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bioRxiv2018

Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 [A]. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

biochemistry↗

bioRxiv2018

Evidence of an Unusual Poly(A) RNA Signature Detected by High-throughput Chemical Mapping

Homopolymeric adenosine RNA plays numerous roles in both cells and non-cellular genetic material, and for lack of evidence to the contrary, it is generally accepted to form a random coil under physiological conditions. However, chemical mapping data generated by the Eterna Massive Open Laboratory indicates that a poly (A) sequence of length seven or more, at pH 8.0 and MgCl concentrations of 10 mM, develops unexpected protection to selective 2-hydroxyl acylation read out by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing. This protection first appears in poly(A) sequences of length 7 and grows to its maximum strength at length ~10. In a long poly(A) sequence, substitution of a single A by any other nucleotide disrupts the protection, but only for the 6 or so nucleotides on the 5 side of the substitution. The authors are grateful for pre-publication comments; please use https://docs.google.com/document/d/14972Q36IDTYMglwMXTOrqd4P9orQ6-P3bPbCuITdv6A.

biochemistry↗

bioRxiv2018

Serum glycoprotein biomarker validation for esophageal adenocarcinoma and application to Barrett’s surveillance

BACKGROUND & AIMSEsophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barretts esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance for BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker panel for BE surveillance.\n\nMETHODSSerum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and USA (1 clinic) using lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS). The area under receiver operating characteristic curve was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multi-marker panel for BE surveillance.\n\nRESULTSDifferent glycoforms of complement C9 (C9), gelsolin (GSN), serum paraoxonase/arylesterase 1 (PON1) and serum paraoxonase/lactonase 3 (PON3) were validated as diagnostic glycoprotein biomarker candidates for EAC across both cohorts. A panel of 10 serum glycoproteins accurately discriminated BE patients not requiring intervention [BE+/-low grade dysplasia] from those requiring intervention [BE with high grade dysplasia (BE-HGD) or EAC]. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed towards EAC, levels of serum C9 glycoforms were increased with disease progression.\n\nCONCLUSIONSFurther prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted. A first-line BE surveillance blood test may be developed based on these findings.\n\nAbbreviations

biochemistry↗

bioRxiv2018

Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest Peptide

Secretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation channel complex. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER-targeting signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation initiation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient, versus inefficient (poorly hydrophobic) signal sequences, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phases event in which the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products. Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.

biochemistry↗

bioRxiv2018

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Amber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

biochemistry↗

bioRxiv2018

Deactivation and Mislocalization of a kinase protein induced by a single amino acid mutation on the proton transport catalytic aspartic acid

Rhoptry protein 18 (ROP18) is a major determinant of strain-specific virulence in Toxoplasma gondii. The kinase activity of ROP18 is required for acute virulence, while the aspartate in the catalytic loop of ROP18 is considered essential for phosphoryl transfer. We showed that a single amino acid mutation at the catalytic aspartate residue (D409A mutation) significantly altered ROP18 kinase activity in vitro, and abolished ROP18-mediated ATF6{beta} degradation. Furthermore, the investigated single amino acid mutation in ROP18 led to alternation of subcellular localization of ROP18 protein. Structural modeling analysis suggests that these phenotypes might be associated with D409A mutation induced conformation changes in ROP18.\n\nOur findings demonstrate that a single amino acid mutation on the proton transport catalytic aspartic acid induced conformational alternations in ROP18 resulting in functional changes associated with ROP18 protein.

biochemistry↗

bioRxiv2018

Identifying small molecule binding sites for epigenetic proteins at domain-domain interfaces

Epigenetics is of rapidly growing field in drug discovery. Of particular interest is the role of post-translational modifications to histone and the proteins that read, write, and erase such modifications. The development of inhibitors for reader domains has focused on single domains. One of the major difficulties of designing inhibitors for reader domains, is that with the notable exception of bromodomains, they tend not to possess a well enclosed binding site amenable to small molecule inhibition. As many of the proteins in epigenetic regulation have multiple domains there are opportunities for designing inhibitors that bind at a domain-domain interface which provide a more suitable interaction pocket. Examination of X-ray structures of multiple domains involved in recognizing and modifying post-translational histone marks using the SiteMap algorithm identified potential binding sites at domain-domain interfaces. For the tandem plant homeodomain-bromodomain of SP100C, a potential inter-domain site identified computationally was validated experimentally by the discovery of ligands by X-ray crystallographic fragment screening.

biochemistry↗

bioRxiv2018

Structural elements required for coupling ion and substrate transport in the neurotransmitter transporter homolog LeuT.

