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bioRxiv2018

Identification and characterization of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

AbstractNitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

biochemistry↗

bioRxiv2018

A method for faster purification of serine proteinases from Bothrops alternatus and Bothrops moojeni snake venoms

Snake venoms are important sources of substances with a variety of pharmacological activities. Among the different proteins present in these venoms, snake venom serine proteinases (SVSPs) have important effects on the hemostatic system that influence the hemodynamic properties of blood. Bothrops genus snakes presented their venom richly composed of SVSPs thrombin-like, and the isolation of these enzymes is of great interest. In 1994, the Center for the Study of Venoms and Venomous Animals (CEVAP) - UNESP standardized the fibrin sealant derived from snake venom, replacing the bovine thrombin by gyroxin thrombin-like enzyme from Crotalus durissus terrificus (Rattlesnake) and human plasma fibrinogen by buffaloes cryoprecipitate. Despite chromatographic techniques for the purification of gyroxin be well grounded in the literature, that income is considered low. Thus, in addition to gyroxin, other thrombin-like enzymes could be employed in the composition of the new fibrin sealant after being standardized to the purifying and chromatographic performance and widely evaluated for biological activities. Therefore, it is extremely important that in our lab is deployed, standardized and validated a method for the chromatographic purification of other thrombin-like enzymes such as found in Bothrops snake venoms. Thus a two-step chromatographic procedure was developed to routinely purify serine proteinases from Bothrops alternatus and B. moojeni snakes venoms to provide new enzymes for improving the CEVAPs heterologous fibrin sealant.

biochemistry↗

bioRxiv2018

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.

biochemistry↗

bioRxiv2018

Structures of DPAGT1 explain glycosylation disease mechanisms and advance TB antibiotic design

Protein glycosylation is a widespread post-translational modification. The first committed step to the lipid-linked glycan used for this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and congenital disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use as antibiotics. However, little is known about the mechanism or the effects of disease-associated mutations in this essential enzyme. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (and thus cause disease) and allow design of non-toxic lipid-altered tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo thereby providing a promising new class of antimicrobial drug.\n\nHighlightsO_LIStructures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis\nC_LIO_LIDPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity\nC_LIO_LIStructures, kinetics and biosynthesis reveal role of lipid in tunicamycin\nC_LIO_LILipid-altered, tunicamycin analogues give non-toxic antibiotics against TB\nC_LI

biochemistry↗

bioRxiv2018

Exonic unpaired elements modulate pre-mRNA structure for splice site recognition

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short ([lsim]70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

biochemistry↗

bioRxiv2018

In vitro assembly of an early spliceosome defining both splice sites

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML - a model pre-mRNA substrate - guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome.

biochemistry↗

bioRxiv2018

Estimating RNA structure chemical probing reactivities from reverse transcriptase stops and mutations.

Chemical probing experiments interrogate RNA structures by creating covalent adducts on RNA molecules in structure-dependent patterns. Adduct positions are then detected through conversion of the modified RNAs into complementary DNA (cDNA) by reverse transcription (RT) as either stops (RT-stops) or mutations (RT-mutations). Statistical analysis of the frequencies of RT-stops and RT-mutations can then be used to estimate a measure of chemical probing reactivity at each nucleotide of an RNA, which reveals properties of the underlying RNA structure. Inspired by recent work that showed that different reverse transcriptase enzymes show distinct biases for detecting adducts as either RT-stops or RT-mutations, here we use a statistical modeling framework to derive an equation for chemical probing reactivity using experimental signatures from both RT-stops and RT-mutations within a single experiment. The resulting formula intuitively matches the expected result from considering reactivity to be defined as the fraction of adduct observed at each position in an RNA at the end of a chemical probing experiment. We discuss assumptions and implementation of the model, as well as ways in which the model may be experimentally validated.

biochemistry↗

bioRxiv2018

Receptor binding and proteolysis do not induce large conformational changes in the SARS-CoV spike

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as a highly transmissible pathogenic human betacoronavirus. The viral spike glycoprotein (S) utilizes angiotensin-converting enzyme 2 (ACE2) as a host protein receptor and mediates fusion of the viral and host membranes, making S essential to viral entry into host cells and host species tropism. As SARS-CoV enters host cells, the viral S undergoes two proteolytic cleavages at S1/S2 and S2 sites necessary for efficient membrane fusion. Here, we present a cryo-EM analysis of the trimeric SARS-CoV S interactions with ACE2 and of the trypsin-cleaved S. Surprisingly, neither binding to ACE2 nor cleavage by trypsin at the S1/S2 cleavage site impart large conformational changes within S or expose the secondary cleavage site, S2. These observations suggest that S2 cleavage does not occur in the S prefusion conformation and that additional triggers may be required.

