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© 2019 The Authors., The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here, we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated in arthropods a paralog protein (SLIMP) that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM and preventing mitochondrial DNA (mtDNA) accumulation. Thus, mitochondrial translation is directly coupled to mtDNA levels by a network based upon a profound structural modification of an animal ARS., This work was supported by grants SVP2014-068398 and BIO2015-64572 from the Spanish Ministry of Economy and Competitiveness (to L.R.d.P.).

Background and Purpose— Determining the underlying cause of stroke is important to optimize secondary prevention treatment. Increased blood levels of natriuretic peptides (B-type natriuretic peptide/N-terminal pro-BNP [BNP/NT-proBNP]) have been repeatedly associated with cardioembolic stroke. Here, we evaluate their clinical value as pathogenic biomarkers for stroke through a literature systematic review and individual participants’ data meta-analysis. Methods— We searched publications in PubMed database until November 2013 that compared BNP and NT-proBNP circulating levels among stroke causes. Standardized individual participants’ data were collected to estimate predictive values of BNP/NT-proBNP for cardioembolic stroke. Dichotomized BNP/NT-proBNP levels were included in logistic regression models together with clinical variables to assess the sensitivity and specificity to identify cardioembolic strokes and the additional value of biomarkers using area under the curve and integrated discrimination improvement index. Results— From 23 selected articles, we collected information of 2834 patients with a defined cause. BNP/NT-proBNP levels were significantly elevated in cardioembolic stroke until 72 hours from symptoms onset. Predictive models showed a sensitivity >90% and specificity >80% when BNP/NT-proBNP were added considering the lowest and the highest quartile, respectively. Both peptides also increased significantly the area under the curve and integrated discrimination improvement index compared with clinical models. Sensitivity, specificity, and precision of the models were validated in 197 patients with initially undetermined stroke with final pathogenic diagnosis after ancillary follow-up. Conclusions— Natriuretic peptides are strongly increased in cardioembolic strokes. Future multicentre prospective studies comparing BNP and NT-proBNP might aid in finding the optimal biomarker, the best time point, and the optimal cutoff points for cardioembolic stroke identification.

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3′–5′ domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing., We thank the following bodies for funding: Ministerio de Economía y Competitividad (MINECO) (T.H.S.: BFU2012-39521, BFU2015-68354, Ayudas para incentivar la incorporación estable de doctores (IED) 2015; L.R.d.P.: BIO2015-64572; T.H.S. and L.R.d.P: institutional funding through the Centres of Excellence Severo Ochoa award and from the CERCA Programme of the Catalan Government; and O.Y.: SAF2011-30578 and BFU2014-57466); the Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), an initiative of Instituto de Investigacion Carlos III (ISCIII) to O.Y.; and the Biotechnology and Biological Sciences Research Council (BBSRC: BB/H019723/1 and BB/M008800/1) to A.J.D. and L.B. S.A. was supported by a Finnish Cultural Society Fellowship, J.S. by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/87025/2012), P.A.K. by an Advanced Postdoc Mobility fellowship from the Swiss National Science Foundation (SNF), I.G.-C. by an Asociación Española Contra el Cáncer (AECC) fellowship, A.A.-F. by a Severo Ochoa FPI fellowship (MINECO; SVP2014068398), and A.A.J. by an EMBO long-term fellowship (ALTF 554-2015).

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARS) is a central reaction in biology. Multiple regulatory pathways utilize the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown. Here we describe a functional network that couples protein synthesis to DNA replication in animal mitochondria. We show that a duplication of the gene coding for mitochondrial seryl-tRNA synthetase (SerRS2) generated a paralog protein (SLIMP) in arthropods that forms a heterodimeric complex with a SerRS2 monomer. This seryl-tRNA synthetase variant is essential for protein synthesis and mitochondrial respiration. In addition, SLIMP interacts with the substrate binding domain of the mitochondrial protease LON, thus stimulating proteolysis of the DNA-binding protein TFAM, and repressing mitochondrial DNA (mtDNA) replication. Thus, mitochondrial translation is directly coupled to mtDNA replication by a network based upon a profound structural modification of an animal ARS.

We disrupted the three conserved disulfide bonds in Xenopus β3 (C127−C144, C154-C170, C191-C248), one at a time, using Cys to Ala/Ser mutations and coexpression with relatively ouabain-resistant (C113Y) Xenopus α1 in oocytes. We assessed function with two-microelectrode voltage clamp in 1µM ouabain to silence endogenous Na/K pumps. Single C127S/A or C154S, or double C127S+C154S β3 mutants yielded steady Na/K pump currents (saturating [Ko]) and transient Na charge movements (without Ko) like those of C113Y α1 coexpressed with wild-type β3, suggesting that neither assembly, trafficking, nor Na/K pump function require intact first and/or second disulfide bonds. In contrast, steady and transient pump currents were both ≥10 fold smaller in Na/K pumps with disrupted third disulfide bond after C113Y α1 coexpression with C191A/S β3. Similarly, in outside-out excised patches, palytoxin-induced pump-channel currents were ≥10 fold smaller for C113Y α1 Na/K pumps coexpressed with β3 C191S than with wild-type β3. Because in uninjected oocytes or oocytes expressing C113Y α1 alone, neither steady nor transient pump current could be detected, and palytoxin elicited no measurable current in outside-out patches, the third disulfide bond appears necessary for efficient Na/K-ATPase assembly and trafficking to the cell membrane but is not essential for assembled pump function. We disrupted salt bridges linking the αTM7-TM8 loop and β-exodomain in Na/K-ATPase structures (α1 E901-β3 K219, α1 E911-β3 R262), by coexpressing E901R(C113Y) or E911R(C113Y) α1 with wild-type β3. In both mutants, steady Na/K pump currents (at saturating [Ko]) and transient Na charge movements were like wild type; but the E901R α1 mutation selectively enhanced apparent Ko affinity ∼2 fold. These results suggest that some β-exodomain structural alterations can be tolerated; but α-β subunit interface interactions closely regulate Ko binding/occlusion steps, though not the slow step that occludes/deoccludes the third Na ion. [HL36783]

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome.