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Results for “Biochemistry”

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bioRxiv2019

Interaction of NPC2 protein with Lysobisphosphatidic Acid is required for normal endolysosomal cholesterol trafficking

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates by the LE/LY phospholipid lysobisphosphatidic acid (LBPA) and in these studies sought to determine their functional relationship in normal LE/LY cholesterol egress. Here we demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Using its precursor phosphatidylglycerol (PG), we show that PG-induced LBPA enrichment results in clearance of accumulated cholesterol from NPC1-deficient cells but is ineffective in cells lacking functional NPC2. Together these studies reveal a heretofore unknown aspect of intracellular cholesterol trafficking, in which NPC2 and LBPA function together in an obligate step of sterol egress from the LE/LY compartment, which appears to be independent of NPC1.

biochemistry↗

bioRxiv2019

LRRC33 is a novel binding and regulating protein of TGF-β1 function in human acute myeloid leukemia cells

Transforming growth factor - {beta}1 (TGF-{beta}1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-{beta}1 requires release of the growth factor from pro-complexes and is regulated through TGF-{beta} binding proteins. Two types of TGF-{beta} binding partners, latent TGF-{beta}-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-{beta}1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-{beta}1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-{beta}1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-{beta}1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-{beta}1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-{beta}1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-{beta} regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy.

biochemistry↗

bioRxiv2019

Flv1-4 proteins function in versatile combinations in O2 photoreduction in cyanobacteria

Flavodiiron proteins (FDPs) constitute a group of modular enzymes widespread in all life Domains. Synechocystis sp. PCC 6803 has four FDPs (Flv1-4) essential for photoprotection of photosynthesis. A direct comparison of the Mehler-like reaction (O2 photoreduction) in high Ci (3% CO2, HC) and low Ci (air level CO2, LC) acclimated cells demonstrated that the Flv1/Flv3 heterodimer is responsible for an efficient steady-state O2 photoreduction under HC, with flv2 and flv4 expression strongly down-regulated. Conversely, under LC conditions Flv1/Flv3 acts only as a transient electron sink due to competing withdrawal of electrons by the highly induced NDH-1 complex. Further, in vivo evidence is provided indicating that Flv2/Flv4 contributes to the Mehler-like reaction when naturally expressed under LC conditions, or when artificially overexpressed under HC. The O2 photoreduction driven by Flv2/Flv4 occurs down-stream of PSI in a coordinated manner with Flv1/Flv3 and supports slow and steady-state O2 photoreduction.

biochemistry↗

bioRxiv2019

Synthetic Dual-Acting Small-Molecule Inhibitors That Target Mycobacterial DNA Replication

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium and a causative agent of tuberculosis (TB), a disease that kills more than 1.5 million people worldwide annually. One of the main reasons for this high mortality rate is the evolution of new Mtb strains that are resistant to available antibiotics. Therefore, new therapeutics for TB are in constant demand. Here we report the development of such inhibitors that target two DNA replication enzymes of Mtb, namely DnaG primase and DNA gyrase, which share a conserved TOPRIM fold near the inhibitors binding site. The molecules were developed on the basis of previously reported inhibitors for T7 DNA primase that bind near the TOPRIM fold. In order to improve the physicochemical properties of the molecules as well as their inhibitory effect on primase and gyrase, 49 novel compounds were synthesized as potential drug candidates in three stages of optimization. The last stage of chemical optimization yielded two novel inhibitors for the fast-growing nonpathogenic model Mycobacterium smegmatis (Msmg).

biochemistry↗

bioRxiv2019

Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism

Iron-sulfur clusters (ISC) are essential in all life forms and carry out many crucial cellular functions. The core machinery for de novo ISC biosynthesis, located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreichs ataxia (FRDA). Recently crystal structures depicting the inactive 3- and 4-way sub-complexes of the ISC biosynthesis machinery, lacking the key activator FXN, have been determined. Here, the 3.2 [A] resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates for the first time in any organism the interactions of FXN with the component proteins. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure sheds light on how FXN facilitates ISC production through unlocking the zinc inhibition and stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and offers an explanation of how FRDA clinical mutations affect complex formation and FXN activation.

