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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 88,878 bioRxiv papers from 381,072 authors.

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in category biochemistry

2,870 results found. For more information, click each entry to expand.

1861: Extraction of a recombinant full-length NADPH-cytochrome P450 oxidoreductase from bacterial membranes: effect of detergents and additives
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Posted to bioRxiv 08 Mar 2018

Extraction of a recombinant full-length NADPH-cytochrome P450 oxidoreductase from bacterial membranes: effect of detergents and additives
249 downloads biochemistry

Sara A. Arafeh, Azamat R. Galiakhmetov, Elizaveta A. Kovrigina, Eric Fellin, Evgenii L Kovrigin

NADPH-cytochrome P450 oxidoreductase (POR) is a membrane protein in the endoplasmic reticulum of eukaryotic cells. POR is as a key reducing partner for a number of cytochrome P450 proteins involved in different metabolic degradation and signaling pathways. Preparation of the full-length recombinant POR expressed in bacteria has been reported and, typically, involved the use of Triton X-100 detergent for extraction of the overexpressed POR from bacterial membranes. However, extraction efficiency is always relatively low hindering structural studies, particularly-the NMR spectroscopy requiring isotopic enrichment. In this paper, we assessed the effect of a variety of detergents and additives on the efficiency of the membrane-extraction step in POR preparation protocol. We evaluated non-ionic detergents with the variable hydrophobicity (Triton X-100, X-114, and X-405) and structure (Triton X-100, TWEEN-20, Brij-35), a zwitterionic/non-ionic detergent combination (Triton X-100 and CHAPS), as well as a range of alkylamines and polyamines as additives to the conventional extraction buffer containing Triton X-100. None of the detergents or detergent-additive combinations yielded better extraction efficiency than the conventional protocol with the Triton X-100. Lack of variation of the extraction yield allows to hypothesize that the conventional protocol extracts all of the available natively-folded monomeric POR while the remaining fraction is possibly an unfolded aggregated POR, which did not insert in the membranes during expression. We propose that the yield of soluble POR may be increased by a careful optimization of expression conditions while monitoring the distribution of POR between soluble and insoluble fractions in the detergent extraction step.

1862: Systems pathology analysis identifies neurodegenerative nature of age-related retinal diseases
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Posted to bioRxiv 15 Jan 2018

Systems pathology analysis identifies neurodegenerative nature of age-related retinal diseases
249 downloads biochemistry

Tiina Öhman, Fitsum Tamene, Helka Göös, Sirpa Loukovaara, Markku Varjosalo

Aging is a phenomenon associated with profound medical implications. Idiopathic epiretinal membrane (iEMR) and macular hole (MH) are the major vision-threatening vitreoretinal diseases affecting millions of aging people globally, making these conditions an important public health issue. The iERM is characterized by fibrous tissue developing on the surface of the macula, leading to biomechanical and biochemical macular damage. MH is a small breakage in the macula associated with many ocular conditions. Although several individual factors and pathways are suggested, a systems pathology level understanding of the molecular mechanisms underlying these disorders is lacking. Therefore, we performed mass spectrometry based label-free quantitative proteomics analysis of the vitreous proteomes from patients with iERM (n=26) and MH (n=21) to identify the key proteins as well as the multiple interconnected biochemical pathways contributing to the development of these diseases. We identified a total of 1014 unique proteins, of which many were linked to inflammation and complement cascade, revealing the inflammational processes in retinal diseases. Additionally, we detected a profound difference in proteomes of the iEMR and MH compared to the non-proliferative diabetic retinopathy. A large number of neuronal proteins were present at higher levels in iERM and MH vitreous, including neuronal adhesion molecules, nervous system development proteins and signalling molecules. This points toward the important role of neurodegeneration component in the pathogenesis of age-related vitreoretinal diseases. Despite of marked similarities, several unique vitreous proteins were identified in both iERM and MH conditions, providing a candidate targets for diagnostic and new therapeutic approaches. Identification of previously reported and novel proteins in human vitreous humor from patient with iERM and MH provide renewed understanding of the pathogenesis of age-related vitreoretinal diseases.

