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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 65,445 bioRxiv papers from 289,895 authors.

Most downloaded bioRxiv papers, all time

in category biochemistry

1,877 results found. For more information, click each entry to expand.

1781: Zinc-independent activation of Toll-like receptor 4 by S100A9
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Posted to bioRxiv 07 Oct 2019

Zinc-independent activation of Toll-like receptor 4 by S100A9
87 downloads biochemistry

Andrea Nicole Loes, Ran Shi, Michael J Harms

The homodimer formed by the protein S100A9 induces inflammation through Toll-like receptor 4 (TLR4), playing critical roles in both healthy and pathological innate immune responses.  The molecular mechanism by which S100A9 activates TLR4 remains unknown.  Previously, the interaction between purified S100A9 and TLR4 was shown to depend on Zn2+; however, the Zn2+ binding site(s) on S100A9 were not identified.  Here, we investigated the role of Zn2+ binding in the pro-inflammatory activity of S100A9.  We found that the S100A9 homodimer was prone to reversible, Zn2+-dependent aggregation in vitro.  Using a combination of site-directed mutagenesis and Isothermal Titration Calorimetry (ITC), we identified multiple residues that contribute to Zn2+ binding in S100A9.  We then used mutagenesis to construct a version of S100A9 with no detectable Zn2+ binding by either ITC or Inductively Coupled Plasma-Mass Spectrometry. This protein did not exhibit aggregation upon addition of saturating Zn2+.  Further, despite the lack of Zn2+-binding, this protein was capable of activating TLR4 in a cell-based functional assay.  We then modified the functional assay so the Zn2+ concentration was exceedingly low relative to the concentration of S100A9 added.  Again, S100A9 was able to activate TLR4.   This reveals that, despite the ability of S100A9 to bind Zn2+, S100A9 does not require Zn2+ to activate TLR4.   Our work represents an important step in clarifying the nature of the interaction between S100A9 and TLR4.

1782: Musculoskeletal fat imaging and quantification by high-resolution metabolite cycling magnetic resonance spectroscopic imaging at 3 T: A fast method to generate separate distribution maps of lipid components
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Posted to bioRxiv 12 Aug 2019

Musculoskeletal fat imaging and quantification by high-resolution metabolite cycling magnetic resonance spectroscopic imaging at 3 T: A fast method to generate separate distribution maps of lipid components
87 downloads biochemistry

Ahmad A Alhulail, Debra A Patterson, Pingyu Xia, Xiaopeng Zhou, Chen Lin, M. Albert Thomas, Ulrike Dydak, Uzay Emir

Purpose: To provide a rapid, non-invasive fat quantification technique capable of producing separate lipid component maps. Methods: The calf muscles in 5 healthy adolescents (age 12-16 years; BMI = 20 ± 3 Kg/m2) were scanned by two different fat fraction (FF) quantification methods. A high-resolution, density-weighted concentric ring trajectory (DW-CRT) metabolite cycling (MC) magnetic resonance spectroscopic imaging (MRSI) technique was implemented to collect data with 0.25 mL resolution within 3 minutes and 16 seconds. For comparative purposes, the standard Dixon technique was performed. The two techniques were compared using structural similarity (SSIM) analysis. Additionally, the difference in the distribution of each lipid over the adolescent calf muscles was assessed based on the MRSI data. Results: The proposed MRSI technique provided individual FF maps for eight musculoskeletal lipids identified by LCModel analysis (L09, L11, L13, L15, L21, L23, L53, and L55) with mean SSIM indices of 0.19, 0.04, 0.03, 0.50, 0.45, 0.04, 0.07, and 0.12, respectively compared to that of Dixon FF map. Further analysis of voxels with zero SSIM demonstrated an increased sensitivity of FF lipid maps from data acquired using this MRSI technique over the standard Dixon technique. The trend of lipid spatial distribution over calf muscles was consistent with previously published findings in adults. Conclusion: The advantages of this MRSI technique make it a useful tool when individual lipid FF maps are desired within a short scanning time.