The coupled transport of ions and substrates allows transporters to accumulate substrates using the energy in transmembrane ion gradients and electrical potentials. During transport, conformational changes that switch accessibility of substrate and ion binding sites from one side of the membrane to the other must be controlled so as to prevent uncoupled movement of ions or substrates. In the Neurotransmitter:Sodium Symporter (NSS) family, Na+ stabilizes the transporter in an outward-open state, thus decreasing the likelihood of uncoupled Na+ transport. In a step essential for coupled transport, substrate binding must overcome the effect of Na+, allowing intracellular substrate and Na+ release from an inward-open state. However, it is unclear which specific elements of the protein mediate this conformational response to substrate binding. Previously, we showed that in the prokaryotic NSS transporter LeuT, the effect of Na+ on conformation occurs at the Na2 site, where it influences conformation by fostering interaction between two domains of the protein (JBC 291: 1456, 2016). Here, we identify a conserved tyrosine residue in the substrate binding site required for substrate to enable conversion to inward-open states by establishing an interaction between the two transporter domains. We further identify additional interactions between the two transporter domains in the extracellular pathway that are required. Together with our previous work on the conformational effect of Na+, these results identify mechanistic components underlying ion-substrate coupling in NSS transporters.

biochemistry↗

bioRxiv2018

Structures reveal opening of the store-operated calcium channel Orai

The store-operated calcium (Ca2+) channel Orai governs Ca2+ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to depletion of Ca2+ within the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the open pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and the exquisite selectivity of the channel for Ca2+. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed intermediate, we propose a sequence for store-operated activation.

biochemistry↗

bioRxiv2018

Conversion of a soluble protein into a potent chaperone in vivo

Protein-folding assistance and aggregation inhibition by cellular factors are largely understood in the context of molecular chaperones. As an alternative and complementary model, we previously proposed that, in general, soluble cellular macromolecules including chaperones with large excluded volume and surface charges exhibit the intrinsic chaperone activity to prevent aggregation of their connected polypeptides, irrespective of the connection types, and thus to aid productive protein folding. As a proof of concept, we here demonstrated that a model soluble protein with an inactive protease domain robustly exerted chaperone activity toward various proteins harboring a short protease-recognition tag of 7 residues in Escherichia coli. The chaperone activity of this protein was similar or even superior to that of representative E. coli chaperones in vivo. Furthermore, in vitro refolding experiments confirmed the in vivo results. Our findings revealed that a soluble protein exhibits the intrinsic chaperone activity, which is manifested, upon binding to aggregation-prone proteins. This study gives new insights into the ubiquitous chaperoning role of cellular macromolecules in protein-folding assistance and aggregation inhibition underlying the maintenance of protein solubility and proteostasis in vivo.

biochemistry↗

bioRxiv2018

Frequent Monitoring of C-peptide Levels in Newly Diagnosed Type 1 Subjects Using Dried Blood Spots Collected at Home

ObjectiveTo evaluate a novel approach to measure {beta}-cell function by frequent testing of C-peptide concentrations in dried blood spots (DBS)\n\nPatientsThirty-two children, aged 7-17 years, recently diagnosed with type 1 diabetes.\n\nDesignMixed-meal-tolerance-test (MMTT) within 6 and again 12 months after diagnosis with paired venous and DBS C-peptide sampling at 0 and 90 minutes. Weekly DBS C-peptide before and after standardized breakfasts collected at home.\n\nResultsDBS and plasma C-peptide levels (n=115) correlated strongly (r=0{middle dot}91; p<0.001). The Bland-Altman plot indicated good agreement. The median number of home-collected DBS cards per participant was 24 over a median of 6.9 months. Repeated DBS C-peptide levels varied considerably within and between subjects. Adjustment for corresponding home glucose measurements reduced the variance permitting accurate description of changes over time. The correlation of the C-peptide slope over time assessed by repeated home DBS versus area under the curve during the two MMTTs was r=0{middle dot}73; p<0.001. Mixed models showed that a 1-month increase of diabetes duration was associated with 17 pmol/l decline in fasting DBS C-peptide, whereas increases of 1 mmol/l in glucose, 1 year older age-at-diagnosis and 100 pmol/l higher baseline plasma C-peptide were associated with 18, 17 and 61 pmol/l higher fasting DBS C-peptide levels, respectively. In addition, glucose responsiveness decreased with longer diabetes duration.\n\nConclusionOur approach permitted frequent assessment of C-peptide, making it feasible to monitor {beta}-cell function at home. Evaluation of changes in the slope of C-peptide using this method may permit short-term evaluation of promising interventions.