1863: A novel redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily
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Posted to bioRxiv 14 Jan 2020

A novel redox-active switch in Fructosamine-3-kinases expands the regulatory repertoire of the protein kinase superfamily
249 downloads biochemistry

Safal Shrestha, Samiksha Katiyar, Carlos E. Sanz-Rodriguez, Nolan R. Kemppinen, Hyun W. Kim, Renuka Kadirvelraj, Charalampos Panagos, Neda Keyhaninejad, Maxwell Colonna, Pradeep Chopra, Dominic P Byrne, Geert J. Boons, Esther van der Knaap, Patrick A Eyers, Arthur S. Edison, Zachary A. Wood, Natarajan Kannan

Aberrant regulation of metabolic kinases by altered redox homeostasis is a major contributing factor in aging and disease such as diabetes. However, the biochemical mechanisms by which metabolic kinases are regulated under oxidative stress is poorly understood. In this study, we demonstrate that the catalytic activity of a conserved family of Fructosamine-3-kinases (FN3Ks), which are evolutionarily related to eukaryotic protein kinases (ePKs), are regulated by redox-active cysteines in the kinase domain. By solving the crystal structure of FN3K homolog from Arabidopsis thaliana (AtFN3K), we demonstrate that it forms an unexpected strand-exchange dimer in which the ATP binding P-loop and adjoining beta strands are swapped between two chains in the dimer. This dimeric configuration is characterized by strained inter-chain disulfide bonds that stabilize the P-loop in an extended conformation. Mutational analysis and solution studies confirm that the strained disulfides function as redox 'switches' to reversibly regulate FN3K activity and dimerization. Consistently, we find that human FN3K (HsFN3K), which contains an equivalent P-loop Cys, is also redox-sensitive, whereas ancestral bacterial FN3K homologs, which lack a P-loop Cys, are not. Furthermore, CRISPR knockout of FN3K in human HepG2 cells results in significant upregulation of redox metabolites including glutathione. We propose that redox regulation evolved progressively in FN3Ks in response to changing cellular redox conditions. Our studies provide important new insights into the origin and evolution of redox regulation in the protein kinase superfamily and open new avenues for targeting HsFN3K in diabetic complications. ### Competing Interest Statement The authors have declared no competing interest.

1864: 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
249 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.

1865: The viral SUMO-targeted Ubiquitin Ligase ICP0 is phosphorylated and activated by host kinase Chk2
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Posted to bioRxiv 21 Jul 2019

The viral SUMO-targeted Ubiquitin Ligase ICP0 is phosphorylated and activated by host kinase Chk2
249 downloads biochemistry

Dambarudhar SS Hembram, Hitendra Negi, Poulomi Biswas, Vasvi Tripathi, Lokesh Bhushan, Divya Shet, Vikas Kumar, Ranabir Das

When the Herpes Simplex virus (HSV) genome enters the nucleus for replication and transcription, phase-segregated nuclear protein bodies called PML Nuclear Bodies (PML NBs) colocalize with the genome and repress it. HSV encodes a SUMO-targeted Ubiquitin ligase ICP0 that degrades PML NBs to alleviate the repression. The molecular mechanism used by ICP0 to target PML NBs is unclear. For reasons unknown, the growth of HSV is dependent on the ATM/Chk2 pathway. Here we identify a bonafide SUMO-Interacting motif in ICP0 (SLS4) that is essential and sufficient to target SUMOylated proteins in PML NBs like PML and Sp100. Phosphorylation of SLS4 creates new salt-bridges between SUMO and SLS4, increases the SUMO/SLS4 affinity and switches ICP0 into a potent STUbL. We also report that ICP0 exploits the kinase Chk2 to phosphorylate SLS4 and enhance its STUbL activity. Our results uncover how a viral STUbL counters antiviral response by exploiting an unprecedented mechanism involving three post-translational modifications; ubiquitination, SUMOylation, and phosphorylation.