1783: Categorical Assignment of Pulmonary Embolism is a Simple and More Accurate Indicator of Right Ventricular Dysfunction and Short Team Mortality
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Posted to bioRxiv 20 May 2019

Categorical Assignment of Pulmonary Embolism is a Simple and More Accurate Indicator of Right Ventricular Dysfunction and Short Team Mortality
87 downloads biochemistry

Yu Lin Chen, Colin Wright, Anthony P. Pietropaoli, Ayman Elbadawi, Joseph Delehanty, Bryan Barrus, Igor Gosev, David Trawick, Dhwani Patel, Scott J Cameron

Several risk stratification tools are available to predict short term mortality in patients with acute pulmonary embolism (PE). Right ventricular (RV) dysfunction, which is common to intermediate and high risk PE, is an independent predictor of mortality and may be a faster and simpler way to assess patient risk in acute care settings. We evaluated 571 patients presenting with acute PE as the primary diagnosis, stratifying them by the Pulmonary Embolism Severity Index (PESI), by the BOVA score, or categorically as low risk (no RV dysfunction by imaging), intermediate risk (RV dysfunction by imaging), or high risk PE (RV dysfunction by imaging with sustained hypotension). Using imaging data to firstly define the presence of RV dysfunction, and plasma cardiac troponin T (cTnT) and NTproBNP as additional evidence for myocardial strain, we evaluated the PESI and BOVA scoring systems compared to categorical assignment of PE as low risk, submassive, and massive PE. Cardiac biomarkers poorly distinguished between PESI classes and BOVA stages in patients with acute PE. Cardiac TnT and NTproBNP easily distinguished low risk from submassive PE with an area under the curve (AUC) of 0.84 (95% C.I. 0.73–0.95, p< 0.0001), and 0.88 (95% C.I. 0.79–0.97, p< 0.0001), respectively, and low risk from massive PE with an area under the curve (AUC) of 0.89 (95% C.I. 0.78–1.00, p< 0.0001), and 0.89 (95% C.I. 0.82–0.95, p< 0.0001), respectively. Predicted short term mortality by PESI score or BOVA stage was lower than the observed mortality for submassive PE by a two fold order of magnitude. These data suggest the presence of RV dysfunction in the context of acute PE is sufficient for the purposes of risk stratification, while more complicated risk stratification algorithms may underestimate short term mortality risk.

1784: Upregulation of photosynthetic capacity and thylakoid membrane protein enhanced tolerance to heat stress in wucai (Brassica campestris L.)
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Posted to bioRxiv 11 Dec 2018

Upregulation of photosynthetic capacity and thylakoid membrane protein enhanced tolerance to heat stress in wucai (Brassica campestris L.)
87 downloads biochemistry

Yujie Yuan, Lingyun Yuan, Chenggang Wang, Mengru Zhao, Yun Dai, Shilei Xie, Shidong Zhu, Jingfeng Hou, Guohu Chen

