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Rxivist combines preprints from bioRxiv with data from Twitter to help you find the papers being discussed in your field. Currently indexing 70,077 bioRxiv papers from 306,093 authors.

Most downloaded bioRxiv papers, all time

in category biochemistry

2,039 results found. For more information, click each entry to expand.

1941: Development of a novel HPTLC-based method for the simultaneous quantification of clinically relevant lipids from cells and tissue extracts
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Posted to bioRxiv 23 Sep 2019

Development of a novel HPTLC-based method for the simultaneous quantification of clinically relevant lipids from cells and tissue extracts
93 downloads biochemistry

Michelle Pinault, Cyrille Guimaraes, Céline Ben Hassen, Jorge L Gutierrez-Pajares, Stéphan Chevalier, Caroline Goupille, Pierre Bernard-Savary, Philippe G. Frank

Lipids such as cholesterol, triglycerides, and fatty acids play important roles in the regulation of cellular metabolism and cellular signaling pathways and, as a consequence, in the development of various diseases. It is therefore important to understand how their metabolism is regulated to better define the components involved in the development of various human diseases. In the present work, we described the development and validation of an HPTLC method allowing the separation and quantification of free cholesterol, cholesteryl esters, non-esterified fatty acids, and triglycerides. This method will be of interest as the quantification of these lipids in one single assay is difficult to perform.

1942: Benzaldehyde Synthases Are Encoded by Cinnamoyl-CoA Reductase Genes in Cucumber (Cucumis sativus L.)
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Posted to bioRxiv 28 Dec 2019

Benzaldehyde Synthases Are Encoded by Cinnamoyl-CoA Reductase Genes in Cucumber (Cucumis sativus L.)
93 downloads biochemistry

Baoxiu Liu, Guo Wei, Zhongyi Hu, Guodong Wang

Benzaldedyde, commonly detected in plant VOC (volatile organic compounds) profiling, is derived from phenylalanine. However, the last enzymatic step for benzaldedyde formation, designated as benzaldehyde synthase, remains elusive for long time. Here, we demonstrated that cinnamoyl-CoA reductases are responsible for benzaldedyde production in cucumber (Cucumis sativus L.). Comprehensive tissue specificity of VOC profiling revealed that benzaldehyde was specifically accumulated in root and flower of cucumber plants. VOC-gene correlation analysis suggested that several CCRs are candidate genes for benzaldehyde production: CsaCCR7 had a root-specific expression pattern while CsaCCR9 and CsaCCR18 showed a flower-specific expression pattern. Enzymatic assay demonstrated that CsaCCR7, CsaCCR9 and CsaCCR18 convert benzoyl-CoA to benzaldehyde. Subcellular localization experiments revealed that CsaCCR7 and CsaCCR18 are localized in cytosol, while CsaCCR9 was localized in peroxisome. In contrast to the long-standing view that CCR enzymes are involved in lignin biosynthesis in plants, it is the first time here to add a new biochemical role of CCR as benzaldehyde synthase in plants.

1943: Detection of active Granzyme A in NK92 cells with fluorescent activity-based probe
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Posted to bioRxiv 10 Dec 2019

Detection of active Granzyme A in NK92 cells with fluorescent activity-based probe
92 downloads biochemistry

Sonia Kołt, Tomasz Janiszewski, Dion Kaiserman, Sylwia Modrzycka, Scott J Snipas, Guy Salvesen, Marcin Drag, Phillip I. Bird, Paulina Kasperkiewicz

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation; and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

1944: Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression
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Posted to bioRxiv 30 Sep 2019

Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression
91 downloads biochemistry

Ditipriya Hazra, Vedrana Andrić, Benoit Palancade, Mathieu Rougemaille, Marc Graille

Timely and accurate expression of the genetic information relies on the integration of environmental cues and the activation of regulatory networks involving transcriptional and post-transcriptional mechanisms. In fission yeast, meiosis-specific transcripts are selectively targeted for degradation during mitosis by the EMC complex, composed of Erh1, the ortholog of human ERH, and the YTH family RNA-binding protein Mmi1. Here, we present the crystal structure of Erh1 and show that it assembles as a homodimer. Mutations of amino acid residues to disrupt Erh1 homodimer formation result in loss-of-function phenotypes, similar to erh1∆ cells: expression of meiotic genes is derepressed in mitotic cells and meiosis progression is severely compromised. Interestingly, formation of Erh1 homodimer is dispensable for interaction with Mmi1, suggesting that only fully assembled EMC complexes consisting of two Mmi1 molecules bridged by an Erh1 dimer are functionally competent. We also show that Erh1 does not contribute to Mmi1-dependent down-regulation of the meiosis regulator Mei2, supporting the notion that Mmi1 performs additional functions beyond EMC. Overall, our results provide a structural basis for the assembly of the EMC complex and highlight its biological relevance in gametogenic gene silencing and meiosis progression.