biochemistry↗

bioRxiv2018

Phosphoserine acidic cluster motifs in the cytoplasmic domains of transmembrane proteins bind distinct basic regions on the μ subunits of clathrin adaptor protein complexes

Protein trafficking in the endosomal system involves the recognition of specific signals within the cytoplasmic domains (CDs) of transmembrane proteins by clathrin adaptors. One such signal is the phosphoserine acidic cluster (PSAC), the prototype of which is in the endoprotease Furin. How PSACs are recognized by clathrin adaptors has been controversial. We reported previously that HIV-1 Vpu, which modulates cellular immunoreceptors, contains a PSAC that binds to the {micro} subunits of clathrin adaptor protein (AP) complexes. Here, we show that the CD of Furin binds the {micro} subunits of AP-1 and AP-2 in a phosphorylation-dependent manner. Moreover, we identify a PSAC in a cytoplasmic loop of the cellular transmembrane Serinc3, an inhibitor of the infectivity of retroviruses. The two serines within the PSAC of Serinc3 are phosphorylated by casein kinase II and mediate interaction with the {micro} subunits in vitro. The sites of these serines vary among mammals in a manner consistent with host-pathogen conflict, yet the Serinc3-PSAC seems dispensible for anti-HIV activity and for counteraction by HIV-1 Nef. The CDs of Vpu, Furin, and the PSAC-containing loop of Serinc3 each bind the subunit of AP-2 ({micro}2) with similar affinities, but they appear to utilize different basic regions on {micro}2. The Serinc3 loop requires a region previously reported to bind the acidic plasma membrane lipid phosphatidylinositol-4,5-bisphosphate. These data suggest that the PSACs within different proteins recognize different basic regions on the {micro} surface, providing the potential to inhibit the activity of viral proteins without necessarily affecting cellular protein trafficking.

biochemistry↗

bioRxiv2018

Crystallographic observation of nonenzymatic RNA primer extension

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5'-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.

biochemistry↗

bioRxiv2018

Influence of SNF1 complex on growth, glucose metabolism and mitochondrial respiration of Saccharomyces cerevisiae

The switch of mitochondrial respiration to fermentation as the main pathway to produce ATP through the increase of glycolytic flux is known as the Crabtree effect. The elucidation of the molecular mechanism of the Crabtree effect may have important applications in ethanol production and lay the groundwork for the Warburg effect, which is essential in the molecular etiology of cancer. A key piece in this mechanism could be Snf1p, which is a protein that participates in the nutritional response that includes glucose metabolism. Thus, this work aimed to recognize the role of the SNF1 complex on the glycolytic flux and mitochondrial respiration, to gain insights about its relationship with the Crabtree effect. Herein, we found that in Saccharomyces cerevisiae cells grown at 1% glucose, mutation of SNF1 gene decreased glycolytic flux, increased NAD(P)H, enhanced HXK2 gene transcription, and decreased mitochondrial respiration. Meanwhile, the same mutation increased the mitochondrial respiration of cells grown at 10% glucose. Moreover, SNF4 gene deletion increased respiration and growth at 1% of glucose. In the case of the GAL83 gene, we did not detect any change in mitochondrial respiration or growth. Altogether, these findings indicate that SNF1 is vital to switch from mitochondrial respiration to fermentation.

biochemistry↗

bioRxiv2018

Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state

Amino acid-dependent activation of mechanistic target of rapamycin complex 1 (mTORC1) is essential to reflect nutrient availabilities in cells for cell growth and metabolism1. Solute carrier 38 family A member 9 (SLC38A9) is the lysosomal transporter responsible for amino acid sensing in the mTORC1 signaling pathway2-4. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by the transmembrane helix 1 (TM1) of SLC38A9 which was anchored at the grove between transmembrane helix 5 and 7 inside the transporter. The key motif WNTMM on TM1, contributing to the anchoring interactions, is highly conserved in various species. Mutations in WNTMM motif abolished arginine transport by SLC38A9. The underlying mechanism of substrate binding is critical for both sensitizing mTORC1 signaling pathway to amino acids and for maintaining amino acid homeostasis across lysosomal membranes2.

