Mba1 (TbMba1) is essential for normal growth of procyclic trypanosomes but redundant in the bloodstream form, which lacks an oxidative phosphorylation system. Proteomic analyses of TbMba1-depleted mitochondria from procyclic cells revealed reduced levels of many components of the oxidative phosphorylation system, most of which belong to the cytochrome c oxidase (Cox) complex, three subunits of which are mitochondrially encoded. However, the integrity of the mitoribosome and its interaction with the inner membrane were not affected. Pull-down experiments showed that TbMba1 forms a dynamic interaction network that includes the trypanosomal Mdm38/Letm1 orthologue and a trypanosome-specific factor that stabilizes the CoxI and CoxII mRNAs. In summary, our study suggests that the function of Mba1 in the biogenesis of membrane subunits of OXPHOS complexes is conserved among yeast, mammals and trypanosomes, which belong to two eukaryotic supergroups.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Trypanosoma brucei brucei , Animales , Fosforilación Oxidativa , Trypanosoma brucei brucei/metabolismo , Proteómica , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.
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Copépodos , Neoplasias Pulmonares , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Copépodos/genética , Copépodos/metabolismo , Edición Génica , Genes Reporteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Neoplasias Pulmonares/genética , RatonesRESUMEN
The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the â¼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.
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Replicación del ADN , ADN Mitocondrial , Genoma Mitocondrial , Membranas Mitocondriales , Proteínas Protozoarias , Trypanosoma brucei brucei , Cuerpos Basales/química , ADN Mitocondrial/genética , Epítopos/química , Flagelos/química , Membranas Mitocondriales/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMEN
BACKGROUND: ADAR1 (adenosine deaminase acting on RNA-1)-mediated adenosine to inosine (A-to-I) RNA editing plays an essential role for distinguishing endogenous from exogenous RNAs, preventing autoinflammatory ADAR1 also regulates cellular processes by recoding specific mRNAs, thereby altering protein functions, but may also act in an editing-independent manner. The specific role of ADAR1 in cardiomyocytes and its mode of action in the heart is not fully understood. To determine the role of ADAR1 in the heart, we used different mutant mouse strains, which allows to distinguish immunogenic, editing-dependent, and editing-independent functions of ADAR1. METHODS: Different Adar1-mutant mouse strains were employed for gene deletion or specific inactivation of ADAR1 enzymatic activity in cardiomyocytes, either alone or in combination with Ifih1 (interferon induced with helicase C domain 1) or Irf7 (interferon regulatory factor 7) gene inactivation. Mutant mice were investigated by immunofluorescence, Western blot, RNAseq, proteomics, and functional MRI analysis. RESULTS: Inactivation of Adar1 in cardiomyocytes resulted in late-onset autoinflammatory myocarditis progressing into dilated cardiomyopathy and heart failure at 6 months of age. Adar1 depletion activated interferon signaling genes but not NFκB (nuclear factor kappa B) signaling or apoptosis and reduced cardiac hypertrophy during pressure overload via induction of Irf7. Additional inactivation of the cytosolic RNA sensor MDA5 (melanoma differentiation-associated gene 5; encoded by the Ifih1 gene) in Adar1 mutant mice prevented activation of interferon signaling gene and delayed heart failure but did not prevent lethality after 8.5 months. In contrast, compound mutants only expressing catalytically inactive ADAR1 in an Ifih1-mutant background were completely normal. Inactivation of Irf7 attenuated the phenotype of Adar1-deficient cardiomyocytes to a similar extent as Ifih1 depletion, identifying IRF7 as the main mediator of autoinflammatory responses caused by the absence of ADAR1 in cardiomyocytes. CONCLUSIONS: Enzymatically active ADAR1 prevents IRF7-mediated autoinflammatory reactions in the heart triggered by endogenous nonedited RNAs. In addition to RNA editing, ADAR1 also serves editing-independent roles in the heart required for long-term cardiac function and survival.
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Adenosina Desaminasa , Insuficiencia Cardíaca , Adenosina/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Animales , Inosina/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferones/metabolismo , Ratones , Ratones Mutantes , FN-kappa B/metabolismo , ARNRESUMEN
Angiogenesis, the process by which endothelial cells (ECs) form new blood vessels from existing ones, is intimately linked to the tissue's metabolic milieu and often occurs at nutrient-deficient sites. However, ECs rely on sufficient metabolic resources to support growth and proliferation. How endothelial nutrient acquisition and usage are regulated is unknown. Here we show that these processes are instructed by Yes-associated protein 1 (YAP)/WW domain-containing transcription regulator 1 (WWTR1/TAZ)-transcriptional enhanced associate domain (TEAD): a transcriptional module whose function is highly responsive to changes in the tissue environment. ECs lacking YAP/TAZ or their transcriptional partners, TEAD1, 2 and 4 fail to divide, resulting in stunted vascular growth in mice. Conversely, activation of TAZ, the more abundant paralogue in ECs, boosts proliferation, leading to vascular hyperplasia. We find that YAP/TAZ promote angiogenesis by fuelling nutrient-dependent mTORC1 signalling. By orchestrating the transcription of a repertoire of cell-surface transporters, including the large neutral amino acid transporter SLC7A5, YAP/TAZ-TEAD stimulate the import of amino acids and other essential nutrients, thereby enabling mTORC1 activation. Dissociating mTORC1 from these nutrient inputs-elicited by the loss of Rag GTPases-inhibits mTORC1 activity and prevents YAP/TAZ-dependent vascular growth. Together, these findings define a pivotal role for YAP/TAZ-TEAD in controlling endothelial mTORC1 and illustrate the essentiality of coordinated nutrient fluxes in the vasculature.