1945: Lipidomics analysis of juveniles’ blue mussels (Mytilus edulis L. 1758), a key economic and ecological species.
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Posted to bioRxiv 13 Sep 2019

Lipidomics analysis of juveniles’ blue mussels (Mytilus edulis L. 1758), a key economic and ecological species.
91 downloads biochemistry

Vincenzo Alessandro Laudicella, Christine Beveridge, Stefano Carboni, Sofia Cota Franco, Mary K Doherty, Nina Long, Elaine Mitchell, Michelle S. Stanley, Philip D. Whitfield, Adam D. Hughes

Blue mussels (Mytilus edulis L.) are important components of coastal ecosystems functioning through benthopelagic coupling and ecosystem engineering. At the same time, mussel production is central in the economy of coastal areas. Therefore, understanding their nutritional, physiological and metabolic processes at key life stages is important for their management, both within food production systems and in wild populations. Lipids are crucial molecules for bivalve growth, but their diversity and roles have been considered from fatty acid (FA) perspective. In this paper, we applied lipidomics to bivalve nutrition. Lipidomics provides a holistic perspective on lipid patterns; by examining the lipidome, important physiological information can be acquired. Here, we use controlled laboratory experiments to elucidate the responses to changes in the diet of newly settled mussels juveniles, one of the most critical life stages. The diets considered in this study are single strains diet of Cylindrotheca fusiformis CCAP 1017/2 – CYL, Isochrysis galbana CCAP 927/1– ISO, Monodopsis subterranean CCAP 848/1 – MONO, Nannochloropsis oceanica CCAP 849/10– NANNO and a commercial algae paste –SP. The diets had a significant effect on spat GR and WI, and according to their efficacy resulted ranked as follows: ISO>NANNO/CYL>SP>MONO. Spat FA composition and neutral lipid content (principally triacylglycerols - TG), were influenced by the diets. Furthermore, untargeted lipidomics also showed shifts in several phospholipid species, with changes related to the essential PUFA available from the diet. TG content, neutral lipids and several TG and FA species were correlated (Spearman R2>0.8 FDR p<0.05) with spat WI, suggesting their possible application as markers of mussel juvenile condition. The availability of dietary essential PUFA deeply modified the spat lipidome both for neutral and for polar lipids. This change in the lipidome could have major impacts on their ecology and their production for food.

1946: Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets
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Posted to bioRxiv 09 May 2019

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets
91 downloads biochemistry

Qingling Tang, Maria T Villar, Antonio Artigues, John P. Thyfault, Udayan Apte, Hao Zhu, Kenneth R. Peterson, Aron W. Fenton

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Screening mutations that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of our screen. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired screening outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

1947: Structural analysis of nonapeptides derived from elastin
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Posted to bioRxiv 07 Dec 2019

Structural analysis of nonapeptides derived from elastin
90 downloads biochemistry

B Hernandez, JM Crowet, J Thiery, SG Kruglik, N Belloy, S Baud, M Dauchez, L Debelle