biochemistry↗

bioRxiv2019

The Ps and Qs of alarmone synthesis in Staphylococcus aureus

During the stringent response, bacteria synthesize guanosine-3,5-bis(diphosphate) (ppGpp) and guanosine-5-triphosphate 3-diphosphate (pppGpp), which act as secondary messengers to promote cellular survival and adaptation. (p)ppGpp alarmones are synthesized and/or hydrolyzed by proteins belonging to the RelA/SpoT Homologue (RSH) family. Many bacteria also encode small alarmone synthetase (SAS) proteins (e.g. RelP, RelQ) which may also be capable of synthesizing a third alarmone: guanosine-5-phosphate 3-diphosphate (pGpp). Here, we report the biochemical properties of the Rel (RSH), RelP and RelQ proteins from Staphylococcus aureus (Sa-Rel, Sa-RelP, Sa-RelQ, respectively). Sa-Rel synthesized pppGpp more efficiently than ppGpp, but lacked the ability to produce pGpp. However, Sa-Rel efficiently hydrolyzed all three alarmones in a Mn(II) ion-dependent manner. The removal of the C-terminal regulatory domain of Sa-Rel increased its rate of (p)ppGpp synthesis ca. 10-fold, but had negligible effects on its rate of (pp)pGpp hydrolysis. Sa-RelP and Sa-RelQ efficiently synthesized pGpp in addition to pppGpp and ppGpp. The alarmone-synthesizing abilities of Sa-RelQ, but not Sa-RelP, were allosterically-stimulated by the addition of pppGpp, ppGpp or pGpp. The respective (pp)pGpp-synthesizing activities of Sa-RelP/Sa-RelQ were compared and contrasted with SAS homologues from Enterococcus faecalis (Ef-RelQ) and Streptococcus mutans (Sm-RelQ, Sm-RelP). Results indicated that EF-RelQ, Sm-RelQ and Sa-RelQ were functionally-equivalent; but exhibited considerable variations in their respective biochemical properties, and the degrees to which alarmones and single-stranded RNA molecules allosterically stimulated their respective alarmone-synthesizing activities. The respective (pp)pGpp-synthesizing capabilities of Sa-RelP and Sm-RelP proteins were inhibited by pGpp, ppGpp and pppGpp. Our results support the premise that RelP and RelQ proteins may synthesize pGpp in addition to (p)ppGpp within S. aureus and other Gram-positive bacterial species.

biochemistry↗

bioRxiv2019

Diflunisal targets the HMGB1/CXCL12 heterocomplex and blocks immune cell recruitment

Extracellular HMGB1 triggers inflammation following infection or injury, and supports tumorigenesis in inflammation-related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide-containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that Diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, Diflunisal does not inhibit TLR4-dependent responses. Our findings clarify the mode of action of Diflunisal, and open the way to the rational design of functionally specific anti-inflammatory drugs.

biochemistry↗

bioRxiv2019

Fragment-based discovery of a new class of inhibitors targeting mycobacterial tRNA modification

Translational frameshift errors are often deleterious to the synthesis of functional proteins as they lead to the production of truncated or inactive proteins. TrmD (tRNA-(N(1)G37) methyltransferase) is an essential tRNA modification enzyme in bacteria that prevents +1 errors in the reading frame during protein translation and has been identified as a therapeutic target for several bacterial infections. Here we validate TrmD as a target in Mycobacterium abscessus and describe the application of a structure-guided fragment-based drug discovery approach for the design of a new class of inhibitors against this enzyme. A fragment library screening followed by structure-guided chemical elaboration of hits led to the development of compounds with potent in vitro TrmD inhibitory activity. Several of these compounds exhibit activity against planktonic M. abscessus and Mycobacterium tuberculosis. The compounds were further active in macrophage infection models against Mycobacterium leprae and M. abscessus suggesting the potential for novel broad-spectrum mycobacterial drugs.