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Células Endoteliales , Transactivadores , Aciltransferasas/metabolismo , Animales , Células Endoteliales/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Nutrientes , Factores de Transcripción de Dominio TEA/metabolismo , Transactivadores/metabolismo , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all eukaryotes.
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Trypanosoma brucei brucei , Trypanosoma , Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Saccharomyces cerevisiae/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.
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Genoma Mitocondrial , Trypanosoma brucei brucei , ADN de Cinetoplasto/genética , ADN de Cinetoplasto/metabolismo , Flagelos/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.
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Trypanosoma brucei brucei , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
Skeletal muscle stem cells (MuSCs), also called satellite cells, are instrumental for postnatal muscle growth and skeletal muscle regeneration. Numerous signaling cascades regulate the fate of MuSCs during muscle regeneration but the molecular mechanism by which MuSCs sense mechanical stimuli remain unclear. Here, we describe that Piezo1, a mechanosensitive ion channel, keeps MuSCs in a quiescent state and prevents senescence. Absence of Piezo1 induces precocious activation of MuSCs, attenuates proliferation, and impairs differentiation, essentially abolishing efficient skeletal muscle regeneration and replenishment of the MuSC pool. Furthermore, we discovered that inactivation of Piezo1 results in compensatory up-regulation of T-type voltage-gated Ca2+ channels, leading to increased Ca2+ influx, which strongly induces NOX4 expression via cPKC. Elevated NOX4 expression in Piezo1-deficient MuSCs increases ROS levels and DNA damage, causing P53-dependent cellular senescence and cell death. The importance of the P53/P21-axis for mediating Piezo1-dependent cellular defects was confirmed by pharmacological inhibition of P53 in Piezo1-deficient mice, which abrogates increased senescence of muscle cells and normalizes muscle regeneration. Our findings uncover an essential role of Piezo1-mediated mechano-signaling in MuSCs for maintaining quiescence and preventing senescence. Reduced mechano-signaling due to decreased physical activity during aging may contribute to the increase of senescent cells and the decline of MuSC numbers in geriatric mice and humans.
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Músculo Esquelético , Proteína p53 Supresora de Tumor , Animales , Diferenciación Celular/genética , Senescencia Celular/genética , Canales Iónicos/genética , Ratones , Músculo Esquelético/fisiología , Mioblastos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
More than 95% of mitochondrial proteins are encoded in the nucleus, synthesised in the cytosol and imported into the organelle. The evolution of mitochondrial protein import systems was therefore a prerequisite for the conversion of the α-proteobacterial mitochondrial ancestor into an organelle. Here, I review that the origin of the mitochondrial outer membrane import receptors can best be understood by convergent evolution. Subsequently, I discuss an evolutionary scenario that was proposed to explain the diversification of the inner membrane carrier protein translocases between yeast and mammals. Finally, I illustrate a scenario that can explain how the two specialised inner membrane protein translocase complexes found in most eukaryotes were reduced to a single multifunctional one in trypanosomes.
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Membranas Mitocondriales , Proteínas Mitocondriales , Animales , Proteínas Portadoras/metabolismo , Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/metabolismoRESUMEN
Introduction: Spinal cord stimulation is emerging as a minimally invasive technique for treatment of persistent spinal pain syndrome (PSPS). Methods: We describe a case series of 25 individuals with PSPS who underwent implantation of a spinal cord stimulator device between 2017 and 2021. Results: There was a significant reduction in mean visual analog scale pain scores in the immediate postoperative phase, (8.61 vs. 2.3, p < 0.001). There were twelve patients who consumed pre-operative opioid, and 75% showed reduction of use with a significantly lower average daily dose (66.8 vs. 26.9 meq/D, p < 0.05). There was a significant reduction in the Oswestry Disability Index during postoperative follow-up visits (p < 0.001). There were no major perioperative or long-term complications from the procedure in follow-up. Conclusion: The analysis of this cohort suggests successful long-term treatment of a diverse set of patients with PSPS who underwent spinal cord stimulation (SCS) and had meaningful improvement in quality of life and reduction in opioid consumption.
RESUMEN
The mitochondrial F1Fo ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F1 moiety is relatively highly conserved in structure and composition, the Fo subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F1Fo ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of Fo subunits, but not those of F1, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to Fo subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.