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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

The structure of an injectisome export gate demonstratesconservation of architecture in the core export gatebetween flagellar and virulence type three secretionsystems

Export of proteins through type three secretion systems (T3SS) is critical for motility and virulence of many major bacterial pathogens. Proteins are exported though a genetically defined export gate complex consisting of three proteins. We have recently shown at 4.2 [A] that the flagellar complex of these three putative membrane proteins (FliPQR in flagellar systems, SctRST in virulence systems) assemble into an extra-membrane helical assembly that likely seeds correct assembly of the rod above. Here we present the structure of an equivalent complex from the more fragile Shigella virulence system at 3.5 [A] by cryo-electron microscopy. This higher resolution structure reveals further detail and confirms the prediction of structural conservation in this core complex. Analysis of particle heterogeneity also reveals details of how the SctS/FliQ subunits sequentially assemble in the complex.

biochemistry↗
bioRxiv2019

A Chemical Probe of CARM1 Alters Epigenetic Plasticity against Breast Cancer Cell Invasion

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73-associated reduction of invasiveness act via altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 and CARM1 knockout alter the epigenetic plasticity with remarkable difference, arguing distinct modes of action between the small-molecule and genetic perturbation. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.

biochemistry↗
bioRxiv2019

Substrate structure and computation guided engineering of a Lipase for Omega-3 fatty acid selectivity

Optimum health benefits of omega-3 fatty acids ({omega}-3 FAs) require it to be concentrated in its natural sources. Fatty acid selectivity of lipase governs the efficacy of the production of lipase-mediated {omega}-3 FAs concentrates. We attempted to improve the fatty acid selectivity of a lipase from thermophilic bacterium Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, six amino acid positions of GTL interacting with the substrate, were identified by docking and were subjected to site-saturation mutagenesis. Three best substitutions were incorporated into GTL(CM-GTL). Hydrolysis of oil by lipase was monitored in a pH-Stat and the fatty acids released at various time points were analyzed by GC-MS.CM-GTL showed a significant improvement in discrimination against DHA during hydrolysis. In the second approach based on rational design, the active site was narrowed by incorporating heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky {omega}-3 FAs. For this purpose, two amino acids surrounding the opening of the acyl pocket were replaced with the next heavier amino acids and the affinities were evaluated in silico. The double mutant, thus deigned, was found to be excellent in discriminating the {omega}-3 FAs during hydrolysis of triglycerides. Engineering the binding pocket of a complex substrate, such as a triglyceride, with the supportive information on substrate structure and its binding modes with the enzyme provided by computational methods, has resulted in designing two efficient lipase variants with improved substrate selectivity.

biochemistry↗
bioRxiv2019

Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasomes unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasomes unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

biochemistry↗
bioRxiv2019

A ‘proton ratchet’ for coupling the membrane potential to protein transport

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism by which PMF can aid transport, and enabling a proton ratchet, wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

biochemistry↗
bioRxiv2019

A combined strategy of neuropeptide predictions and tandem mass spectrometry identifies evolutionarily conserved ancient neuropeptides in the sea anemone Nematostella vectensis

Neuropeptides are a class of bioactive peptides and are responsible for various physiological processes including metabolism, development and reproduction. Although accumulated genome and transcriptome data have reported a number of neuropeptide candidates, it still remains difficult to obtain a comprehensive view of neuropeptide repertoires due to their small and variable nature. Neuropeptide prediction tools usually work only for those peptides for which sequentially related homologs have previously been identified. Recent peptidomics technology has enabled systematic structural identification of neuropeptides by using the combination of liquid chromatography and tandem mass spectrometry. However, obtaining reliable identifications of endogenous peptides is still difficult using a conventional tandem mass spectrometry-based peptide identification approach using protein database because a large search space has to be scanned due to the absence of a cleavage enzyme specification. We developed a pipeline consisting of the prediction of in silico cleaved endogenous neuropeptides followed by peptide-spectrum matching enabling highly sensitive and reliable neuropeptide identification. This approach effectively reduces the search space of peptide-spectrum matching, and thus increases search sensitivity. To identify neuropeptides in Nematostella vectensis, a basal eumetazoan having one of the most primitive nervous systems, we scanned the Nematostella protein database for sequences displaying structural hallmarks of metazoan neuropeptides, including C/N-terminal structures and modifications. Peptide-spectrum matching was performed against the in silico cleaved peptides and successfully identified dozens of neuropeptides at high confidence. The identification of Nematostella neuropeptides structurally related the tachykinin, GnRH/AKH, neuromedin-U/pyrokinin peptide families indicate that these peptides already originated in the eumetazoan ancestor of all animal species, most likely concomitantly with the development of a nervous system.

