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Results for “Biochemistry”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2020

Cryo-electron Microscopy and Exploratory Antisense Targeting of the 28-kDa Frameshift Stimulation Element from the SARS-CoV-2 RNA Genome

Drug discovery campaigns against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are beginning to target the viral RNA genome1, 2. The frameshift stimulation element (FSE) of the SARS-CoV-2 genome is required for balanced expression of essential viral proteins and is highly conserved, making it a potential candidate for antiviral targeting by small molecules and oligonucleotides3-6. To aid global efforts focusing on SARS-CoV-2 frameshifting, we report exploratory results from frameshifting and cellular replication experiments with locked nucleic acid (LNA) antisense oligonucleotides (ASOs), which support the FSE as a therapeutic target but highlight difficulties in achieving strong inactivation. To understand current limitations, we applied cryogenic electron microscopy (cryo-EM) and the Ribosolve7 pipeline to determine a three-dimensional structure of the SARS-CoV-2 FSE, validated through an RNA nanostructure tagging method. This is the smallest macromolecule (88 nt; 28 kDa) resolved by single-particle cryo-EM at subnanometer resolution to date. The tertiary structure model, defined to an estimated accuracy of 5.9 [A], presents a topologically complex fold in which the 5' end threads through a ring formed inside a three-stem pseudoknot. Our results suggest an updated model for SARS-CoV-2 frameshifting as well as binding sites that may be targeted by next generation ASOs and small molecules.

biochemistry↗

bioRxiv2020

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays--with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage in eukaryotic cells.

biochemistry↗

bioRxiv2020

Double Domain Swapping in Human γC and γD Crystallin Drives Early Stages of Aggregation

Human{gamma} D (H{gamma}D) and{gamma} C (H{gamma}C) are double domained crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations of Crys proteins. Using coarse grained protein models and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multi-dimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored pathway involves intermediates where both the domains are partially folded. A native like intermediate (I*), featuring the folded domains and disrupted inter domain contacts, gets populated in all the three pathways. I* forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of H{gamma}D Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/210443v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@fd37e6org.highwire.dtl.DTLVardef@60e7cdorg.highwire.dtl.DTLVardef@128386org.highwire.dtl.DTLVardef@178f240_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract figure C_FIG

biochemistry↗

bioRxiv2020

Carotenogenesis of Staphylococcus aureus: new insights and impact on membrane biophysical properties

Staphyloxanthin (STX) is a saccharolipid derived from a carotenoid in Staphylococcus aureus involved in oxidative-stress tolerance and antimicrobial peptide resistance. In this work, a targeted metabolomics and biophysical study was carried out on native and knock-out S. aureus strains to investigate the biosynthetic pathways of STX and related carotenoids. Identification of 34 metabolites at different growth phases (8, 24 and 48h), reveal shifts of carotenoid populations during progression towards stationary phase. Six of the carotenoids in the STX biosynthetic pathway and three menaquinones (Vitamin K2) were identified in the same chromatogram. Furthermore, other STX homologues with varying acyl chain structures reported herein for the first time, which reveal the extensive enzymatic activity of CrtO/CrtN. Fourier Transform infrared spectroscopy show that STX increases acyl chain order and shifts the cooperative melting of the membrane indicating a more rigid lipid bilayer. This study shows the diversity of carotenoids in S. aureus, and their influence on membrane biophysical properties.

biochemistry↗

bioRxiv2020

Identification and Characterization of a B-Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and [~]3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

biochemistry↗

bioRxiv2020

Sarcolambans are phospholamban- and sarcolipin-like regulators of the sarcoplasmic reticulum calcium pump SERCA

From insects to humans, calcium signaling is essential for life. An important part of this process is the sarco-endoplasmic reticulum calcium pump SERCA, which maintains low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, this is a tightly controlled system where SERCA interacts with tissuespecific regulatory subunits such as phospholamban in cardiac muscle and sarcolipin in skeletal muscle. With the recent discovery of the sarcolambans, the family of calcium pump regulatory subunits also appears to be ancient, spanning more than 550 million years of evolutionary divergence from insects to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and range from vaguely phospholamban-like to vaguely sarcolipin-like. Here, our goal was to investigate select sarcolamban peptides for their ability to regulate calcium pump activity. For a side-by-side comparison of diverse sarcolamban peptides, we tested them against mammalian skeletal muscle SERCA1a. This allowed us to determine if the sarcolamban peptides mimic phospholamban and sarcolipin in their regulatory activities. Four sarcolamban peptides were chosen from different invertebrate species. Of these, we were able to express and purify sarcolamban peptides from bumble bee, water flea, and tadpole shrimp. Sarcolamban peptides were co-reconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. While all peptides were super-inhibitors of SERCA, they exhibited either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations were used to reveal novel features of insect versus mammalian calcium pumps and the sarcolamban regulatory subunits.

