The role of caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) in regulation of mitochondrial ribosome biogenesis in mammals

CLPP (caseinolytic mitochondrial matrix peptidase proteolytic subunit) is a highly conserved serine protease. Molecular and structural studies in E. coli and other prokaryotes have revealed CLPP specific substrates and the mechanisms underlying their identification and subsequent degradation. These studies showed that ClpXP is involved in DNA damage repair, stationary-phase gene expression, and ssrA-mediated protein quality control. Similarly, diverse roles for the eukaryotic CLPP have been suggested. In the filamentous fungus Podospora anserine Clpp depletion promotes longevity. In Caenorhabditis elegans it has been demonstrated that CLPP have a central role in mediating the UPRmt signals. Loss of function CLPP mutations in humans cause Perrault syndrome that results in ovarian failure and sensorineural hearing loss accompanied with shorter stature. Despite this we still have a very limited knowledge about the functional role of eukaryotic CLPP, its specific substrates and underlying molecular mechanism. In order to decipher the in vivo role of CLPP in mammals we have developed a CLPP deficient mouse model (Clpp-/-). Interestingly, only about half of Clpp knockout mice according to Mendelian proportion (12,5%) are born from intercrossing of Clpp+/- mice. These mice are infertile and born ~ 30% smaller than littermates. CLPP deficient mice faithfully replicate the phenotypes observed in human patients. On the molecular level CLPP deficiency leads to an early specific decrease in Complex I activity, followed by a decrease in Complex IV activity later in life. Furthermore, we observed a decrease in mitochondrial translation, which is compensated for by upregulation of mitochondrial transcription. This suggests a direct or indirect role of CLPP in the process of mitochondrial protein synthesis. Gradient sedimentation analysis demonstrates an increase in the steady state levels of small ribosomal subunits, while large ribosomal subunits and monosomes are present in almost normal levels. We also observed an impairment of 12S rRNA assembly into monosomes leading to lower loading of mt- mRNAs. This indicates complications in the function of monosomes. Search for CLPXP substrates and interactors revealed two candidates that are likely to be involved in this process. We show that ERAL1 is one of the substrates of CLPP that is likely causing defective 12S rRNA assembly into the small ribosomal subunit. Additionally, p32, a CLPP interactor is permanently bound to the mitoribosomes. We believe that through interaction with CLPXP, these proteins are involved in resolution of stalled ribosomes. We are currently working further on elucidating the molecular mechanism underlying impaired mitochondrial translation

Mechanism of membrane-tethered mitochondrial protein synthesis

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.Peer reviewe

Characterization and partial purification of DNase in Batrachochytrium dendrobatidis

The pathogen Batrachochytrium dendrobatidis (Bd) has been associated with amphibian declines in multiple continents, including western North America. Preliminary studies done in our laboratory have shown that Bd can form a biofilm and is active in the biofilm. We have identified DNase activity in Bd that may play a role in degrading the biofilm matrix and helping the organism to reinfect. DNase may also facilitate the supply of nutrients to the organism. Here we show the presence of this activity using qualitative and quantitative methods. DNase tests were performed using methyl green DNA containing plates and zymography. DNase activity is highest within a pH range of 5.5 to 7.5 and in the presence of Ca2+ and Mg2+ divalent cations. We have partially purified the enzyme and have seen that the enzyme may be present in 3 isoforms

Mitochondrial ribosomes in cancer

Mitochondria play fundamental roles in the regulation of life and death of eukaryotic cells. They mediate aerobic energy conversion through the oxidative phosphorylation (OXPHOS) system, and harbor and control the intrinsic pathway of apoptosis. As a descendant of a bacterial endosymbiont, mitochondria retain a vestige of their original genome (mtDNA), and its corresponding full gene expression machinery. Proteins encoded in the mtDNA, all components of the multimeric OXPHOS enzymes, are synthesized in specialized mitochondrial ribosomes (mitoribosomes). Mitoribosomes are therefore essential in the regulation of cellular respiration. Additionally, an increasing body of literature has been reporting an alternative role for several mitochondrial ribosomal proteins as apoptosis-inducing factors. No surprisingly, the expression of genes encoding for mitoribosomal proteins, mitoribosome assembly factors and mitochondrial translation factors is modified in numerous cancers, a trait that has been linked to tumorigenesis and metastasis. In this article, we will review the current knowledge regarding the dual function of mitoribosome components in protein synthesis and apoptosis and their association with cancer susceptibility and development. We will also highlight recent developments in targeting mitochondrial ribosomes for the treatment of cancer