biochemistry↗

bioRxiv2019

A molecular model for self-assembly of synaptonemal complex protein SYCE3

The synaptonemal complex (SC) is a supramolecular protein assembly that mediates homologous chromosome synapsis during meiosis. This zipper-like structure assembles in a continuous manner between homologous chromosome axes, enforcing a 100-nm separation along their entire length, and providing the necessary three-dimensional framework for crossover formation. The mammalian SC is formed of eight components - SYCP1-3, SYCE1-3, TEX12 and SIX6OS1 - arranged in transverse and longitudinal structures. These largely -helical coiled-coil proteins undergo heterotypic interactions, coupled with recursive self-assembly of SYCP1, SYCE2-TEX12, and SYCP2-SYCP3, to achieve the vast supramolecular structure of the SC. Here, we report a novel self-assembly mechanism of SC central element component SYCE3, identified through multi-angle light scattering and small-angle X-ray scattering. SYCE3 adopts a dimeric four-helical bundle structure that acts as the building block for concentration-dependent self-assembly into a series of discrete higher order oligomers. This is achieved through staggered lateral interactions between self-assembly surfaces of SYCE3 dimers, and their end-on interaction through intermolecular domain-swap between dimer folds. These mechanisms combine to achieve potentially limitless SYCE3 assembly, which particularly favours formation of dodecamers of three laterally associated domain-swap tetramers. Our findings extend the family of self-assembling proteins within the SC and provide novel means for structural stabilisation of the SC central element.

biochemistry↗

bioRxiv2019

Glycosylation of Zika Virus Is Important in Host-Virus Interaction and Pathogenesis

Zika virus (ZIKV) is a global public health issue due to its association with severe developmental disorders in infants and neurological disorders in adults. Because ZIKV uses glycosylation of its envelope (E) protein to interact with host cell receptors to facilitate entry, these interactions could also be important for designing therapeutics and vaccines. Due to a lack of information about Asn-linked (N-glycans) on ZIKV E, we analyzed ZIKV E of various strains derived from different cells. ZIKV E proteins are extensively modified with oligomannose-, hybrid- and complex-N-glycans of a highly heterogeneous nature. Host cell-surface glycans correlated strongly with the glycomic features of ZIKV E. Mechanistically, we discovered that ZIKV N-glycans are important in viral pathogenesis, as mannose-specific C-type lectins DC-SIGN and L-SIGN mediate cell entry of ZIKV. Our findings represent the first detailed mapping of N-glycans on ZIKV E of various strains and their functional significance.

biochemistry↗

bioRxiv2019

Protein secondary structure determines the temporal relationship between folding and disulfide formation

How and when disulfides form in proteins during their folding is a fundamental question in cell biology. Two models describe the relationship between disulfide formation and folding, the folded precursor model, in which formation of nascent structure occurs prior to the disulfides and the quasi-stochastic model where disulfides form prior to complete domain folding. Here we investigate oxidative folding within a cellular milieu of three structurally diverse substrates in order to understand the folding mechanisms required to achieve correct cysteine coupling. We use a eukaryotic translation system in which we can manipulate the redox conditions and produce stalled translation intermediates representative of different stages of translocation. We identify different disulfide bonded isomers by non-reducing SDS-PAGE. Using this approach, we determined whether each substrate followed a folding driven or disulfide driven mechanism. Our results demonstrate that the folding model is substrate-dependent with disulfides forming prior to complete domain folding in a cysteine-rich domain lacking secondary structure, whereas disulfide formation was absent in proteins with defined structural elements. In addition, we demonstrate the presence and rearrangement of non-native disulfides specifically in substrates following the quasi-stochastic model. These findings demonstrate why non-native disulfides are prevented from forming in proteins with well-defined secondary structure. Significance statementA third of human proteins contain structural elements called disulfide bonds that are often crucial for stability and function. Disulfides form between cysteines in the specialised environment of the endoplasmic reticulum (ER), during the complex process of protein folding. Many proteins contain multiple cysteines that can potentially form correct or incorrect cysteine pairings. To investigate how correct disulfide pairs are formed in a biological context, we developed an experimental approach to assess disulfide formation and rearrangement as proteins enter the ER. We found that a disulfide-dense protein domain with atypical secondary structure undergoes disulfide orchestrated folding as it enters the ER and is prone to incorrect disulfide formation. In contrast, proteins with defined secondary structure form folding dependent, native disulfides. These findings show how different mechanisms of disulfide formation can be rationalised from structural features of the folding domains.

biochemistry↗

bioRxiv2019

C-type lectins CTL4 and CTLMA2: conserved heterodimeric structure and glycan specificity in Anopheles mosquitoes