1866: Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation
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Posted to bioRxiv 27 Nov 2017

Dimerization of the Pragmin pseudo-kinase regulates protein tyrosine phosphorylation
248 downloads biochemistry

Céline Lecointre, Valérie simon, Clément kerneur, Frédéric Allemand, Aurélie Fournet, Ingrid Montarras, Jean-Luc Pons, Muriel Gelin, Constance Brignatz, Serge Urbach, Gilles Labesse, Serge Roche

The pseudo-kinase and signaling protein Pragmin has been linked to cancer by regulating protein tyrosine phosphorylation via unknown mechanisms. Here we present the crystal structure of the Pragmin 906-1368 amino acids C-terminus, which encompasses its kinase domain. We show that Pragmin contains a classical protein kinase fold devoid of catalytic activity. A particular inhibitory triad, conserved in a Pragmin/SgK269/PEAK1/C19orf35 superfamily, tightly holds the catalytic lysine (K997) to prevent ATP binding. By proteomics, we discovered that this pseudo-kinase uses the tyrosine kinase CSK to induce protein tyrosine phosphorylation in human cells. Interestingly, the protein kinase domain is bordered by N- and C-terminal extensions forming an original dimerization domain that regulates Pragmin self-association and stimulates CSK activity. A1329E mutation in the C-terminal extension destabilizes Pragmin dimerization and reduces CSK activation. Thus, our results reveal a new dimerization mechanism by which a pseudo-kinase can induce protein tyrosine phosphorylation.

1867: Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2
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Posted to bioRxiv 16 May 2019

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2
248 downloads biochemistry

Laurel M. Pegram, Jennifer C. Liddle, Yao Xiao, Maria Hoh, Johannes Rudolph, Dylan B. Iverson, Guy P. Vigers, Darin Smith, Hailong Zhang, Weiru Wang, John G. Moffat, Natalie G. Ahn

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dual-phosphorylation induces global motions involving exchange between two states, "L" and "R". We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L⇌R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L⇌R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L⇌R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.

1868: The kynurenine pathway is essential for rhodoquinone biosynthesis in Caenorhabditis elegans
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Posted to bioRxiv 22 May 2019

The kynurenine pathway is essential for rhodoquinone biosynthesis in Caenorhabditis elegans
248 downloads biochemistry

Paloma M. Roberts Buceta, Laura Romanelli-Cedrez, Shannon J. Babcock, Helen Xun, Miranda L. VonPaige, Thomas W. Higley, Tyler D. Schlatter, Dakota C. Davis, Julia A. Drexelius, John C. Culver, Inés Carrera, Jennifer N. Shepherd, Gustavo Salinas

A key metabolic adaptation for some species that face hypoxia as part of their life-cycle involves an alternative electron transport chain in which rhodoquinone (RQ) is required for fumarate reduction and ATP production. RQ biosynthesis in bacteria and protists requires ubiquinone (Q) as a precursor. In contrast, Q is not a precursor for RQ biosynthesis in animals such as parasitic helminths, and this pathway has remained elusive. We used Caenorhabditis elegans as a model animal to elucidate several key steps in RQ biosynthesis. Through RNA interference and a series of mutants, we found that arylamine metabolites from the kynurenine pathway are essential precursors for RQ biosynthesis de novo . Deletion of kynu-1 , which encodes a kynureninase that converts L-kynurenine (KYN) into anthranilic acid (AA), and 3-hydroxykynurenine (HKYN) into 3-hydroxyanthranilic acid (3HAA), completely abolishes RQ biosynthesis, but does not affect Q levels. Deletion of kmo-1 , which encodes a kynurenine 3-monooxygenase that converts KYN to HKYN, drastically reduces RQ, but not Q levels. Knockdown of the Q biosynthetic genes, coq-5 and coq-6 , affects both Q and RQ levels demonstrating that common enzymes are used in both biosynthetic pathways. Our study reveals that two pathways for RQ biosynthesis have independently evolved. In contrast to bacteria, where amination is the last step in RQ biosynthesis, worms begin with the arylamine precursor, AA or 3HAA. Since RQ is absent in mammalian hosts of helminths, inhibition of RQ biosynthesis may have broad implications for targeting parasitic infections which cause important neglected tropical diseases. * ETC : electron transport chain HA : anthranilic acid 3HAA : 3-hydroxyanthranilic acid HKYN : 3-hydroxykynurenine KYN : kynurenine Q : ubiquinone RQ : rhodoquinone.