Role of GTPases in Driving Mitoribosome Assembly

Mitoribosomes catalyze essential protein synthesis within mitochondria. Mitoribosome biogenesis is assisted by an increasing number of assembly factors, among which guanosine triphosphate hydrolases (GTPases) are the most abundant class. Here, we review recent progress in our understanding of mitoribosome assembly GTPases. We describe their shared and specific features and mechanisms of action, compare them with their bacterial counterparts, and discuss their possible roles in the assembly of small or large mitoribosomal subunits and the formation of the monosome by establishing quality-control checkpoints during these processes. Furthermore, following the recent unification of the nomenclature for the mitoribosomal proteins, we also propose a unified nomenclature for mitoribosome assembly GTPases. Mitoribosome assembly involves at least six GTP hydrolases (GTPases) belonging to several conserved families.Mitoribosome assembly GTPases act to facilitate rRNA folding, and recruit mitoribosomal proteins and assembly factors to the assembly pathway.Maturation of the large mitoribosomal subunit (mtLSU) requires the assistance of several GTPases acting at late stages, when they function as antiassociation or quality-control factors to ensure joining of the mature small mitoribosomal subunit (mtSSU) and mtLSU into functional ribosomes.Impaired mitoribosome assembly GTPase function leads to defective mitochondrial protein synthesis and human disease.A novel unifying nomenclature for mitoribosome assembly GTPases is proposed

Electric Field-Driven Spatial Information Capture of Dissipative Biocondensate States

The theory behind origin of life to Darwinian evolution considers emergence of dissipative structures driven by the flow of energy across all length scales. To this end, developing and deeper understanding of non-equilibrium self-assembly processes under continuous supply of energy is a demanding matter, both in fundamental and application (for e.g. developing dynamic materials) viewpoint. Herein, we demonstrate transient self-assembly of a DNA-histone condensate where trypsin (already present in the system) hydrolyse histone resulting disassembly. As the process is short-lived, the information of intermediate states between complete assembly and disassembly remains uncaptured in absence of any external energy. We show that performing the process under electric field of varying strength results fractionation of myriad of short-lived states which appears as band in different zone. Deconvolution and capturing of many hidden self-assembling species of similar components but of different compositions which otherwise never be formed in absence of electric energy, will be of immense importance in applied non-equilibrium thermodynamics

Abstract 3775: Inhibition of mitochondrial translation by the marine natural product elatol shows potent antileukemia activity

Abstract Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Recent studies have shown that some forms of hematologic malignancies (Ashton et al., 2018) and solid tumors (Molina et al., 2018) have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation. Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins leading to selective lethality in hematologic malignancies reliant on oxidative phosphorylation (Norberg et al., 2017). Combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant chronic myelogenous leukemia (CML), both in vitro and in vivo (Kuntz et al., 2017). We have previously reported that elatol, the major compound from algae Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary CML cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells treated with single-digit micromolar concentrations of elatol for short periods revealed strong downregulation of mitochondrion-encoded proteins (no transcriptional effect). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 2-10x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines. Through sedimentation property analysis using sucrose gradients, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. Moreover, elatol treatment leads to activation of the integrated stress response (ISR) evidenced by induction of ATF4 and CHOP. We found that elatol activates the ISR through mitochondrial stress in which cleaved DELE1 binds to HRI to phosphorylate eIF2α, a mechanism described in Fessler et al., 2020. In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis and mechanistically different from chloramphenicol. Tigecycline’s compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol’s greatly improved potency provides a potential starting point for further optimization of this paradigm. Citation Format: Tyler A. Cunningham, Priyanka Maiti, Antonio Barrientos, Jonathan H. Schatz. Inhibition of mitochondrial translation by the marine natural product elatol shows potent antileukemia activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3775