biochemistry↗
bioRxiv2019

Pat1 activates late steps in mRNA decay by multiple mechanisms

Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression and mRNA decay. A critical step in all 5-3 mRNA decay pathways is removal of the 5 cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5-3 decay, the Pat1/Lsm1-7 complex engages mRNA at the 3 end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5 end through an unknown mechanism. We reconstitute Pat1 with 5 and 3 decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Secondly, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping mRNP that promotes 5-3 mRNA degradation.

biochemistry↗
bioRxiv2019

Single subunit degradation of WIZ, a lenalidomide- and pomalidomide dependent substrate of E3 ubiquitin ligase CRL4CRBN

Immunomodulators (IMiDs) are an effective class of drugs used to treat blood cancers. IMiDs are believed to work by recruiting protein targets containing a {beta}-hairpin motif for ubiquitination by E3 ubiquitin ligase complexes composed of cereblon (CRBN), Cullin-4a (CUL4a), DNA Damage Binding protein-1 (DDB1), and Ring Box-1 (RBX1). The ubiquitinated protein is subsequently degraded by the proteasome. By characterizing the repertoire of proteins that show an increased physical association with CRBN after IMiD treatment, we identified a novel IMiD substrate, Widely Interspaced Zinc Finger Motifs (WIZ). WIZ contains a C2H2 zinc finger domain, like several other substrates that were previously characterized. We demonstrate that IMiDs stabilize physical association of WIZ with CRBN, deplete WIZ steady state protein levels in a way that is dependent on E3 ligase activity, and enhance the rate of its degradation. Notably, proteins that assemble with WIZ are co-recruited to CRBN by IMiDs but are not degraded, illustrating the potential of targeted protein degradation to eliminate individual subunits of a protein complex. These findings suggest that systematic characterization of the full repertoire of proteins that are targeted for degradation by IMiD compounds will be required to better understand their biological effects.\n\nSynopsisProteolysis Targeting Chimeras (PROTACs) can be used to precisely target a subunit of a transcriptional complex for degradation in subpopulations of cells.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC=\"FIGDIR/small/595389_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@186fed8org.highwire.dtl.DTLVardef@1c522f9org.highwire.dtl.DTLVardef@17b19baorg.highwire.dtl.DTLVardef@d626ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2019

An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP

AMPylation is an inactivating modification that matches the activity of the major endoplasmic reticulum (ER) chaperone BiP to the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICDs activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface or in residues tracing an inhibitory relay from the dimer interface to the enzymes active site favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically-propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Whereas, a reciprocal signal propagated from the nucleotide binding site, provides a mechanism for coupling the oligomeric-state and enzymatic activity of FICD to the energy status of the ER.\n\nImpact StatementUnique amongst known chaperones, the endoplasmic reticulum (ER)-localized Hsp70, BiP, is subject to transient inactivation under conditions of low ER stress by reversible, covalent modification - AMPylation. The enzyme responsible for this modification, FICD, is in fact a bifunctional enzyme with a single active site capable of both AMPylation and deAMPylation. Here we elucidate, by biochemical, biophysical and structural means, the mechanism by which this enzyme is able to switch enzymatic modality: by regulation of its oligomeric state. The oligomeric state-dependent reciprocal regulation of FICD activity is, in turn, sensitive to the ATP/ADP ratio. This allosteric pathway potentially facilitates the sensing of unfolded protein load in the ER and permits the transduction of this signal into a post-translational buffering of ER chaperone activity.