biochemistry↗

bioRxiv2018

Structural Analysis of Inhibitor Binding to CAMKK1 Identifies Features Necessary for Design of Specific Inhibitors

The calcium/calmodulin-dependent protein kinases (CAMKKs) are upstream activators of CAMK1 and CAMK4 signalling and have important functions in neural development, maintenance and signalling, as well as in other aspects of biology such as Ca2+ signalling in the cardiovascular system. To support the development of specific inhibitors of CAMKKs we have determined the crystal structure of CAMKK1 with two ATP-competitive inhibitors. The structures reveal small but exploitable differences between CAMKK1 and CAMKK2, despite the high sequence identity, which could be used in the generation of specific inhibitors. Screening of a kinase inhibitor library revealed molecules that bind potently to CAMKK1. Isothermal titration calorimetry revealed that the most potent inhibitors had binding energies largely dependent on favourable enthalpy. Together, the data provide a foundation for future inhibitor development activities.

biochemistry↗

bioRxiv2018

A Con Artist: Phenylphenoxybenzamide is not a Glycosyltransferase Inhibitor

To combat bacterial resistance against antibiotics, glycosyltransferase inhibiting molecules, which block the synthesis of the pre-cursor of the bacterial cell wall, need to be discovered and developed. In this study, we demonstrate that phenylphenoxybenzamide, a salicylanilide, is not a glycosyltransferase inhibiting molecule, despite claims in literature to the contrary, and through our work show that glycosyltransferase construct choice and detergent choice are crucial parameters to consider when designing glycosyltransferase assays that aim to discover and develop molecules that inhibit these types of enzymes.

biochemistry↗

bioRxiv2018

Knockdown of GAS5 restores ox-LDL-induced impaired autophagy flux via upregulating miR-26a in human endothelial cells

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced endothelial cell (EC) injury and autophagy dysfunction play a vital role in the development of atherosclerosis. LncRNAs have been identified to participate in the regulation of pathogenesis of atherosclerosis. However, it remains largely undefined whether growth-arrest specific transcript 5 (GAS5) could influence ox-LDL-induced autophagy dysfunction in ECs.\n\nMethodsThe expressions of GAS5 and miR-26a in the plasma samples of patients with atherosclerosis and ox-LDL-treated human aortic endothelial cells (HAECs) were detected by qRT-PCR. Luciferase reporter assay, RNA immunoprecipitation (RIP), and RNA pull down were performed to validate whether GAS5 could directly interact with miR-26a. The effects of ox-LDL, GAS5 or combined with miR-26a on apoptosis and autophagy were evaluated by flow cytometry analysis and western blot, respectively.\n\nResultsGAS5 expression was upregulated and miR-26a was downregulated in the plasma samples of patients with atherosclerosis and ox-LDL-treated HAECs. There was reciprocal inhibition between GAS5 and miR-26a expressions in ox-LDL-treated HAECs. We further demonstrated that GAS5 directly interacted with miR-26a in ox-LDL-treated HAECs. Additionally, ox-LDL administration induced apoptosis and impaired autophagy flux in HAECs. Rescue experiments demonstrated that GAS5 knockdown restored ox-LDL-induced impaired autophagy flux by upregulating miR-26a in HAECs.\n\nConclusionKnockdown of GAS5 restores ox-LDL-induced impaired autophagy flux via upregulating miR-26a in human endothelial cells, revealing a novel regulatory mechanism for ox-LDL-induced impaired autophagy flux in ECs through ceRNA crosstalk.

biochemistry↗

bioRxiv2018

Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS

The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.

biochemistry↗

bioRxiv2018

Dynamics of Plasma Lipidome in Progression to Islet Autoimmunity and Type 1 Diabetes: Type 1 Diabetes Prediction and Prevention Study (DIPP)

Type 1 diabetes (T1D) is one of the most prevalent autoimmune diseases among children in Western countries. Earlier metabolomics studies suggest that T1D is preceded by dysregulation of lipid metabolism. Here we used a lipidomics approach to analyze molecular lipids in a prospective series of 428 plasma samples from 40 children who progressed to T1D (PT1D), 40 children who developed at least a single islet autoantibody but did not progress to T1D during the follow-up (P1Ab) and 40 matched controls (CTR). Sphingomyelins were found to be persistently downregulated in PT1D when compared to the P1Ab and CTR groups. Triacylglycerols and phosphatidylcholines were mainly downregulated in PT1D as compared to P1Ab at the age of 3 months. Our study suggests that children who progressed to islet autoimmunity or overt T1D are characterized by distinct lipidomic signatures, which may be helpful in the identification of at-risk children before the initiation of autoimmunity.

biochemistry↗

bioRxiv2018

LIkelihood-based Fits of Folding Transitions (LIFFT) for Biomolecule Mapping Data