Inhibition of Mitochondrial Translation By the Marine Natural Product Elatol Shows Potent Antileukemia Activity

Abstract Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Relapse/refractory disease that results from treatment with targeted signaling inhibitors is a major hurdle in obtaining curative responses. Interestingly, work over the past decade or more has shown that chronic myelogenous leukemia (CML) stem cells (CD34+CD38-) are resistant to targeted signaling inhibitors, such as the BCR-ABL kinase class of inhibitors, often a problematic source of resistance leading to residual disease that may precipitate later progression (Hamilton et al., 2012). Recent studies have shown that some forms of lymphoma and leukemia cell have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation (Ashton et al., 2018). Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins due to the similarity of bacterial and mitochondrial ribosomes, leading to selective lethality in hematologic malignancies reliant on enhanced oxidative phosphorylation (Norberg et al., 2017). Indeed, it was established that CML stem cells are reliant on upregulated oxidative phosphorylation, and combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant CML, both in vitro and in animal models (Kuntz et al., 2017). We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic myelogenous leukemia cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong downregulation of mitochondrion-encoded proteins as in Figure 1, (with no effect on mitochondrial transcription). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines when compared at 72h. Additionally, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Tigecycline's compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provide a potential starting point for further optimization of this paradigm. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare

Molecular Docking Study of drug molecules from Drug Bank database against COVID-19 Mpro protein

Aims: SARS-CoV-2 which is NovelCoronavirushas been disseminated all over the world and causing Coronavirus disease (COVID-19) resulting in many deaths as well as economic loss in several countries.This virus is showinga considerable amount of high morbidity and mortality.Currently, no drugs are available againstSARS-CoV-2. Therefore,for the treatment of disease, researchers are looking fornew drugs that can treat the disease and prevent it to be spread.In this regard,drug repurposingmay help scientists for treating and preventing infections associated with SARS-CoV-2. Drug repurposingis a strategy that can identify new targets for existing drugs that are already approved for the treatment of a disease. Main methods: In this study, we present a virtual screening procedure employing deep lerning regression method in 9101 drugs from Drug bank database against the target Main protease (Mpro) for the treatment of COVID-19. 500 screened compounds were subjected to docking. Key findings: Among those 500 drugs, 10 best drugs were selected, which had better binding energy as compared to the reference molecule. Based on the Binding energy score, we can suggest that the identified drug may be considered for therapeutic development against the virus. Significance: Drug repurposing has many advantages as it could shorten the time and reduce the cost of new drug discovery. This research will help to get new drugs against COVID-19 and help humans against this pandemic disease. Keyword- Drug Repurposing, Deep learning, Molecular Docking, COVID-19, Drug bank database, MPr

Human GTPBP10 is required for mitoribosome maturation

Most steps on the biogenesis of the mitochondrial ribosome (mitoribosome) occur near the mitochondrial DNA nucleoid, in RNA granules, which contain dedicated RNA metabolism and mitoribosome assembly factors. Here, analysis of the RNA granule proteome identified the presence of a set of small GTPases that belong to conserved families of ribosome assembly factors. We show that GTPBP10, a member of the conserved Obg family of P-loop small G proteins, is a mitochondrial protein and have used gene-editing technologies to create a HEK293T cell line KO for GTPBP10 . The absence of GTPBP10 leads to attenuated mtLSU and mtSSU levels and the virtual absence of the 55S monosome, which entirely prevents mitochondrial protein synthesis. We show that a fraction of GTPBP10 cosediments with the large mitoribosome subunit and the monosome. GTPBP10 physically interacts with the 16S rRNA , but not with the 12S rRNA , and crosslinks with several mtLSU proteins. Additionally, GTPBP10 is indirectly required for efficient processing of the 12S-16S rRNA precursor transcript, which could explain the mtSSU accumulation defect. We propose that GTPBP10 primarily ensures proper mtLSU maturation and ultimately serves to coordinate mtSSU and mtLSU accumulation then providing a quality control check-point function during mtLSU assembly that minimizes premature subunit joining