biochemistry↗
bioRxiv2019

Association between thyroid hormones and HOMA-IR in Saudi-obese women with metabolic syndrome

BackgroundThe obesity epidemic is a pressing global health concern, as obesity rates continue to climb worldwide. The current study was aimed mainly to evaluate the correlation between thyroid hormones and homeostatic model assessment of insulin resistance in Saudi obese women with metabolic syndrome.\n\nMethods100 obese women aged 25 to 55 years were clinically evaluated, from which 72 women were diagnosed with the metabolic syndrome and 28 without metabolic syndrome. Insulin resistance was quantified using the homeostatic model assessment of insulin resistance method and the resulting values were analyzed for association with demographic, clinical, and metabolic parameters.\n\nResultsThis analysis revealed that body mass index, systolic blood pressure, and biochemical parameters and fasting insulin showed statistically higher levels in the group with metabolic syndrome compared to the group without metabolic syndrome. Similarly, values of waist circumference, fat ratio, cholesterol, free thyroxine, free triiodothyronine and homeostatic model assessment of insulin resistance results were higher in the group with metabolic syndrome as compared to the group without metabolic syndrome. Correlation analysis revealed positive association of thyroid-stimulating hormone with waist circumference (P=0.01), total cholesterol (P=0.002), fasting insulin (P=0.03) and homeostatic model assessment of insulin resistance results (P<0.01), and negatively associated with diastolic blood pressure (P=0.013) and age (P=0.05). Free thyroxine was positively associated with triglyceride level (P=0.003) and negatively associated with homeostatic model assessment of insulin resistance values (P=0.035) and fasting insulin. Free triiodothyronine was positively associated with body mass index (P=0.032) and waist circumference (P= 0.006) and negatively with age (P=0.004) and total cholesterol (P=0.001).\n\nHomeostatic model assessment of insulin resistance test revealed elevated level with positive association of body mass index, waist circumference, biochemical parameters and thyroid-stimulating hormone in insulin resistant obese women. Higher level of free triiodothyronine was found to be associated with low insulin sensitivity.

biochemistry↗
bioRxiv2019

The biochemical activities of the Saccharomyces cerevisiae Pif1 helicase are regulated by its N-terminal domain

PIF1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many PIF1 helicase are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 over-expression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1{Delta}N) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1{Delta}N was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of PIF1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

biochemistry↗
bioRxiv2019

RNA base pairing complexity in living cells visualized by correlated chemical probing

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

biochemistry↗
bioRxiv2019

De novo design of proteins with two isoenergetic but structurally divergent ground states

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used to de novo design proteins that fold to a single state with a deep free energy minima (Huang et al., 2016), and to reengineer natural proteins to alter their dynamics (Davey et al., 2017) or fold (Alexander et al., 2009), the de novo design of closely related sequences which adopt well-defined, but structurally divergent structures remains an outstanding challenge. Here, we design closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations -- one short ([~]66 [A] height) and the other long ([~]100 [A] height) -- reminiscent of the conformational transition of viral fusion proteins (Ivanovic et al., 2013; Podbilewicz, 2014; Skehel and Wiley, 2000). Crystallographic and NMR spectroscopic characterization show that both the short and long state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large scale conformational switches between structurally divergent forms.