The hot climate of southern China from late summer to early fall is one of the major factors limiting the yield and quality of wucai (Brassica campestris L.). Under high temperature stress, heat-tolerant cultivars presented moderate injury to the photosynthesic apparatus, less inhibition of photochemical activity, better osmotic adjustment and antioxidant defences capacity compared to heat-sensitive cultivars. To study the effects of high temperature on the growth and development of wucai, plants of WS-1 (heat-tolerant) and WS-6 (heat-sensitive) were exposed to four heat stress treatments in growth chambers for 3 days. Chloroplasts of two cultivars evaluated for photosynthetic characteristics, fatty acid composition and differentially expressed proteins of thylakoid membrane. Larger decreases in growth and photosynthetic parameters occurred under heat stress in WS-6, compared with WS-1. In addition, WS-6 showed an obviouse K point in O-J-I-P steps under extremely high temperature, which indicated OEC had been damaged. WS-1 showed higher of maximum quantum yield of primary PSII photochemistry, number of active reaction centres per cross section of PSII, average absorbed photon flux per cross section of PSII, maximum trapped exciton flux per cross section of PSII, electron transport flux from QA to QB per cross section of PSII and performance index on absorption basis which indicated greater heat stability in terms of PSII function under higher temperature. Compared to WS-6, WS-1 showed higher membrane stability and photochemical efficiency, and greater increase of saturated fatty acid, especially palmitic acid under heat stress. WS-1 had higher recovery rate compared to WS-6 after 41℃ heat stress treatment. Additionally, two-dimensional blue native/SDS-PAGE analysis of chloroplast was carried out to compare the differentially expressed proteins between two cultivars. We obtained seven major protein complexes included supercomplexes, PSI-LHCII/PSII monomer, PSII monomer, CP43 less PSII/ATP synthase, LHCII trimer, LHCII monomer and ATP synthase after first dimentional seperation in both cultivars after the first dimensional separation. Then ten differential membrane proteins included light-harvesting Chl a/b-binding protein , ATP synthase subunit alpha, ATP synthase subunit beta, photosystem I P700 chlorophyll a apoprotein A2, photosystem II CP43 reaction center protein, photosystem II D2 protein and photosystem II OS have been found between WS-1 and WS-6. These differentially proteins in cellular membranes could contribute to the differential level of heat tolerance between two wucai cultivars. Our results demonstrated that the heat-tolerant cultivar WS-1 had a greater capacity for photosynthesis and membrane stability by upregulating proteins abundance including light harvesting (light-harvesting Chl a/b-binding protein), energy metabolism (ATPase), and proteins of PSII reaction center (D2, CP43) under heat stress.

1785: Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase
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Posted to bioRxiv 05 Sep 2019

Molecular Mechanism Underlying Inhibition of Intrinsic ATPase Activity in a Ski2-like RNA Helicase
86 downloads biochemistry

Eva Absmeier, Karine F. Santos, Markus C. Wahl

RNA-dependent NTPases can act as RNA/RNA-protein remodeling enzymes and typically exhibit low NTPase activity in the absence of RNA/RNA-protein substrates. How futile intrinsic NTP hydrolysis is prevented is frequently not known. The ATPase/RNA helicase Brr2 belongs to the Ski2-like family of nucleic acid-dependent NTPases and is an integral component of the spliceosome. Comprehensive nucleotide binding and hydrolysis studies are not available for a member of the Ski2-like family. We present crystal structures of Chaetomium thermophilum Brr2 in the apo, ADP-bound and ATPyS-bound states, revealing nucleotide-induced conformational changes and a hitherto unknown ATPyS binding mode. Our results in conjunction with Brr2 structures in other molecular contexts reveal multiple molecular mechanisms that contribute to the inhibition of intrinsic ATPase activity, including an N-terminal region that restrains the RecA-like domains in an open conformation and exclusion of an attacking water molecule, and suggest how RNA substrate binding can lead to ATPase stimulation.

1786: Solution and gas-phase modifiers effect on heme proteins environment and conformational space
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Posted to bioRxiv 27 Jun 2018

Solution and gas-phase modifiers effect on heme proteins environment and conformational space
86 downloads biochemistry

D. Butcher, J. Miksovska, M. E. Ridgeway, M. A. Park, Francisco Fernandez-Lima

The molecular environment is known to impact the secondary and tertiary structure of biomolecules, shifting the equilibrium between different conformational and oligomerization states. In the present study, the effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100 - 500 ms) using trapped ion mobility spectrometry - mass spectrometry. Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) depending on the protein. In the presence of gas-phase modifiers (i.e., N2 containing methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed. We attribute these changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas-phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

1787: Rats Sniff Off Toxic Air
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Posted to bioRxiv 01 Sep 2019