AbstractThe C-type lectins CTL4 and CTLMA2 cooperatively influence Plasmodium infection in the malaria vector Anopheles. Here we report the purification and biochemical characterization of CTL4 and CTLMA2 from An. gambiae and An. albimanus. CTL4 and CTLMA2 are known to form a disulfide-bridged heterodimer via an N-terminal tri-cysteine CXCPC motif. We demonstrate in vitro that CTL4 and CTLMA2 intermolecular disulfide formation is promiscuous within this motif. Furthermore, CTL4 and CTLMA2 exhibit charge complementarity that promotes the formation of higher oligomeric states at physiological pH. Both lectins bind specific sugars, with an apparent preference for glycosaminoglycan motifs comprising {beta}1-3/{beta}1-4 linkages between glucose (Glc), galactose (Gal) and their respective hexosamines. Small-angle x-ray scattering data supports a compact heterodimer between the CTL domains. Recombinant CTL4/CTLMA2 is functional in vivo, reversing the enhancement of phenoloxidase activity in dsCTL4-treated mosquitoes. We propose these molecular features underline a common function for CTL4/CTLMA2 in mosquitoes, with species and strain-specific variation in degrees of activity in response to Plasmodium infection. Author SummaryMosquitoes of the genus Anopheles are vectors for the single-celled parasite Plasmodium, the causative agent of malaria. Mosquitoes, like all insects, utilize the process of melanization for both wound healing and defense against pathogens. CTL4 and CTLMA2 are two proteins found in Anopheles mosquitoes that act as inhibitors of melanization, so understanding their molecular function is important to understanding the immune response of Anopheles mosquitoes to Plasmodium infection. We have purified CTL4 and CTLMA2 from two species of Anopheles and studied their molecular properties with a variety of biochemical and biophysical techniques. We also verified that our purified protein is functional by injecting it into mosquitoes. We learned that CTL4 and CTLMA2 are joined together by a disulfide bond between any one of three cysteine residues near the N-terminus of each protein. The CTL4/CTLMA2 complex is compact, but can associate into larger structures in solution, probably because of a loop in each protein that carries an opposite charge. The proteins cooperatively bind calcium and sugars, specifically glycosaminoglycan sugars, which are typically present in the connective tissues of insects. This information will aid in further investigations of the function of CTL4 and CTLMA2.

biochemistry↗

bioRxiv2019

Post-transcriptional regulation of Nrf2-mRNA by the mRNA-binding proteins HuR and AUF1

The Nrf2-driven antioxidant response (AR) is a target of covalent drugs and bioactive native electrophiles. However, much of our understanding of AR has centered on protein-level regulation of Nrf2. Here we report a post-transcriptional modality to directly regulate Nrf2-mRNA. Our initial studies focused on the effects of the key mRNA-binding protein (mRBP) HuR on global transcriptomic changes incurred upon oxidant or electrophile stimulation. These data led us to discover a novel role of HuR in regulating Nrf2/AR, and in the process we further identified the related mRBP AUF1 as an additional novel Nrf2/AR regulator. Both mRBPs regulate AR by direct interaction with the Nrf2 transcript. Our data showed that HuR enhances Nrf2-mRNA maturation and promotes its nuclear export; whereas AUF1 stabilizes Nrf2-mRNA. Both mRBPs target the 3'-UTR of Nrf2-mRNA. Using an AR-reporter zebrafish strain, we document that this post-transcriptional control of AR is conserved at the whole-vertebrate level.

biochemistry↗

bioRxiv2019

Sensitive determination of proteolytic proteoforms in limited microscale proteome samples

Protein N-termini reveal fundamental regulatory mechanisms and their perturbation in disease. Current terminome identification approaches are limited to whole organs or expandable cultured cells. We present a robust, sensitive, scalable and automatable method for system-wide identification of thousands of N-termini from minute samples. Identification of distinct N- terminal profiles in sorted immune cells, subcellular compartments, clinical biopsies, plasma from pediatric cancer patients, and protease substrates in Arabidopsis seedlings demonstrate broad applicability.