biochemistry↗

bioRxiv2020

Multistep changes in amyloid structure that are induced by cross-seeding on a rugged energy landscape

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures depending on species, and each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new-type amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin fibrils. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition (SVD) analysis of the UV-Vis absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.

biochemistry↗

bioRxiv2020

Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not physiologically relevant for their uncoupling activity.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocyte. In the presence of long-chain fatty acids (LCFA), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Several atomic models of UCP1 and UCP2 have been obtained by NMR in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiment on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of S. cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S and K56S/K269S mutants did not display any default in activation, which shows that the NMR experiments in DPC detergent are not relevant to understand UCP1 function.

biochemistry↗

bioRxiv2020

INTS13 Mutations Causing a Developmental Ciliopathy Disrupt Integrator Complex Assembly

Oral-facial-digital syndromes (OFD) are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. To date, mutations in 12 ciliary-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline mutations in INTS13, a subunit of the Integrator complex. This 14-component complex associates with RNAPII and can cleave nascent RNA to modulate gene expression. We determined that INTS13 utilizes a discrete domain within its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline INTS13 mutations. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos lead to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions within Integrator components.

biochemistry↗

bioRxiv2020

A phosphoproteomic approach reveals that PKD3 controls phenylalanine and tyrosine metabolism

Members of the Protein Kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified over three-hundred putative targets of PKD3. Among them phenylalanine hydroxylase (PAH). PAH catalyses the conversion of phenylalanine to tyrosine and its activity is regulated by, phenylalanine concentration and glucagon-induced signaling. Consistently, we showed that PKD3 is activated by glucagon and promotes tyrosine levels in primary hepatocytes and liver of mice. Taken together, our comprehensive proteomic approach established that PKD3 determine the rate of phenylalanine to tyrosine conversion in the liver. Therefore, our data indicate that PKD3 might play a role in development of diseases related to the defective tyrosine and phenylalanine metabolism.

biochemistry↗

bioRxiv2020

Mapping specificity, entropy, allosteric changes and substrates in blood proteases by a high-throughput protease screen

Proteases are among the largest protein families in eukaryotic phylae with more than 500 genetically encoded proteases in humans. By cleaving a wide range of target proteins, proteases are critical regulators of a vast number of biochemical processes including apoptosis and blood coagulation. Over the last 20 years, knowledge of proteases has been drastically expanded by the development of proteomic approaches to identify and quantify proteases and their substrates. In spite of their merits, some of these methods are laborious, not scalable or incompatible with native environments. Consequentially, a large number of proteases remain poorly characterized. Here, we introduce a simple proteomic method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter and their analysis in a mass spectrometer. The method is significantly faster, cheaper, technically less demanding, easily multiplexed and produces accurate protease fingerprints in near-native conditions. By using the blood cascade proteases as a case study we obtained protease substrate profiles of unprecedented depth that can be reliably used to map specificity, entropy and allosteric changes of the protease and to design fluorescent probes and predict physiological substrates. The native protease characterization method is comparable in performance, but largely exceeds the throughput of current alternatives.

biochemistry↗

bioRxiv2020

Discovery of membrane-permeating cyclic peptides via mRNA display

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and non-disruptively controlling intracellular or intraembryonic processes.

biochemistry↗

bioRxiv2020

Biochemical characterization of recombinant UDP-sugar pyrophosphorylase and galactinol synthase from Brachypodium distachyon