1869: Global analysis of protein degradation in prion infected cells
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Posted to bioRxiv 23 Jul 2019

Global analysis of protein degradation in prion infected cells
247 downloads biochemistry

Charles Hutti, Kevin A Welle, Jennifer R Hryhorenko, Sina Ghaemmaghami

Prion diseases are rare neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates have the potential to globally influence protein degradation kinetics within an infected cell. We analyzed the proteome-wide effect of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The analysis quantified the degradation rates of more than 4,700 proteins in prion-infected and uninfected cells. As expected, the degradation rate of the prion protein is significantly decreased upon aggregation in infected cells. In contrast, the degradation kinetics of the remainder of the N2a proteome generally increases upon prion infection. This effect occurs concurrently with increases in the cellular activities of autophagy and lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.

1870: Molecular control of gene expression by Brucella BaaR, an IclR-family repressor
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Posted to bioRxiv 22 Jan 2018

Molecular control of gene expression by Brucella BaaR, an IclR-family repressor
247 downloads biochemistry

Julien Herrou, Daniel M. Czyż, Aretha Fiebig, Jonathan W Willett, Youngchang Kim, Ruiying Wu, Gyorgy Babnigg, Sean Crosson

The Brucella abortus general stress response sigma factor, σE1, directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection. Characterizing the functions of σE1-regulated genes therefore contributes to understanding of B. abortus physiology and infection biology. Transcription of the IclR family regulator, Bab2_0215, is indirectly activated by σE1 but its function remains undefined. We present a structural and functional characterization of Bab2_0215, which we have named Brucella adipic acid activated regulator (BaaR). BaaR adopts a classic IclR-family fold and directly regulates the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and represses transcription. We identified three carboxylic acids (adipic acid tetradecanedioic acid, ε-aminocaproic acid) and a lactone (ε-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhance transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid requires the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a set of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program regulated downstream of σE1.

1871: A canstatin-derived peptide provides insight into the role of Capillary Morphogenesis Gene 2 in angiogenic regulation and matrix uptake
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Posted to bioRxiv 17 Jul 2019

A canstatin-derived peptide provides insight into the role of Capillary Morphogenesis Gene 2 in angiogenic regulation and matrix uptake
247 downloads biochemistry

Jordan G. Finnell, Tsz-Ming Tsang, Lorna Cryan, Samuel Garrard, Sai-Lun Lee, P. Christine Ackroyd, Michael S. Rogers, Kenneth A. Christensen

Capillary Morphogenesis Gene 2 protein (CMG2) is a transmembrane, integrin-like receptor and the primary receptor for the anthrax toxin. In addition to its role as an anthrax toxin receptor, CMG2 has been repeatedly shown to play a role in angiogenic processes. However, the molecular mechanism mediating observed CMG2-related angiogenic effects has not been fully elucidated. Previous studies have found that CMG2 binds type IV collagen (Col-IV), a key component of the vascular basement membrane, as well as other ECM proteins. Currently, no link has been made between these CMG2-ECM interactions and angiogenesis; however, ECM fragments are known to play a role in regulating angiogenesis. Here, we further characterize the CMG2-Col-IV interaction and explore the effect of this interaction on angiogenesis. Using a peptide array, we observed that CMG2 preferentially binds peptide fragments of the NC1 (non-collagenous domain 1) domains of Col-IV. These domains are also known as the fragments arresten (from the α1 chain) and canstatin (from the α2 chain) and have documented antiangiogenic properties. A second peptide array was probed to map a putative binding epitope. A top hit from the initial array, a canstatin-derived peptide, binds to the CMG2 ligand-binding von Willebrand factor A (vWA) domain with sub-micromolar affinity (peptide S16, Kd = 400 ± 200 nM). This peptide competes with anthrax protective antigen (PA) for CMG2 binding, and does not bind CMG2 in the presence of EDTA. Together these data suggest that, like PA, S16 interacts with CMG2 at the metal-ion dependent adhesion site (MIDAS) of its vWA domain. We demonstrate that CMG2 specifically mediates endocytic uptake of S16, since CMG2-/- endothelial cells show markedly reduced S16 uptake, without reducing total endocytosis. Furthermore, we show that S16 reduces endothelial migration but not cell proliferation. Taken together, our data demonstrate that a Col IV-derived anti-angiogenic peptide acts via CMG2, suggesting a possible link between CMG2-Col IV interactions and angiogenesis.