Rats Sniff Off Toxic Air
86 downloads biochemistry

Haoxuan Chen, Xinyue Li, Maosheng Yao

Breathing air is a fundamental human need, yet its safety, e.g., when challenged by various harmful or lethal substances, is often not properly guarded. Currently, air toxicity is monitored only for single or limited number of known toxicants, thus failing to fully warn against possible hazardous air. Here, using a photoionization detector (PID) or GC-MS/FID we found that within minutes living rats emitted distinctive profiles of volatile organic compounds (VOCs) via breath when exposed to various airborne toxicants such as endotoxin, O3, ricin, and CO2. Compared to background indoor air, when exposed to ricin or endotoxin aerosols breath-borne VOC levels, especially that of carbon disulfide, were shown to decrease, while elevated levels were observed for O3 and CO2 exposures. Principal component analysis (PCA) revealed a clear contrast in breath-borne VOCs profiles of rats among different toxicant exposures. MicroRNA regulations such as miR-33, miR-146a and miR-155 from rats' blood samples also suggested varying mechanisms used by the rats in combating different air toxicant challenges. By integrating living rats, breath sampling, and VOC online detection, we pioneered a system that can real-time monitor air toxicity without the need of detecting specific species. Importantly, rats were shown to be able to sniff off toxic air.

1788: Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors
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Posted to bioRxiv 06 Jul 2019

Gsα stimulation of mammalian adenylate cyclases regulated by their hexahelical membrane anchors
86 downloads biochemistry

Anubha Seth, Manuel Finkbeiner, Julia Grischin, Joachim E. Schultz

Nine mammalian adenylate cyclase (mAC) isoforms are pseudoheterodimers with two dissimilar hexahelical membrane-anchors, isoform-specifically conserved for more than half a billion years. In the past, we have postulated a receptor function for these membrane anchors. Here, we exchanged both membrane anchors of the human AC isoform 2 (hAC2) with the hexahelical membrane domain of the quorum-sensing receptor from Vibrio, CqsS, which has a known ligand, Cholera-Autoinducer-1 (CAI-1). In the chimera, cyclase activity was stimulated by Gsα, whereas CAI-1 by itself had no effect. Surprisingly, CAI-1 inhibited Gsα stimulation, shifting the concentration-response curve and attenuating the maximal response. Extending these data we report that Gsα stimulation of hAC2 expressed in Sf9 insect cells is inhibited concentration-dependently by human serum whereas human serum albumin does not. The data establish the existence of an allosteric linkage in mACs, in which the membrane anchors, as receptors, can transduce extracellular signals to the catalytic dimer, regulating the extent of mAC stimulation by G-protein-coupled receptor-signaling. A general mechanistic model of AC regulation is presented which is compatible with all known regulatory inputs into mammalian ACs, i.e. stimulation by Gsα as well as modulations by secondary modifications. The data allow to unequivocally designate the membrane anchors of mammalian ACs as orphan receptors, and thus establish a new level of AC regulation in mammals. This opens the possibility to start a targeted ligand search.

1789: A Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos
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Posted to bioRxiv 01 Nov 2019

A Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos
85 downloads biochemistry

Paul P. Geurink, Raymond Kooij, Aysegul Sapmaz, Sijia Liu, Bo-Tao Xin, George M C Janssen, Peter A van Veelen, Peter ten Dijke, Huib Ovaa

Many reagents have been emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement allowing investigations of the function of individual enzymes in a cellular context. We here report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and whole animals. The probe labels active Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1-2% of total brain protein. UCHL1 variants have been linked with the neurodegenerative disorders Parkinsons and Alzheimers disease. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1 specific research tools exist. We show that the reagents reported here are specific for UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 irreversibly in an activity-dependent manner. Its use is demonstrated by labelling of UCHL1 both in vitro and in cells. We furthermore show that this probe can report UCHL1 activity during the development of zebrafish embryos.

1790: Development of a lipid-based delivery system for the chemotherapeutic compound SN-38
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Posted to bioRxiv 04 Oct 2019

Development of a lipid-based delivery system for the chemotherapeutic compound SN-38
85 downloads biochemistry