Raffinose (Raf) protects plant cells during seed desiccation and under different abiotic stress conditions. The biosynthesis of Raf starts with the production of UDP-galactose by UDP-sugar pyrophosphorylase (USPPase) and continues with the synthesis of galactinol by galactinol synthase (GolSase). Galactinol is then used by Raf synthase to produce Raf. In this work, we report the biochemical characterization of USPPase (BdiUSPPase) and GolSase 1 (BdiGolSase1) from Brachypodium distachyon. The catalytic efficiency of BdiUSPPase was similar with galactose 1-phosphate and glucose 1-phosphate, but 5-to 17-fold lower with other sugar 1-phosphates. The catalytic efficiency of BdiGolSase1 with UDP-galactose was three orders of magnitude higher than with UDP-glucose. A structural model of BdiGolSase1 allowed us to determine the residues putatively involved in the binding of substrates. Among these, we found that Cys261 lies within the putative catalytic pocket. BdiGolSase1 was inactivated by oxidation with diamide and H2O2. The activity of the diamide-oxidized enzyme was recovered by reduction with dithiothreitol or E. coli thioredoxin, suggesting that BdiGolSase1 is redox-regulated.

biochemistry↗

bioRxiv2020

Increased isoDGR motifs in plasma fibronectin are associated with atherosclerosis through facilitation of vascular fibrosis

Abnormal matrix deposition on vessels and recruitment of inflammatory cells into the arterial wall are critical events in atherosclerotic plaque formation. Fibronectin protein is a key matrix component that exhibits high levels of deamidation in atherosclerotic plaques and blood plasma, but it is unclear how this structural change impacts on endothelial function or modifies interactions with recruited leukocytes. This study aimed to determine how deamidation-induced isoDGR motifs in fibronectin influence extracellular matrix accumulation on endothelial cells, and to investigate possible effects on integrin outside-in signalling in matrix-bound monocytes which are key mediators of human atherosclerosis. Blood plasma fibrinogen and fibronectin displayed marked accumulation of isoDGR motifs in ischemic heart disease (IHD) as determined by ELISA analysis of patients undergoing coronary artery bypass grafting compared with age-matched healthy controls. Biochemical and functional assays confirmed that isoDGR-containing fibronectin promoted activation of integrin {beta}1 in monocytes and facilitated protein deposition and fibrillogenesis on endothelial cell layers. In addition, isoDGR interactions with integrins on the monocyte cell surface triggered an ERK:AP-1 signalling cascade that induced potent secretion of chemotactic mediators (including CCL2, CCL4, IL-8, and TNF), that promoted further leukocyte recruitment to the assembling plaque. Fibronectin deamidation forms isoDGR motifs that increase binding to {beta}1 integrins on the surface of endothelial cells and monocytes. Subsequent activation of integrin outside-in signalling pathways elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix and likely constitutes a key early event in progression to IHD.

biochemistry↗

bioRxiv2020

Characterization of Plasmodium falciparum NEDD8 and identification of cullins as its substrates

A variety of post-translational modifications of Plasmodium falciparum proteins, including phosphorylation and ubiquitination, are shown to have key regulatory roles. The neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is a ubiquitin-like modifier of cullin-RING E3 ubiquitin ligases, which regulate diverse cellular processes, including the cell-cycle. Although neddylation pathway is conserved in eukaryotes, it is yet to be characterized in Plasmodium and related apicomplexan parasites. Towards studying the neddylation pathway in malaria parasites, we characterized P. falciparum NEDD8 (PfNEDD8) and identified cullins as its physiological substrates. PfNEDD8 is a 76 amino acid residue protein without the C-terminal tail, indicating that it can be readily conjugated. The wild type and mutant (Gly75Gly76 mutated to Ala75Ala76) PfNEDD8 were expressed in P. falciparum. Western blot of wild type PfNEDD8-expressing parasites indicated multiple high molecular weight conjugates, which were absent in the parasites expressing the mutant, indicating conjugation of NEDD8 to proteins through Gly76. Immunoprecipitation followed by mass spectrometry of wild type PfNEDD8-expressing parasites identified several proteins, including two putative cullins. Furthermore, we expressed PfNEDD8 in mutant S. cerevisiae strains that lacked endogenous NEDD8 ({Delta}rub1) or NEDD8 conjugating E2 enzyme ({Delta}Ubc12). The western blot of complemented strains and mass spectrometry of PfNEDD8 immunoprecipitate showed conjugation of PfNEDD8 to S. cerevisiae cullin cdc53, demonstrating functional conservation and cullins as the physiological substrates of PfNEDD8. The characterization of PfNEDD8 and identification of cullins as its substrates make ground for investigation of specific roles and drug target potential of neddylation pathway in malaria parasites.

biochemistry↗

bioRxiv2020

Hypothetical structure for energy transformation. Evolution of cellular structures for energy transformation