1872: Substrate-assisted Enzymatic Formation of Lysinoalanine in Duramycin
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Posted to bioRxiv 08 Jul 2018

Substrate-assisted Enzymatic Formation of Lysinoalanine in Duramycin
247 downloads biochemistry

Linna An, Dillon P. Cogan, Claudio D. Navo, Gonzalo Jiménez-Osés, Satish K. Nair, Wilfred A. van der Donk

Duramycin is a heavily post-translationally modified peptide that binds phosphatidylethanolamine. It has been investigated as an antibiotic, inhibitor of viral entry, therapeutic for cystic fibrosis, and tumor and vasculature imaging agent. Duramycin contains a β-hydroxylated Asp (Hya) and four macrocycles, including an essential lysinoalanine (Lal) crosslink. The mechanism of Lal formation is not known. We here show that Lal is installed stereospecifically by DurN via addition of Lys19 to a dehydroalanine. The structure of DurN reveals an unusual dimer with a new fold. Surprisingly, in the structure of duramycin bound to DurN, no residues of the enzyme are near the Lal. Instead, Hya15 of the substrate makes interactions with Lal suggesting it acts as a base to deprotonate Lys19 during catalysis. Biochemical data suggest that DurN preorganizes the reactive conformation of the substrate, such that the Hya15 of the substrate can serve as the catalytic base for Lal formation.

1873: The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles
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Posted to bioRxiv 10 Oct 2017

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles
247 downloads biochemistry

Y. Rose Citron, Carey J. Fagerstrom, Bettina Keszthelyi, Bo Huang, Nasser M. Rusan, Mark J S Kelly, David Agard

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes a significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution interactions are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results we suggest a model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.

1874: A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5
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Posted to bioRxiv 23 Jan 2020

A Highly Selective Chemical Probe for Activin Receptor-like Kinases ALK4 and ALK5
247 downloads biochemistry

Thomas Hanke, Jong Fu Wong, Benedict-Tilmann Berger, Ismahan Abdi, Lena Marie Berger, Roberta Tesch, Claudia Tredup, Alex N Bullock, Susanne Müller, Stefan Knapp

The transforming growth factor beta-receptor I/activin receptor-like kinase 5 (TGFBR1/ALK5) and its close homologue ALK4 are receptor protein kinases associated with the development of diverse diseases, including cancer, fibrosis, heart diseases and dysfunctional immune response. Therefore, ALK4/5 are among the most studied kinases and several inhibitors have been developed. However, current commercially available inhibitors either lack selectivity or have not been comprehensively characterized, limiting their value for studying ALK4/5 function in cellular systems. To this end, we report the characterization of the 2-oxo-imidazopyridine, TP-008, a potent chemical probe with dual activity for ALK4 and ALK5 as well as the development of a matching negative control compound. TP-008 has excellent cellular potency and strongly abrogates phosphorylation of the substrate SMAD2 (mothers against decapentaplegic homolog 2). Thus, this chemical probe offers an excellent tool for mechanistic studies on the ALK4/5 signaling pathway and the contribution of these targets to disease.