Christophe Danelon, Alicia Soler Canton, Niels van den Broek

SN-38 is a chemotherapeutic compound with potent antitumor effects. However, its clinical application is currently limited due to its poor solubility and low stability at physiological pH. Liposomes and cyclodextrins have been long studied for the solubilization and delivery of hydrophobic compounds. Aiming to combine the advantages from both systems, we attempted to develop an SN-38-in-cyclodextrin-in-liposome formulation. We found that the encapsulation of SN-38-SBE-β-CD inclusion complexes in the lumen of liposomes was not possible, owing to the disassembly of liposomes and the formation of lipid nanoparticles, as revealed by size exclusion chromatography and single nanoparticle fluorescence microscopy. Interestingly, the retention time of SN-38 inside SN-38-SBE-β-CD-lipid nanoparticles is higher than in liposomes, whereby SN-38 was directly loaded into the lipid film. The toxicity of purified SN-38-SBE-β-CD-lipid nanoparticles was assayed in cultured cancer cells, showing no therapeutic advantage compared to bulk SN-38-SBE-β-CD complexes. Further formulation optimization, in particular an increased concentration of the nanoparticles, will be necessary to obtain cytotoxicity effects. Moreover, the results highlight the value of fluorescence imaging of single, surface-immobilized nanoparticles, in the development of liposomal delivery systems such as drug-in-cyclodextrin-in-liposomes.

1791: Relationship between epicardial and perivascular fatty tissue and adipokine-cytokine level in coronary artery disease patients
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Posted to bioRxiv 14 Nov 2018

Relationship between epicardial and perivascular fatty tissue and adipokine-cytokine level in coronary artery disease patients
85 downloads biochemistry

Olga Gruzdeva, Evgenya Uchasova, Yulia Dyleva, Daria Borodkina, Olga Akbasheva, Viktoria Karetnikova, Natalia Brel, Kokov Alexander, Olga Barbarash

The aim of this study was to determine the relationship between the thickness of EAT and PVAT and the adipokine-cytokine profile of patients with coronary heart disease, which can be of significant importance for predicting the course of CVD. 84 patients with CVD, were assessed and divided into two groups based on the presence of visceral obesity (VO). In VO patients, the thickness of the epicardial deposits of the left and right ventricles were 1.75 and 1.43 times greater, respectively, than in patients without VO. For patients with VO, the prevalence of the volume of the left anterior descending artery was 10% higher, and the middle third of the envelope artery was 28% higher, when compared to patients without VO. When evaluating inflammatory status, it was established that the concentration of TNF-α and IL-1β, leptin in the blood serum of patients with VO exceeded the values of patients without VO. Level of proinflammatory IL-10 was 2-times lower in patients with VO. The findings of this study show that the increase of EAT and PVAT are independent risk factors of CVD, as well as a possible model for the assessment of drug effectiveness for CVD.

1792: Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells
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Posted to bioRxiv 07 Oct 2019

Non-Canonical Binding of a Small Molecule to Sortilin Alters Cellular Trafficking of ApoB and PCSK9 in Liver Derived Cells
85 downloads biochemistry

Robert P. Sparks, Andres S. Arango, Zachary L Aboff, Jermaine L Jenkins, Wayne C Guida, Emad Tajkhorshid, Charles E Sparks, Janet D Sparks, Rutilio A Fratti

Sortilin regulates hepatic exocytosis and endocytosis of ApoB containing lipoproteins (ApoB-Lp) and mediates the secretion of the subtilase PCSK9. To elucidate connections between these pathways, we previously identified a small molecule (cpd984) that binds to a non-canonical site on Sortilin. In hepatic cells cpd984 augments ApoB-Lp secretion, increases cellular PCSK9 levels, and reduces LDLR expression indicative of reduced secretion of PCSK9. We have shown that insulin-induced ApoB-Lp degradation occurs through Vps34-dependent autophagy. Here we show that the specific Vps34 inhibitor PIK-III enhances ApoB-100 secretion, reducing cellular levels of PCSK9 and Sortilin resulting in reduced LDLR expression, which implicates a role for autophagy in PCSK9 secretion. Results suggest that Sortilin is central to both PCSK9 and ApoB-100 secretion. Finally, we found that cpd984 in yeast blocks CPY secretion while increasing vacuolar homotypic fusion in a Vps10-dependent manner, indicating an evolutionarily conserved mechanism required for lysosomal protease trafficking.