Elastin-derived peptides are released from the extracellular matrix remodeling by numerous proteases and seem to regulate many biological processes, notably cancer progression. The canonical elastin peptide is VGVAPG which harbors the XGXXPG consensus pattern allowing interaction with the elastin receptor complex located at the surface of cells. Besides these elastokines, another class of peptides has been identified. This group of bioactive elastin peptides presents the XGXPGXGXG consensus sequence but the reason for their bioactivity remains unexplained. In order to better understand their nature and structure-function relationships, herein we searched the current databases for this nonapeptide motif and observed that the XGXPGXGXG elastin peptides define a specific group of tandemly repeated patterns. Further, we focused on four tandemly repeated human elastin nonapeptides, i.e. AGIPGLGVG, VGVPGLGVG, AGVPGLGVG and AGVPGFGAG. These peptides were analysed by means of optical spectroscopies and molecular dynamics. UV-circular dichroism and Raman spectra are consistent with a conformational equilibrium between beta-turn, beta-strand and random chain secondary elements in aqueous media. This equilibrium was found to be concentration-independent. Quantitative analysis of their conformations suggested that turns corresponded to half of the total population of structural elements while the remaining half was equally distributed between beta-strand and unordered chains. These distributions were confirmed by molecular dynamics simulations. Altogether, our data suggest that these peptides harbor a type II beta-turn located in their central part. We hypothesize that this structural element could explain their specific bioactivity.

1948: The temperature dependence of platelet metabolism
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Posted to bioRxiv 14 Oct 2019

The temperature dependence of platelet metabolism
89 downloads biochemistry

F. Jóhannsson, J.T. Yurkovich, S. Guðmundsson, Ó. E. Sigurjónsson, Ó. Rolfsson

Temperature plays a fundamental role in biology, influencing cellular function, affecting chemical reaction rates, molecular structures, and interactions. While the temperature dependence of many biochemical reactions is well defined in vitro, the effect of temperature on metabolic function at the network level is not well understood but remains an important challenge in optimizing the storage of cells and tissues at lower temperatures. Here, we have used time-course metabolomics data and systems biology approaches to characterize the effects of storage temperature on human platelets (PLTs) in platelet additive solution. We observed that changes to the metabolome with storage time do not simply scale with temperature but instead display complex temperature dependence, with only a small subset of metabolites following an Arrhenius-type. Investigation of PLT energy metabolism through integration with computational modeling revealed that oxidative metabolism is more sensitive to temperature changes than is glycolysis. The increased contribution of glycolysis to ATP turnover at lower temperature indicates a stronger glycolytic phenotype with decreasing storage temperature. More broadly, these results demonstrate that the temperature dependence of the PLT metabolic network is not uniform, suggesting that efforts to improve the health of stored PLTs could be targeted at specific pathways.

1949: High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the "inserted" state of the ATP synthase ϵ-subunit
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Posted to bioRxiv 28 Aug 2019

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the "inserted" state of the ATP synthase ϵ-subunit
89 downloads biochemistry

Yang Liu, Jiayu Yu, Mengyuan Wang, Qingfang Zeng, Xinmiao Fu, Zengyi Chang

ATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of α3β3γδϵab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (α3β3γδϵ) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the ϵ-subunit could adopt either an "inserted" or "non-inserted" state (with or without interacting with the α/β-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an "inserted" state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the ϵ-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the "inserted" and "non-inserted" states of the ϵ-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

1950: FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici
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Posted to bioRxiv 02 Oct 2019

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici
89 downloads biochemistry

Yashwanth Ashok, Mirko M. Maksimainen, Tuija Kallio, Pekka Kilpeläinen, Lari Lehtiö

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared to Aerococcus viridans lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

1951: Substrate recognition by the Pseudomonas aeruginosa EF-Tu methyltransferase EftM
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Posted to bioRxiv 23 Sep 2019

Substrate recognition by the Pseudomonas aeruginosa EF-Tu methyltransferase EftM
89 downloads biochemistry

Emily G. Kuiper, Debayan Dey, Paige A. LaMore, Joshua P. Owings, Samantha M. Prezioso, Joanna B. Goldberg, Graeme L. Conn

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of serious infections in individuals with cystic fibrosis, compromised immune systems, and severe burns. During infection, P. aeruginosa adhesion to host epithelial cells is enhanced by surface exposed translation elongation factor EF-Tu carrying a Lys5 trimethylation. This modification is incorporated by the S-adenosyl-L-methionine-dependent methyltransferase EftM. Thus, EF-Tu modification by EftM may represent a novel target to restrict the establishment of P. aeruginosa infections in vulnerable individuals. Here, we extend our understanding of EftM action by defining the molecular mechanism of EF-Tu substrate recognition by this enzyme. First, following the observation that EftM can bind to EF-Tu lacking an N-terminal peptide (encompassing the Lys5 target site), an EftM homology model was generated and used in protein-protein docking studies to predict EftM:EF-Tu interactions. The predicted protein-protein interface was then experimentally validated using site-directed mutagenesis of residues in both proteins coupled with binding and methyltransferase activity assays. We also show that EftM is unable to methylate the isolated N-terminal EF-Tu peptide and that binding-induced conformational changes in EftM are likely needed to allow placement of the first 5-6 amino acids of EF-Tu into the conserved peptide binding channel. In this channel, a group of residues that are highly conserved in EftM family proteins position the N-terminal sequence to facilitate modification of Lys5. Our findings provide detailed insights into substrate recognition by this lysine methyltransferase, paving the way for a deeper understanding of EftM's mechanism of action on EF-Tu.