SummaryBiomolecules shift their structures as a function of temperature and concentrations of protons, ions, small molecules, proteins, and nucleic acids. These transitions impact or underlie biological function and are being monitored at increasingly high throughput. For example, folding transitions for large collections of RNAs can now be monitored at single residue resolution by chemical mapping techniques. LIkelihood-based Fits of Folding Transitions (LIFFT) quantifies these data through well-defined thermodynamic models. LIFFT implements a Bayesian framework that takes into account data at all measured residues and enables visual assessment of modeling uncertainties that can be overlooked in least-squares fits. The framework is appropriate for multimodal techniques ranging from chemical mapping including multi-wavelength spectroscopy.\n\nAvailabilityFreely available MATLAB package at https://ribokit.stanford.edu/LIFFT/.\n\nContactrhiju@stanford.edu\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

biochemistry↗

bioRxiv2018

Visualizing conformational dynamics of proteins in solution and at the cell membrane

Conformational dynamics underlie enzyme function, yet are generally inaccessible via traditional structural approaches. FRET has the potential to measure conformational dynamics in vitro and in intact cells, but technical barriers have thus far limited its accuracy, particularly in membrane proteins. Here, we combine amber codon suppression to introduce a donor fluorescent noncanonical amino acid with a new, biocompatible approach for labeling proteins with acceptor transition metals in a method called ACCuRET (Anap Cyclen-Cu2+ resonance energy transfer). We show that ACCuRET measures absolute distances and distance changes with high precision and accuracy using maltose binding protein as a benchmark. Using cell unroofing, we show that ACCuRET can accurately measure rearrangements of proteins in native membranes. Finally, we implement a computational method for correcting the measured distances for the distance distributions observed in proteins. ACCuRET thus provides a flexible, powerful method for measuring conformational dynamics in both soluble proteins and membrane proteins.

biochemistry↗

bioRxiv2018

Distinct RanBP1 nuclear export and cargo dissociation mechanisms between fungi and animals

Ran binding protein 1 (RanBP1), the primary effector of nuclear GTPase Ran, is a cytoplasmic-enriched and nuclear-cytoplasmic shuttling protein, playing important roles in nuclear transport through preventing RanGTP from being trapped with karyopherin proteins and dissociating cargoes from nuclear export factor CRM1. Much of what we know about RanBP1 is learned from fungi. Here we show that animal RanBP1 has distinct cargo dissociation and nuclear export mechanisms. In contrast to CRM1-RanGTP sequestration mechanism of cargo dissociation in fungi, animal RanBP1 solely sequesters RanGTP from nuclear export complexes. In fungi, RanBP1, CRM1 and RanGTP form a 1:1:1 nuclear export complex; in contrast, animal RanBP1, CRM1 and RanGTP form a 1:1:2 nuclear export complex. The key feature for the two mechanistic changes from fungi to animals is the loss of affinity between RanBP1-RanGTP and CRM1, since residues mediating their interaction in fungi are not conserved in animals. The biological significances of these different mechanisms in fungi and animals are also studied and discussed. Our study illustrates how orthologous proteins may play conserved functions through distinct routes, and may provide directions for design of antifungal medicines.

biochemistry↗

bioRxiv2018

The practical value of an explicit response model in quantitative uses of Biacore™ biosensors

Suppose we aim to use data obtained by studying a biomolecular interaction system with a surface plasmon resonance (SPR) biosensor in quantifying some system feature. We assume a parametric mathematical model for biosensor response due to sources of mass such as analyte-ligand complexes. Some parameters represent interaction features, such as rate constants. Whenever we attempt to estimate parameters from data, we may obtain multiple estimates, regardless of the amount and quality of data. Inconveniently, we may be unable to distinguish between alternatives. This is problematic when alternative parameter values lead to very different predictions of system behaviour for a situation where we lack data. Anticipating this issue prior to data collection allows us to redesign the combination of planned experiments and model, replacing a certain failure to achieve our studys aim with the possibility of success. The literature on SPR biosensors (and computational biology more generally) has paid little attention to this matter. In order to remedy this, it is appropriate to begin with a consideration of the assumed models. These are rarely specified completely, causing ambiguity that impedes scrutiny of their properties and comparison with other models. We demonstrate this by reviewing some model types seen in the Biacore biosensor literature. We propose to eliminate model ambiguity by providing a suitable framework for specifing models for biosensor data. This framework will aid future efforts to compose models for data arising from particular interaction mechanisms in a form that is amenable to scrutiny. We expect that the issues raised here will have relevance to the modelling of data obtained from other apparatus employed in quantifying binding behaviour.

biochemistry↗

bioRxiv2018

New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3-phosphoadenosine 5-phosphosulphate) to the 2-O position of -L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.\n\nSUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

biochemistry↗