1793: Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection.
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Posted to bioRxiv 31 Jul 2019

Anti-inflammatory Role of Curcumin in LPS Treated A549 cells at Global Proteome level and on Mycobacterial infection.
85 downloads biochemistry

Suchita Singh, Rakesh Arya, Rhishikesh R Bargaje, Mrinal Kumar Das, Subia Akram, Hossain Md. Faruquee, Rajendra Kumar Behera, Ranjan Kumar Nanda, Anurag Agrawal

A diet derived agent Curcumin (Diferuloylmethane), demonstrated its clinical application in inflammation, infection and cancer conditions. Nevertheless, its impact on the proteome of epithelial cells of non-small cell lung carcinoma (NSCLC) still needs further attention. We employed a stable isotope labeling method for cell culture (SILAC) based relative quantitative proteomics and informatics analysis to comprehend global proteome change in A549 cells treated to curcumin and/or Lipopolysaccharide (LPS). Pretreated A549 cells were infected with Mycobacterium tuberculosis H37Rv strain to monitor bacterial entry and load. With exposure to curcumin and LPS, out of the 1492 identified proteins, 305 and 346 proteins showed deregulation respectively. The expression of BID and AIFM1 mitochondrial proteins which play critical role in apoptotic pathway was deregulated in curcumin treated cells. A549 cells treated with curcumin showed higher active mitochondria intensity with respect to LPS treatment. Simultaneous treatment of curcumin and LPS neutralized the effect of LPS. Mycobacterial H37Rv strain incubated with pretreated A549 cells, showed successful internalization with varied bacterial load. Treatment with both curcumin and LPS had similar bacterial load as control cells. This study generated evidence to deepen our understanding on anti-inflammatory role of curcumin and identified targets might be useful as drug targets.

1794: Structural analysis of mycobacterial homoserine transacetylases central to methionine biosynthesis reveals druggable active site
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Posted to bioRxiv 17 Oct 2019

Structural analysis of mycobacterial homoserine transacetylases central to methionine biosynthesis reveals druggable active site
84 downloads biochemistry

Catherine T. Chaton, Emily S. Rodriguez, Robert W. Reed, Jian Li, Cameron W. Kenner, Konstantin V Korotkov

Mycobacterium tuberculosis is cause of the world's most deadly infectious disease. Efforts are underway to target the methionine biosynthesis pathway, as it is not part of the host metabolism. The homoserine transacetylase MetA converts L-homoserine to O-acetyl-L-homoserine at the committed step of this pathway. In order to facilitate structure-based drug design, we determined the high-resolution crystal structures of three MetA proteins, including M. tuberculosis (MtMetA), Mycolicibacterium abscessus (MaMetA), and Mycolicibacterium hassiacum (MhMetA). Comparison to other homoserine transacetylase family-members reveals a high degree of structural conservation. Utilizing homologous structures with bound cofactors, we analyzed the potential ligandability of MetA. The deep active-site tunnel surrounding the catalytic serine yielded a number of consensus clusters during mapping, suggesting that MtMetA is highly druggable.

1795: The effects of terminal tagging on homomeric interactions of the sigma 1 receptor
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Posted to bioRxiv 15 Aug 2019

The effects of terminal tagging on homomeric interactions of the sigma 1 receptor
84 downloads biochemistry

Hideaki Yano, Leanne Liu, Sett Naing, Lei Shi

The sigma 1 receptor (σ1R) has been implicated in cancers, neurological disorders, and substance use disorders. Yet, its molecular and cellular functions have not been well-understood. Recent crystal structures of σ1R reveal a single N-terminal transmembrane segment and C-terminal ligand-binding domain, and a trimeric organization. Nevertheless, outstanding issues surrounding the functional or pharmacological relevance of σ1R oligomerization remain, such as the minimal protomeric unit and the differentially altered oligomerization states by different classes of ligands. Western blot (WB) assays have been widely used to investigate protein oligomerizations. However, the unique topology of σ1R renders several intertwined challenges in WB. Here we describe a WB protocol without temperature denaturization to study the ligand binding effects on the oligomerization state of σ1R. Using this approach, we observed unexpected ladder-like incremental migration pattern of σ1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of intact σ1R construct and the C-terminally tagged σ1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged σ1R constructs show opposite trends to that of the intact construct, suggesting distorted elicitation of the ligand binding effects on oligomerization. Together, our findings indicate that the N-terminus plays an important role in eliciting the impacts of bound ligands, whereas the C-terminus is amenable for modifications for biochemical studies.