1952: S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli
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Posted to bioRxiv 30 Nov 2019

S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli
88 downloads biochemistry

Fiorella Faienza, Matteo Lambrughi, Salvatore Rizza, Chiara Pecorari, Paola Giglio, Juan Salamanca Viloria, Maria Francesca Allega, Giovanni Chiappetta, Joëlle Vinh, Francesca Pacello, Andrea Battistoni, Andrea Rasola, Elena Papaleo, Giuseppe Filomeni

The mitochondrial chaperone TRAP1 has been involved in several mitochondrial functions, and modulation of its expression/activity has been suggested to play a role in the metabolic reprogramming distinctive of cancer cells. TRAP1 posttranslational modifications, i.e. phosphorylation, can modify its capability to bind to different client proteins and modulate its oncogenic activity. Recently, it has been also demonstrated that TRAP1 is S-nitrosylated at Cys501, a redox modification associated with its degradation via the proteasome. Here we report molecular dynamics simulations of TRAP1, together with analysis of long-range structural communication, providing a model according to which Cys501 S-nitrosylation induces conformational changes to distal sites in the structure of the protein. The modification is also predicted to alter open and closing motions for the chaperone function. By means of colorimetric assays and site directed mutagenesis aimed at generating C501S variant, we also experimentally confirmed that selective S-nitrosylation of Cys501 decreases ATPase activity of recombinant TRAP1. Coherently, C501S mutant was more active and conferred protection to cell death induced by staurosporine. Overall, our results provide the first in silico, in vitro and cellular evidence of the relevance of Cys501 S-nitrosylation in TRAP1 biology.

1953: Ibotenic acid biosynthesis in the fly agaric is initiated by glutamate hydroxylation
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Posted to bioRxiv 18 Nov 2019

Ibotenic acid biosynthesis in the fly agaric is initiated by glutamate hydroxylation
88 downloads biochemistry

Sebastian Obermaier, Michael Müller

The fly agaric, Amanita muscaria, is widely known for its content of the psychoactive metabolites ibotenic acid and muscimol. 50 years ago, their biosynthesis was hypothesized to start with 3 hydroxyglutamate. Here, we build on this hypothesis by the identification and recombinant production of a glutamate hydroxylase from A. muscaria. The corresponding gene is surrounded by six other genes, which we link to ibotenic acid production using recent genetic data. Our data provide new insights into a decades-old question concerning a centuries-old drug.

1954: Inhibition of the mitochondrial permeability transition exerts sex-specific stimulatory effect on fracture repair
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Posted to bioRxiv 18 Sep 2019

Inhibition of the mitochondrial permeability transition exerts sex-specific stimulatory effect on fracture repair
88 downloads biochemistry

Brianna H Shares, Tzong-Jen Sheu, Kevin Schilling, Rubens Sautchuk, Ananta Paine, Gisela Beutner, Laura C Shum, Charles O Smith, Emma Gira, Edward Brown, Hani Awad, Roman A Eliseev

Bone fracture is accompanied by mechanical stresses and inflammation; conditions that impair mitochondria via the phenomenon of permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening exacerbates inflammation and cell death and, thus can disrupt fracture repair. Here we tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. Our data indicate that osteoblast activity, bone formation, and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice when compared to controls during fracture repair. These effects were observed in male but not female mice, thus showing sexual dimorphism. Pharmacological inhibition of CypD with NIM811 in male mice also stimulated fracture repair. In addition, CypD knock-out or pharmacological inhibition produced pro-osteogenic effect in isolated bone marrow osteoprogenitors. This in vitro effect was associated with higher mitochondrial respiration and increased β-catenin activity regulated by mitochondria-dependent acetylation. Our findings implicate a sex-specific role of MPTP in bone fracture and suggest CypD inhibition as a modality to promote fracture repair.

