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Results for “Biochemistry”

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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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 molecular basis of hypercontractility caused by the hypertrophic cardiomyopathy mutations R403Q and R663H

Hypertrophic cardiomyopathy (HCM) mutations in {beta}-cardiac myosin and myosin binding protein-C (MyBP-C) cause hypercontractility of the heart. We show that hypercontractility caused by the HCM myosin mutation R663H cannot be explained by changes in the fundamental parameters such as actin-activated ATPase, intrinsic force, velocity of pure actin or regulated thin filaments, or the pCa50 of the velocity of regulated thin filaments. The same conclusion was made earlier for the HCM myosin mutation R403Q (Nag et al. 2015). Using enzymatic assays for the number of functionally-available heads in purified human {beta}-cardiac myosin preparations, we provide evidence that both R403Q and R663H HCM myosin mutations cause hypercontractility by increasing the number of functionally-accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of myosin binding protein-C.

biochemistry↗

bioRxiv2019

NMR Methods for Quantitative Isotopomer Rates in Real-Time Metabolism of Cells

Tracer-based metabolism is becoming increasingly important to study metabolic mechanisms in cells. NMR offers several approaches to measure label incorporation in metabolites, including 13C and 1H-detected spectra. The latter are generally more sensitive but quantification depends on the proton carbon 1JCH coupling constant which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites and quantification of 1H-edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer-based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Here we compare 13C-filtered and 13C-edited methods for quantification with a special focus towards application in real-time NMR of cancer cells under near-physiological conditions. We find an approach using a double-filter most suitable and sufficiently robust to reliably obtain 13C-incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24h.

biochemistry↗

bioRxiv2019

Drying enhances signal intensity for global GC-MS metabolomics.

We report here that a straightforward change of standard derivatization procedure for GC-MS metabolomics is leading to a strong increase in metabolite signal intensity. Drying samples between methoxymation and trimethylsilylation significantly increased signals by two-to tenfold in extracts of yeast cells, plant and animal tissue and human urine. This easy step reduces the cost of sample material and the need for expensive new hardware.

biochemistry↗

bioRxiv2019

Recombinant production and purification of the human protein Tau

Tau protein is a microtubule-stabilizing protein whose aggregation is linked to Alzheimers Disease and other forms of dementia. Tau biology is at the heart of cytoskeletal dynamics and neurodegenerative mechanisms, making it a crucial protein to study. Tau purification, however, is challenging as Tau is disordered, which makes it difficult to produce in recombinant system and is degradation-prone. It is thus challenging to obtain pure and stable preparations of Tau. Here, we present a fast and robust protocol to purify Tau recombinantly in Escherichia coli. Our protocol allows purifyig Tau either tag-less free or FLAG-tagged at its N-terminus. By exploiting a cleavable affinity tag and two anion exchange columns, we obtained Tau is of high purity, stable and suitable for in vitro studies, including aggregation experiments that resemble neurodegenerative processes.

biochemistry↗

bioRxiv2019

The architect of virus assembly: the portal protein complex nucleates procapsid assembly in bacteriophage P22

The genetic material of tailed dsDNA bacteriophages, herpesviruses and adenoviruses is packaged into a precursor capsid through a 12-mer ring-shaped protein complex called the portal protein, located at a unique 5-fold vertex. In several phages and viruses, including T4, {Phi}29, and HSV-1, the dodecameric portal protein forms a nucleation complex with scaffolding proteins to initiate procapsid assembly, thereby ensuring incorporation of only one portal complex per capsid. However, for bacteriophage P22, the role of its portal protein in initiation of procapsid assembly is unclear. We recently developed an in vitro P22 assembly assay where portal protein is co-assembled into procapsid-like particles. We also showed that scaffolding protein catalyzes oligomerization of monomeric portal protein into 12-mer rings, and possibly forming a scaffolding-protein nucleation complex that results in one portal complex per P22 procapsid. Here, we present evidence substantiating that P22 portal protein, similar to the other dsDNA viruses, can act as an assembly nucleator. We find that the presence of P22 portal protein is able to increase the rate of particle assembly. Additionally, we show that P22 portal protein proper contributes to proper morphology of the assembled particles. Our results highlight a key function of portal protein as an assembly initiator, a feature likely conserved among these classes of dsDNA viruses.

biochemistry↗

bioRxiv2019

A non-enzymatic function associated with a putative histone demethylase licenses epigenetic inheritance