biochemistry↗

bioRxiv2018

Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo

RNAi-based therapeutics show promising clinical data for treatment of liver-associated disorders. However, siRNA delivery into extra-hepatic tissues remains an obstacle, limiting the use of siRNA-based therapies. Here we report on a first example of chemical engineering of lipophilic conjugates to enable extra-hepatic delivery. We synthesized a panel of fifteen lipophilic siRNA and evaluated the impact of their chemical configuration on siRNA tissue distribution profile. Generally, lipophilic conjugates allow siRNA distribution to a wide range of tissues, where the degree of lipophilicity defines the ratio of liver/spleen to kidney distribution. In addition to primary clearance tissues, several conjugates achieve significant siRNA distribution to lung, heart, adrenal glands, fat, muscle. siRNA tissue accumulation leads to productive silencing, shown with two independent targets. siRNA concentrations necessary for productive silencing are tissue and conjugate dependent, varying significantly from 5 to 200 ng/mg. The collection of conjugated siRNA described here enables functional gene modulation in vivo in lung, muscle, fat, heart, adrenal glands opening these tissues for future therapeutic intervention.

biochemistry↗

bioRxiv2018

The formation of intramolecular secondary structure brings mRNA ends in close proximity

A number of protein factors regulate protein synthesis by bridging mRNA ends or untranslated regions (UTRs). Using experimental and computational approaches, we show that mRNAs from various organisms, including humans, have an intrinsic propensity to fold into structures in which the 5 end and 3 end are [≤] 7 nm apart irrespective of mRNA length. Computational estimates performed for [~]22,000 human transcripts indicate that the inherent proximity of the ends is a universal property of most, if not all, mRNA sequences. Only specific RNA sequences, which have low sequence complexity and are devoid of guanosines, are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. Our results suggest that the intrinsic proximity of mRNA ends may facilitate binding of translation factors that bridge mRNA 5 and 3 UTRs. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in mRNAs in research and biotechnology.

biochemistry↗

bioRxiv2018

Topovectorial mechanisms control the juxtamembrane proteolytic processing of Nrf1 to remove its N-terminal polypeptides during maturation of the CNC-bZIP factor

The topobiological behaviour of Nrf1 dictates its post-translational modification and its ability to transactivate target genes. Here, we have elucidated that topovectorial mechanisms control the juxtamembrane processing of Nrf1 on the cyto/nucleoplasmic side of endoplasmic reticulum (ER), whereupon it is cleaved and degraded to remove various lengths of its N-terminal domain (NTD, also refold into a UBL module) and acidic domain-1 (AD1) to yield multiple isoforms. Notably, an N-terminal [~]12.5-kDa polypeptide of Nrf1 arises from selective cleavage at an NHB2-adjoining region within NTD, whilst other longer UBL-containing isoforms may arise from proteolytic processing of the protein within AD1 around PEST1 and Neh2L degrons. The susceptibility of Nrf1 to proteolysis is determined by dynamic repositioning of potential UBL-adjacent degrons and cleavage sites from the ER lumen through p97-driven retrotranslocation and -independent pathways into the cyto/nucleoplasm. These repositioned degrons and cleavage sites within NTD and AD1 of Nrf1 are coming into their bona fide functionality, thereby enabling it to be selectively processed by cytosolic DDI-1/2 proteases and also degraded via 26S proteasomes. The resultant proteolytic processing of Nrf1 gives rise to a mature [~]85-kDa CNC-bZIP transcription factor, which regulates transcriptional expression of cognate target genes. Furthermore, putative ubiquitination of Nrf1 is not a prerequisite necessary for involvement of p97 in the client processing. Overall, the regulated juxtamembrane proteolysis (RJP) of Nrf1, though occurring in close proximity to the ER, is distinctive from the mechanism that regulates the intramembrane proteolytic (RIP) processing of ATF6 and SREBP1.

biochemistry↗