1796: Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
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Posted to bioRxiv 27 Sep 2019

Dissecting the structural and functional roles of a vicinal iron-binding site in encapsulated ferritins
84 downloads biochemistry

Cecilia Piergentili, Didi He, Jennifer Ross, Will A Stanley, Laurène Adam, C Logan Mackay, Kevin J Waldron, Dave J Clarke, Jon Marles-Wright

The Encapsulated ferritin-like proteins belong to the universally distributed ferritin superfamily, which function as iron detoxification and storage systems. The encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question due to differences in organisation of the ferroxidase catalytic site and secondary metal binding sites vicinal to this. We have previously identified a putative metal binding site on the inner surface of Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase centre. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron entry site by means of enzymatic assays, mass-spectrometry, and X-ray crystallography. We show that catalysis occurs in the ferroxidase centre and suggest a dual role for the secondary site as an electrostatic trap guiding ferrous ions toward the ferroxidase centre and, at the same time, acting as a barrier protecting the ferroxidase site against non-cognate inhibiting species. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, while enhancing the ferritin ability to undergo catalysis, does not influence the specific function of the secondary site.

1797: Plasma non-esterified fatty acids contribute to increased coagulability in type-2 diabetes through altered plasma zinc speciation
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Posted to bioRxiv 28 Aug 2019

Plasma non-esterified fatty acids contribute to increased coagulability in type-2 diabetes through altered plasma zinc speciation
83 downloads biochemistry

Amélie I. S. Sobczak, Kondwani G. H. Katundu, Fladia A. Phoenix, Siavash Khazaipoul, Ruitao Yu, Fanuel Lampiao, Fiona Stefanowicz, Claudia A. Blindauer, Samantha J. Pitt, Terry K. Smith, Ramzi A. Ajjan, Alan J. Stewart

Zn2+ is an essential regulator of coagulation and its availability in plasma is fine-tuned through buffering by human serum albumin (HSA). Non-esterified fatty acids (NEFAs) transported by HSA reduce its ability to bind/buffer Zn2+. This is important as plasma NEFA levels are elevated in type-2 diabetes mellitus (T2DM) and other diseases with an increased risk of developing thrombotic complications. The presence of 5 mol. eq. of myristate, palmitate, stearate, palmitoleate and palmitelaidate reduced Zn2+ binding to HSA. Addition of myristate and Zn2+ increased thrombin-induced platelet aggregation in platelet-rich plasma and increased fibrin clot density and clot time in a purified protein system. The concentrations of key saturated (myristate, palmitate, stearate) and monounsaturated (oleate, vaccinate) NEFAs positively correlated with clot density in subjects with T2DM (and controls). Collectively, these data strongly support the concept that elevated NEFA levels contribute to an increased thrombotic risk in T2DM through dysregulation of plasma zinc speciation.

1798: HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE
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Posted to bioRxiv 29 Aug 2019

HOPS recognizes each SNARE, assembling ternary trans-complexes for sudden fusion upon engagement with the 4th SNARE
83 downloads biochemistry

Hongki Song, Amy Orr, Max Harner, William T Wickner

Vacuole fusion requires SNAREs, Sec17/18, a Rab, and HOPS. We find that co-incubation of HOPS, proteoliposomes bearing the Rab and R-SNARE, and proteoliposomes with the Rab and any two Q-SNAREs yields a trans complex which includes these 3 SNAREs. The missing Q-SNARE then triggers a burst of fusion, indicating that each HOPS, R-, and QxQy-SNARE trans -complex is an activated intermediate for functional Qz-SNARE incorporation. HOPS can assemble activated fusion intermediates because it recognizes each of the four SNAREs, binding them independently. HOPS-dependent fusion is saturable for each Q-SNARE, indicating saturable functional sites on HOPS. Though a nonspecific tether allows fusion with pre-assembled Q-SNAREs, only HOPS catalyzes fusion when the Q-SNAREs are not pre-assembled by ushering each Q-SNARE into a functional complex. In contrast, there is little spontaneous functional assembly of the 3 Q-SNAREs. HOPS thus recognizes each of the 4 SNAREs to assemble a versatile set of activated fusion intermediates.