1875: Functionalized Mesoporous Silicas Direct Structural Polymorphism of Amyloid-β Fibrils
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Posted to bioRxiv 14 Jan 2020

Functionalized Mesoporous Silicas Direct Structural Polymorphism of Amyloid-β Fibrils
247 downloads biochemistry

Michael J. Lucas, Henry S Pan, Eric J Verbeke, Lauren J. Webb, David W. Taylor, Benjamin K. Keitz

The aggregation of Amyloid-β (Aβ) is associated with the onset of Alzheimers Disease (AD) and involves a complex kinetic pathway as monomers self-assemble into fibrils. A central feature of amyloid fibrils is the existence of multiple structural polymorphs, which complicates the development of disease-relevant structure-function relationships. Developing these relationships requires new methods to control fibril structure. In this work, we demonstrate that mesoporous silicas (SBA-15) functionalized with hydrophobic (SBA-PFDTS) and hydrophilic groups (SBA-PEG) direct the aggregation kinetics and resulting structure of Aβ1-40 fibrils. The hydrophilic SBA-PEG had little effect on amyloid kinetics while as-synthesized and hydrophobic SBA-PFDTS accelerated aggregation kinetics. Subsequently, we quantified the relative population of fibril structures formed in the presence of each material using electron microscopy. Fibrils formed from Aβ1-40 exposed to SBA-PEG were structurally similar to control fibrils. In contrast, Aβ1-40 incubated with SBA-15 or SBA-PFDTS formed fibrils with shorter cross-over distances that were more structurally representative of fibrils found in AD patient-derived samples. Overall, these results suggest that mesoporous silicas and other exogenous materials are promising scaffolds for the de novo production of specific fibril polymorphs of Aβ1-40 and other amyloidogenic proteins.

1876: Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless
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Posted to bioRxiv 09 Nov 2018

Structure, dynamics and roX2-lncRNA binding of tandem double-stranded RNA binding domains dsRBD1,2 of Drosophila helicase Maleless
246 downloads biochemistry

Pravin Kumar Ankush Jagtap, Marisa Müller, Pawel Masiewicz, Sören von Bülow, Nele Merret Hollmann, Bernd Simon, Andreas W Thomae, Peter B. Becker, Janosch Hennig

Maleless (MLE) is an evolutionary conserved member of the DExH family of helicases in Drosophila. Besides its function in RNA editing and presumably siRNA processing, MLE is best known for its role in remodelling non-coding roX RNA in the context of X chromosome dosage compensation in male flies. MLE and its human orthologue, DHX9 contain two tandem double-stranded RNA binding domains (dsRBDs) located at the N-terminal region. The two dsRBDs are essential for localization of MLE at the X-territory and it is presumed that this involves binding roX secondary structures. However, for dsRBD1 roX RNA binding has so far not been described. Here, we determined the solution NMR structure of dsRBD1 and dsRBD2 of MLE in tandem and investigated its role in double-stranded RNA (dsRNA) binding. Our NMR data show that both dsRBDs act as independent structural modules in solution and are canonical, non-sequence-specific dsRBDs featuring non-canonical KKxAK RNA binding motifs. NMR titrations combined with filter binding experiments document the contribution of dsRBD1 to dsRNA binding in vitro. Curiously, dsRBD1 mutants in which dsRNA binding in vitro is strongly compromised do not affect roX2 RNA binding and MLE localization in cells. These data suggest alternative functions for dsRBD1 in vivo.

1877: Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases
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Posted to bioRxiv 30 May 2019

Multiplexable fluorescence lifetime imaging (FLIM) probes for Abl and Src-family kinases
246 downloads biochemistry

Nur P. Damayanti, Sampreeti Jena, Jackie Tan, Joseph M. K. Irudayaraj, L. L. Parker

Most commonly employed strategies to map kinase activities in live cells require expression of genetically-encoded proteins (e.g. FRET sensors). In this work, we describe development and preliminary application of a set of cell-penetrating, fluorophore labelled peptide substrates for fluorescence lifetime imaging (FLIM) of Abl and Src-family kinase activities. These probes do not rely on FRET pairs or genetically-encoded protein expression. We also demonstrate image-by-image and pixel-by-pixel quantification of probe phosphorylation ratio, suggesting that this strategy will be useful for detailed mapping of single cell and subcellular kinase activity in live cells.