The present paper represents a hypothetical structure, the structure for energy transformation (SET), which might be responsible for the proper energy transformation steps leading to the continuous production of H+ and ATP in living cells. We predict that the electron flow is realized through the electron flow device (EFD). We suppose that there are several versions of the SET. Two of them are described below [Structure of Aerobic Glycolysis (SET-AG) and Structure of Oxidative Phosphorylation (SET-OP)]. The hypothesis is based on the atom properties of the protonated adenine molecule and the docking computations of molecular mechanics involved, suggesting that two ascorbate molecules may occupy the empty NADPH pocket, preferably binding to the adenine binding site. We hypothesize that the adenine originates from uric acid (UA), resulting in an ATP-UA-ADP-ATP-UA cycle. It would also mean that UA is one of the oxygen sources in aerobic glycolysis. We also suppose that the EFD contains the well-known molecules of the Nicotinamide Adenine Dinucleotide (NAD) and Flavin Adenine Dinucleotide (FAD) supplemented with two additional UA-originated adenine molecules, two L-ascorbic acids, and two D-glucose molecules. Based on all this, we surmise that a tetra adenine octo phosphate ring (TAR) exists, in which the UA originated adenine molecules form a ring. The molecules are linked to each other through the N7-C2 and C8-N1 atoms of the adenine molecules by H2PO4e- molecules. The four N10 atoms of the adenine molecules bind one flavin, one nicotinamide, and two L-ascorbic acid molecules. Six D-glucose molecules complete one Unit of the structure. Both Fe-S and cytochrome clusters, as well as dehydrogenases, ensure the continuous operation of the Unit. The synchronized function of the three-stoke, three twin-cylinder engine results in continuous energy, ATP, and H+ production. Eukaryotic cells are equipped with the SET-AG and the SET-OP; thus, they can live in an anoxic and oxygenized environment. It is hoped that the SET concept developed here will help the better understanding the way of action regarding the cancer treatment with Vitamin C or glucose deprivation.

biochemistry↗

bioRxiv2020

The answer lies in the energy: how simple atomistic molecular dynamics simulations may hold the key to epitope prediction on the fully glycosylated SARS-CoV-2 spike protein

Betacoronavirus SARS-CoV-2 is posing a major threat to human health and its diffusion around the world is having dire socioeconomical consequences. Thanks to the scientific communitys unprecedented efforts, the atomic structure of several viral proteins has been promptly resolved. As the crucial mediator of host cell infection, the heavily glycosylated trimeric viral Spike protein (S) has been attracting the most attention and is at the center of efforts to develop antivirals, vaccines, and diagnostic solutions. Herein, we use an energy-decomposition approach to identify antigenic domains and antibody binding sites on the fully glycosylated S protein. Crucially, all that is required by our method are unbiased atomistic molecular dynamics simulations; no prior knowledge of binding properties or ad hoc combinations of parameters/measures extracted from simulations is needed. Our method simply exploits the analysis of energy interactions between all intra-protomer aminoacid and monosaccharide residue pairs, and cross-compares them with structural information (i.e., residueresidue proximity), identifying potential immunogenic regions as those groups of spatially contiguous residues with poor energetic coupling to the rest of the protein. Our results are validated by several experimentally confirmed structures of the S protein in complex with anti- or nanobodies. We identify poorly coupled sub-domains: on the one hand this indicates their role in hosting (several) epitopes, and on the other hand indicates their involvement in large functional conformational transitions. Finally, we detect two distinct behaviors of the glycan shield: glycans with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes; those with weaker coupling could themselves be poised for antibody recognition. Predicted Immunoreactive regions can be used to develop optimized antigens (recombinant subdomains, synthetic (glyco)peptidomimetics) for therapeutic applications.

biochemistry↗

bioRxiv2020

Local translation in synaptic mitochondria influences synaptic transmission

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system, and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we addressed this by devising an imaging approach to analyze mitochondrial translation, by following the incorporation of clickable non-canonical amino acids. We applied this method to multiple cell types, including hippocampal neurons, where we found ample evidence for mitochondrial translation in both dendrites and axons. Translation levels were surprisingly heterogeneous, were typically stronger in axons, and were independent of their distance from the cell soma, where mitochondria presumably descent from. Presynaptic mitochondrial translation correlated with local synaptic activity, and blocking mitochondria translation reduced synaptic function. Overall, these findings demonstrate that mitochondrial gene expression in neurons is intimately linked to neuronal function.

biochemistry↗