1799: Thrombospondin module 1 domain (TSP1) of the matricellular protein CCN3 shows an atypical disulfide pattern and incomplete CWR layers
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Posted to bioRxiv 23 Sep 2019

Thrombospondin module 1 domain (TSP1) of the matricellular protein CCN3 shows an atypical disulfide pattern and incomplete CWR layers
83 downloads biochemistry

Emma-Ruoqi Xu, Aleix Lafita, Alex Bateman, Marko Hyvonen

Members of the CCN (Cyr61/CTGF/Nov) family are a group of matricellular regulatory proteins, essential to a wide range of functional pathways in cell signalling. Through interacting with extracellular matrix components and growth factors via one of its four domains, the CCN proteins are involved in critical biological processes such as angiogenesis, cell proliferation, bone development, fibrogenesis, and tumorigenesis. We present here the crystal structure of the thrombospondin module 1 (TSP1) domain of CCN3 (previously known as Nov), which shares a similar three-stranded fold with the thrombospondin type 1 repeats of thrombospondin-1 and Spondin-1, but with variations in the disulfide connectivity. Moreover, the CCN3 TSP1 lacks the typical pi-stacked ladder of charged and aromatic residues on one side of the domain, as seen in other TSP1 domains. Using conservation analysis among orthologous domains, we show that a charged cluster in the centre of the domain is the most conserved site and predict it to be a potential functional epitope for heparan sulphate binding. This variant TSP1 domain has also been used to revise the sequence determinants of TSP1 domains and derive improved Pfam sequence profiles for identification of novel TSP1 domains in more than 10,000 proteins across diverse phyla. Synopsis The first structure of a thrombospondin module 1 domain (TSP1) from a CCN family matricellular protein has been determined by X-ray crystallography. The structure shows a typical three-stranded fold, but with an incomplete pi-stacked structure that is usually found in these domains. The structure reveals highest conservation in the positively charged central segment, which we predict to be a binding site for heparan sulphates. The atypical features of this domain have been used to revise the definition of the TSP1 domains and identify a number of new domains in sequence databases.

1800: Partial metal ion saturation of C2 domains primes Syt1-membrane interactions
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Posted to bioRxiv 18 Oct 2019

Partial metal ion saturation of C2 domains primes Syt1-membrane interactions
82 downloads biochemistry

Sachin Katti, Sarah B. Nyenhuis, Bin Her, David S. Cafiso, Tatyana I Igumenova

Synaptotagmin 1 (Syt1) is an integral membrane protein that acts as a Ca2+ sensor of neurotransmitter release. How the Ca2+-sensing function of Syt1 is coupled to its interactions with anionic membranes and synaptic fusion machinery is not well understood. Here, we investigated the dynamics and membrane-binding properties of Syt1 under conditions where its highest affinity Ca2+ sites, which are thought to drive the initial membrane recruitment, are selectively populated by divalent metal ions. To create such protein states for the Ca2+-sensing C2 domains of Syt1, we exploited the unique chemistry of Pb2+, a xenobiotic metal ion that is isostructural and isofunctional to Ca2+. NMR experiments revealed that binding of a single metal ion results in the loss of conformational plasticity of the C2 domain loop regions that are involved in both coordinating metal ions and membrane interactions. In the C2A domain, a single metal ion is sufficient to drive its weak association with PtdSer-containing membranes; in C2B, it enhances the interactions with the signaling lipid PtdIns(4,5)P2. In full-length Syt1, both C2 domains associate with PtdSer-containing membranes, with the depth of insertion modulated by the occupancy of the metal ion sites. Our data suggest that Syt1 adopts a shallow membrane-bound state upon initial recruitment of its C2 domains to the membranes. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, 'prime' Syt1 for binding a full complement of metal ions required for activation of protein function.

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