biochemistry↗

bioRxiv2019

Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

Inorganic polyphosphates (polyPs) are long polymers of orthophosphate units (Pi), linked by energy-rich phosphoanhydride bonds. Conserved histidine -helical (CHAD) domains of unknown biochemical function are often located at the C-terminus of polyP-metabolizing triphosphate tunnel metalloenzymes (TTMs), or can be found as stand-alone proteins in bacterial operons harboring polyP kinases or phosphatases. Here we report that bacterial, archaeal and eukaryotic CHAD domains are specific polyP binding modules. Crystal structures reveal that CHAD domains are formed by two four-helix bundles, giving rise to a central cavity surrounded by two conserved basic surface patches. Different CHAD domains bind polyPs with dissociation constants ranging from the nano-to mid-micromolar range, but not DNA or other Pi-containing ligands. A 2.1 [A] CHAD - polyP complex structure reveals the phosphate polymer binding across a central pore and along the two basic patches. Mutational analysis of CHAD - polyP interface residues validates the complex structure and reveals that CHAD domains evolved to bind long-chain polyPs. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. In plants, CHAD domains bind polyP in vivo and localize to the nucleus and nucleolus, suggesting that plants harbor polyP stores in these compartments. We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues.\n\nSignificanceA domain of unknown function termed CHAD, present in all kingdoms of life, is characterized as a specific inorganic polyphosphate binding domain. The small size of the domain and its high specificity for inorganic polyphosphates suggest that it could be used as a tool to locate inorganic polyphosphate stores in pro- and eukaryotic cells and tissues.

biochemistry↗

bioRxiv2019

The AAA+ ATPase TorsinA polymerizes into hollow tubes with a helical periodicity of 8.5 subunits per turn

TorsinA is an ER-resident AAA+ ATPase, whose single residue deletion of glutamate E303 results in the genetic neuromuscular disease primary dystonia. TorsinA is a highly unusual AAA+ ATPase in that it needs an external activator. Also, it appears not to thread a peptide substrate through a narrow central channel, in contrast to its closest structural homologs. Here, we examined the oligomerization of TorsinA to get closer to a molecular understanding of the still enigmatic function of it. We observe TorsinA to form helical filaments, which we analyzed by cryo-electron microscopy using helical reconstruction. The 4.4 [A] structure reveals long hollow tubes with a helical periodicity of 8.5 subunits per turn, and an inner cavity of [~]4 nm diameter. We further show that the protein is able to induce tubulation of membranes in vitro, an observation that may reflect an entirely new characteristic of AAA+ ATPases. We discuss the implications of these observations for TorsinA function.

biochemistry↗

bioRxiv2019

Na/K-ATPase Activity and Ketone Body Metabolism in Long-term Diabetic Rats

The long-term (34 weeks) effect of streptozotocin induced diabetes was assessed in Wistar rats.\n\nNa+/K+-ATPase activity was measured by ouabain inhibitable 86Rb+-uptake into erythrocytes. No difference in the rate of Rb+-uptake, the Km for Rb+ or the Ki for ouabain was detected between normal and diabetic rats. Thus, the change in Na+/K+-ATPase activity repeatedly described in short-term studies may not translate into a long term physiologically relevant change in ion flux through the sodium pump.\n\nRats excrete ketone bodies mainly as {beta}-hydroxybutyrate. This compound does not show up with nitroprusside sodium based test sticks, it can however be detected by coupled spectrophotometric assay with hydroxybutyrate dehydrogenase.\n\nAlmost half of the diabetic animals reverted to a non-diabetic state during the experiment, followed by at least partial reversal of secondary diabetic damage.\n\nAbbreviations used

biochemistry↗

bioRxiv2019

The reactivity of an unusual amidase may explain colibactin’s DNA cross-linking activity

Certain commensal and pathogenic bacteria produce colibactin, a small molecule genotoxin that causes interstrand cross-links in host cell DNA. Though colibactin has been found to alkylate DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and {beta}-keto thioester substrates in vitro and in vivo. The substrate specificity of ClbL strongly supports a role for this enzyme in terminating the colibactin NRPS-PKS assembly line. This transformation would incorporate two electrophilic cyclopropane warheads into the final natural product scaffold. Overall, this work provides a biosynthetic explanation for colibactins DNA crosslinking activity and paves the way for further study of its chemical structure.

biochemistry↗