1955: A Novel Process Maintaining Glycerophospholipid Homeostasis in Mammalian Cells
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Posted to bioRxiv 13 Nov 2019

A Novel Process Maintaining Glycerophospholipid Homeostasis in Mammalian Cells
88 downloads biochemistry

Martin Hermansson, Satu Hänninen, Matti Kjellberg, Pentti Somerharju

Glycerophospholipid (GPL) homeostasis in eukaryotic cells is thought to be maintained via biosynthesis, degradation and acyl chain remodeling. Here we provide evidence for an additional process termed head-group remodeling where other GPLs, when in excess, are rapidly converted to phosphatidylcholine and triacylglycerol. Mass spectrometric studies showed the formation of diacylglycerol, but not phosphatidic acid, from the exogenous GPL thus indicating that the first step is catalyzed by a phospholipase C-type enzyme. Consistently, triacylglycerol formation was significantly inhibited by the knock-down of several PLCs, but not phospholipase Ds. Second, we found that each exogenous GPL strongly inhibited the synthesis of the corresponding endogenous GPL class. Based on these and previous data we hypothesize how mammalian cells could coordinate the multiple processes contributing to GPL homeostasis in mammalian cells. In conclusion, this study provides the first evidence that head group remodeling plays an important role in GPL homeostasis in mammalian cells.

1956: The "adductome": a limited repertoire of adducted proteins in human cells
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Posted to bioRxiv 11 Dec 2019

The "adductome": a limited repertoire of adducted proteins in human cells
87 downloads biochemistry

Kostantin Kiianitsa, Nancy Maizels

Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCFR-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r=0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins - the "adductome" - is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

1957: Human hepatic tryptophan 2,3-dioxygenase ubiquitin-dependent protein degradation: The critical role of its exosite as the molecular lynchpin of its substrate-mediated protein stabilization
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Posted to bioRxiv 04 Oct 2019

Human hepatic tryptophan 2,3-dioxygenase ubiquitin-dependent protein degradation: The critical role of its exosite as the molecular lynchpin of its substrate-mediated protein stabilization
87 downloads biochemistry

Sung-Mi Kim, Yi Liu, YongQiang Wang, Shay Karkashon, Ariel Lewis-Ballester, Syun-Ru Yeh, Maria Almira Correia

Hepatic tryptophan 2,3-dioxygenase (TDO) is a cytoplasmic homotetrameric hemoprotein and the rate-limiting enzyme in the irreversible degradation of the essential amino acid L-tryptophan (L-Trp) to N-formylkynurenine, thus controlling the flux of L-Trp into its serotonergic and kynureninic/NAD pathways. TDO has long been recognized to be substrate-inducible via protein stabilization, but the molecular mechanism of this stabilization has remained elusive. Recent elucidation of human TDO (hTDO) crystal structure has identified a high-affinity (Kd ≈ 0.5 μM) Trp-binding exosite in each of its 4 monomeric subunits. Mutation of the Glu105, Trp208 and Arg211 comprising this exosite not only abolished the high-affinity L-Trp binding, but also accelerated the ubiquitin-dependent proteasomal degradation of hTDO. We have further characterized this hTDO degradation by documenting that its ubiquitination by gp78/AMFR and CHIP E2/E3 ligase complexes occurs on external Lys-residues within or vicinal to acidic Asp/Glu and phosphorylated pSer/pThr (DEpSpT)-clusters. Furthermore, we have identified the unstructured hTDO N- and C-termini as imparting relatively high proteolytic instability, as their deletion (ΔNC) markedly prolonged hTDO t1/2. Additionally, although previous studies reported that upon hepatic heme-depletion, the heme-free apoTDO turns over with a t1/2 ≈ 2.2 h relative to the t1/2 of 7.7 h of holoTDO, mutating the axial heme-ligating His328 to Ala has the opposite effect of prolonging hTDO t1/2. Most importantly, introducing the exosite mutation into the ΔNC-deleted or H328A-mutant completely abolished their prolonged half-lives irrespective of L-Trp presence or absence, thereby revealing that the exosite is the molecular lynchpin that defines L-Trp-mediated TDO induction via protein stabilization.