biochemistry↗
bioRxiv2019

Fast and unbiased purification of RNA-protein complexes after UV cross-linking

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 hrs) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

biochemistry↗
bioRxiv2019

Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome

Although receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several different G protein-coupled receptors (GPCRs), potential direct interactions among the three known RAMPs and hundreds of GPCRs has never been investigated. We engineered three epitope-tagged RAMPs and 23 epitope-tagged GPCRs, focusing on the secretin-like family of GPCRs, and developed a suspension bead array (SBA) immunoassay designed to detect RAMP-GPCR complexes. We then used 64 antibodies raised against native RAMPs and GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to detect and measure all possible combinations of interaction among the 23 GPCRs and three RAMPs. The results of the SBA assay provide a complete interactome of secretin-like GPCRs with RAMPs. We demonstrate direct interaction of previously reported secretin-like GPCRs whose functions are modulated by RAMPs. We also discovered novel sets of GPCR-RAMP interacting pairs, and found additional secretin-like GPCRs, chemokine receptors and orphan receptors that interact with RAMPs. Using in situ roximity ligation assay, we verified a subset of these novel GPCR-RAMP interactions in cell membranes. In total, we found GPCR-RAMP interactions for the majority of the 23 GPCRs tested. Each GPCR interacted with either all three RAMPs or with RAMP2 and RAMP3, with the exception of one GPCR that interacted with just RAMP3. In summary, we describe an SBA strategy that will be useful to search for GPCR-RAMP interactions in cell lines and tissues, and conclude that GPCR-RAMP interactions are more common than previously appreciated.

biochemistry↗
bioRxiv2019

To enrich or not to enrich: Enhancing (glyco)peptide ionization using the CaptiveSpray nanoBooster ™

The CaptiveSpray source ensures a stable spray and excellent nano ESI performance facilitated by a vortex gas that sweeps around the emitter spray tip to support liquid desolvation and focus the Taylor cone. Enriching the vortex gas with dopant solvents provides tremendous opportunities to increase ionization efficiency, in particular for hydrophilic compounds such as glycopeptides. How this CaptiveSpray nanobooster benefits their analysis, however, has to date not been systematically studied.\n\nWe evaluated various dopant solvents such as (i) acetone (ii) acetonitrile (iii) methanol (iv) ethanol and (v) isopropanol for their ability to enhance glycopeptide ionization. Using a synthetic IgG2 glycopeptide as a standard, acetonitrile provided a five-fold increase in signal intensities and resulted in an overall charge state increase compared to conventional CaptiveSpray ionization. This trend remained the same when tryptic IgG (glyco)peptides were analyzed and allowed highly sensitive detection of glycopeptides even without any enrichment. While acetone dopant gas enhanced glycopeptide ionization by doubling glycopeptide signal intensities, all other tested solvents resulted either in ion suppression or adduct formation. This is in agreement with and can be explained by their individual physio-chemical properties of the solvents. Finally, by omitting glycopeptide enrichment steps, we established a bias-free human Immunoglobulin G (IgG) subclass specific glycosylation profile applying the optimized CaptiveSpray nanoBooster nano-LC-ESI-MS/MS analysis conditions.

biochemistry↗
bioRxiv2019

A meiosis-specific AAA+ assembly reveals repurposing of ORC during budding yeast gametogenesis

ORC (Orc1-6) is an AAA+ complex that loads the AAA+ MCM helicase to replication origins. Orc1, a subunit of ORC, functionally interacts with budding yeast Pch2, a meiosis-specific AAA+ protein. Pch2 regulates several chromosomal events of gametogenesis, but mechanisms that dictate Pch2 function remain poorly understood. We demonstrate that ORC directly interacts with an AAA+ Pch2 hexamer. The ORC-Pch2 assembly is established without Cdc6, a factor crucial for ORC-MCM binding. Biochemical analysis suggests that Pch2 utilizes ORCs Cdc6-binding interface and employs its non-enzymatic NH2-terminal domain and AAA+ core to engage ORC. In contrast to phenotypes observed upon Orc1 impairment, nuclear depletion of other subunits of ORC does not lead to Pch2-like phenotypes, indicating that ORC integrity per se is not required to support Pch2 function. We thus reveal functional interplay between Pch2 and ORC, and uncover the repurposing of ORC to establish a non-canonical and meiosis-specific AAA+ assembly.

biochemistry↗