1878: Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration
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Posted to bioRxiv 10 Sep 2019

Metabolite therapy guided by liquid biopsy proteomics delays retinal neurodegeneration
246 downloads biochemistry

Katherine J. Wert, Gabriel Velez, Kanchustambham Vijayalakshmi, Vishnu Shankar, Jesse D. Sengillo, Richard N. Zare, Alexander G. Bassuk, Stephen H. Tsang, Vinit B. Mahajan

Neurodegenerative diseases are debilitating, incurable disorders caused by progressive neuronal cell death. Retinitis pigmentosa (RP) is a blinding neurodegenerative disease that results in retinal photoreceptor cell death and progresses to the loss of the entire neural retinal network. We previously found that proteomic analysis of the adjacent vitreous serves as way to indirectly biopsy the neural retina and identify changes in the retinal proteome. We therefore analyzed protein expression in liquid vitreous biopsies from autosomal recessive retinitis pigmentosa (arRP) patients with PDE6A mutations and arRP mice with Pde6ɑ mutations. Proteomic analysis of retina and vitreous samples identified molecular pathways affected at the onset of photoreceptor cell death. Based on affected molecular pathways, arRP mice were treated with a ketogenic diet or metabolites involved in fatty-acid synthesis, oxidative phosphorylation, and the tricarboxylic acid (TCA) cycle. Dietary supplementation of a single metabolite, ɑ-ketoglutarate, increased docosahexaeonic acid (DHA) levels, provided neuroprotection, and enhanced visual function in arRP mice. A ketogenic diet delayed photoreceptor cell loss, while vitamin B supplementation had a limited effect. Finally, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) revealed restoration of key metabolites that correlated with our proteomic findings: pyrimidine and purine metabolism (uridine, dihydrouridine, and thymidine), glutamine and glutamate (glutamine/glutamate conversion), and succinic and aconitic acid (TCA cycle). This study demonstrates that replenishing TCA cycle metabolites via oral supplementation prolongs vision and provides a neuroprotective effect on the photoreceptor cells and inner retinal network.

1879: Indirect Sexual Selection Drives Rapid Sperm Protein Evolution
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Posted to bioRxiv 29 Jun 2019

Indirect Sexual Selection Drives Rapid Sperm Protein Evolution
246 downloads biochemistry

Damien B. Wilburn, Lisa M. Tuttle, Rachel E. Klevit, Willie J. Swanson

Sexual selection can explain rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if they are not directly involved in fertilization. Here we demonstrate that FITZAP, an intrinsically disordered sperm protein in the marine mollusk abalone, exploits differences in the intracellular and oceanic ionic environments to package the fertilization protein lysin at extraordinary concentrations inside sperm by forming Fuzzy Interacting Transient Zwitterion (FITZ) complexes. FITZAP binds lysin at the same protein interface as its egg receptor VERL, and as sexual selection rapidly alters the lysin-VERL interface, FITZAP coevolves rapidly to maintain lysin binding. Consequently, FITZAP-lysin interactions exhibit a similar species-specificity as lysin-VERL interactions. Thus, tethered molecular arms races driven by sexual selection can generally explain rapid sperm protein evolution. One Sentence Summary Structural study of sperm proteins reveals a novel protein packaging/dispersion system embedded in a coevolutionary arms race.

1880: Mechanistic insights into NAD synthase NMNAT chaperoning phosphorylated Tau from pathological aggregation
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Posted to bioRxiv 10 Oct 2019

Mechanistic insights into NAD synthase NMNAT chaperoning phosphorylated Tau from pathological aggregation
246 downloads biochemistry

Xiaojuan Ma, Jinxia Lu, Yi Zhu, Jingfei Xie, Chong Li, Austin Shin, Jiali Qiang, Jiaqi Liu, Shuai Dou, Yi Xiao, Chuchu Wang, Chunyu Jia, Houfang Long, Yanshan Fang, Lin Jiang, Yaoyang Zhang, Shengnan Zhang, R. Grace Zhai, Cong Liu, Dan Li

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining multiple approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD metabolism and Tau homeostasis in aging and neurodegeneration.

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