1958: Natively Oxidized Amino Acid Residues in the Spinach PS I-LHC I Supercomplex
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Posted to bioRxiv 31 Oct 2019

Natively Oxidized Amino Acid Residues in the Spinach PS I-LHC I Supercomplex
86 downloads biochemistry

Ravindra Kale, Larry Sallans, Laurie K. Frankel, Terry M. Bricker

Reactive oxygen species (ROS) production is an unavoidable byproduct of electron transport under aerobic conditions. Photosystem II (PS II), the cytochrome b6/f complex and Photosystem I (PS I) are all demonstrated sources of ROS. It has been proposed that PS I produces substantial levels of a variety of ROS including O2·-,1O2, H2O2 and, possibly, ·OH, however, the site(s) of ROS production within PS I has been the subject of significant debate. We hypothesize that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in spinach PS I which was isolated from field-grown spinach. The modified residues were identified by high-resolution tandem mass spectrometry. As expected, many of the modified residues lie on the surface of the complex. However, a well-defined group of oxidized residues, both buried and surface-exposed, lead from the chl a' of P700 to the surface of PS I. These residues (PsaB: 609F, 611E, 617M, 619W, 620L, and PsaF: 139L, 142A and 143D) may identify a preferred route for ROS, probably 1O2, to egress the complex from the vicinity of P700. Additionally, two buried residues located in close proximity to A1B (PsaB:712H and 714S) were modified, which may be consistent with A1B being a source of O2·-. Surprisingly, no oxidatively modified residues were identified in close proximity to the 4Fe-FS clusters FX, FA or FB. These cofactors had been identified as a principal targets for ROS damage in the photosystem. Finally, a large number of residues located in the hydrophobic cores of Lhca1-Lhca4 are oxidatively modified. These appear to be the result of 1O2 production by the distal antennae for the photosystem.

1959: Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails
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Posted to bioRxiv 23 Dec 2019

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails
85 downloads biochemistry

M.L. Giglio, S. Ituarte, V. Milesi, M.S. Dreon, T.R. Brola, J. Caramelo, J.C.H. Ip, S. Maté, J.W. Qiu, L.H. Otero, H. Heras

The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles but MACPF but pore-forming toxic function are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 15 Å resolution determined by negative stain electron microscopy (NS-EM) and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. Tachylectin is a six-bladed β-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin B delivery subunit would bind to target membranes, and then its MACPF A toxic subunit disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions give rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.

1960: Topology of the U12-U6atac snRNA complex of the minor spliceosome and binding by NTC-related protein RBM22
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Posted to bioRxiv 31 Oct 2019

Topology of the U12-U6atac snRNA complex of the minor spliceosome and binding by NTC-related protein RBM22
85 downloads biochemistry

Joanna Ciavarella, William Perea, Nancy L Greenbaum

Splicing of precursor messenger RNA is catalyzed by the spliceosome, a dynamic ribonucleoprotein assembly composed of five small nuclear (sn)RNAs and >100 proteins. RNA components catalyze the two transesterification reactions, but proteins perform critical roles in assembly and rearrangement. The catalytic core comprises a paired complex involving U2 and U6 snRNAs for the major form of the spliceosome and U12 and U6atac snRNAs for the minor variant (~0.3% of all spliceosomes in higher eukaryotes); the latter performs identical chemistry, despite limited sequence conservation outside key catalytic elements, and lack of the multi-stem central junction found in the U2-U6 snRNA complex. Here we use solution NMR techniques to show that base pairing patterns of the U12-U6atac snRNA complex of both human and Arabidopsis share key elements with the major spliceosome's U2-U6 snRNA complex; probing of the single-stranded segment opposing termini of the snRNAs indicates elongation in this region in place of the stacked base pairs at the base of the U6 intramolecular stem loop in the U2-U6 snRNA complex. Binding affinity of RBM22, a protein implicated in remodeling human U2-U6 snRNA prior to catalysis, to U12-U6atac was analyzed by electrophoretic mobility shift assays in which we monitored migration of both protein and RNA components in the same gel. Results indicate that RBM22 binds the U2-U6 and U12-U6atac snRNA complexes specifically and with Kd = 3.5 μM and 8.2 μM, respectively. Similar affinity between RBM22 and each RNA complex suggests that the protein performs the same role in both spliceosomes.

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