H3K9 methylation (H3K9me) specifies the establishment and maintenance of transcriptionally silent epigenetic states or heterochromatin. The enzymatic erasure of histone modifications is widely assumed to be the primary mechanism that resets epigenetic states during and after DNA replication. Here, we demonstrate that a putative histone-demethylase Epe1 in fission yeast, regulates epigenetic inheritance through a non-enzymatic process. Mutations that map to its putative catalytic domain disrupt its interaction with Swi6HP1 and heterochromatin specific pattern of localization without any requirement for enzymatic activity. Epe1 and Swi6HP1 form an inhibitory complex which displaces histone deacetylases from sites of heterochromatin formation. Sequence-specific recruitment of a histone deacetylase, Clr3 renders heterochromatin refractory to the anti-silencing functions of Epe1 and licenses the inheritance of epigenetic states in cis. Epigenetic inheritance solely depends on the read-write activity of the H3K9 methyltransferase Clr4 which competes with genome-wide nucleosome turnover processes in the absence of enzymatic erasure.

biochemistry↗

bioRxiv2019

How the initiating ribosome copes with (p)ppGpp to translate mRNAs

During host colonization, bacteria use the alarmone (p)ppGpp to reshape its proteome by acting pleiotropically on RNA and protein synthesis. Here, we elucidate how the translation Initiation Factor 2 (IF2) senses the cellular ppGpp to GTP ratio and regulates the progression towards protein synthesis. Our results show that the affinity of GTP and the inhibitory concentration of ppGpp for 30S-bound IF2 vary depending on the programmed mRNA. Highly translated mRNAs enhanced GTP affinity for 30S complexes, resulting in fast transitions to elongation of protein synthesis. Less demanded mRNAs allowed ppGpp to compete with GTP for IF2, stalling 30S complexes until exchange of the mRNA enhances the affinity for GTP. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp, and that bacteria can exploit to modulate its proteome as a function of the nutritional shift happening during infection.

biochemistry↗

bioRxiv2019

Novel Methodology to Detect Physical Interaction of Inositol Phospholipid with Potassium Ion Channel Using Luminescence Resonance Energy Transfer

Membrane proteins like ion channels are located within a lipid environment. While the bulk lipids provide a supporting architecture for proteins, some lipids also play important signaling roles in modulating protein activity. This modulation can occur through direct interaction with proteins without involving second messenger pathways. Phosphoinositides are one such lipids, which have been implicated in interactions with a diverse range of proteins and such interactions mediate a variety of cellular functions. Towards understanding how these lipids play their roles in regulating protein functions It is necessary to detect interactions between proteins and lipids and to identify binding sites on proteins. However, detection of protein-lipid interactions has been difficult especially in their native environment because only a fraction of lipids are bound to proteins while the bulk is freely diffusing. The purpose of this study is to develop an experimental method capable of identifying protein-lipid interactions in membrane environments. The strategy involves luminescence energy transfer to locally illuminate lipids around proteins thereby suppressing the large background of light from unbound lipid molecules. The approach is applied to an inward rectifier potassium ion channel for its interactions with phosphoinositide lipids. Experiments show that it can successfully detect phosphoinositides bound to proteins, and when combined with mutagenic approaches, can further identify binding site. The approach thus provides a valuable methodology for probing protein-lipid interactions in a native-like lipid environment.

biochemistry↗

bioRxiv2019

The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from a cis-splicing intein

Protein trans-splicing catalyzed by split inteins has increasingly become useful as a protein engineering tool. The 1.0 [A]-resolution crystal structure of a variant from naturally split gp41-1 intein, identified from the environmental metagenomic sequence data, revealed an improved pseudo-C2-symmetry commonly found in the Hedgehog/Intein (HINT) superfamily with extensive charge-charge interactions between the split N-and C-terminal intein fragments. We successfully created orthogonal split inteins by engineering a similar charge network in the same region of a cis-splicing intein. The same strategy could be applicable for creating novel natural-like split inteins from other, more prevalent cis-splicing inteins.

biochemistry↗

bioRxiv2019

Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF

Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organise chromosomes, thereby facilitating chromosome segregation. They utilise cycles of ATP binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contacts. We have analysed changes in the architecture of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis. This builds upon previous work showing that MukF kleisin directs formation of a MukBEF tripartite ring as a consequence of functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively (Zawadzka et al., 2018). Using both model truncated substrates and complexes containing full length MukB, we now demonstrate formation of MukBEF dimers of dimers, dependent on MukF dimerization, MukB head-engagement and MukE, which plays an essential role in organizing MukBEF complexes.

biochemistry↗

bioRxiv2019

Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the Gβ subunit

G protein-coupled receptors (GPCRs) are the largest class of integral membrane proteins and represent key targets for pharmacological research. GPCRs modulate cell physiology by engaging and activating a diversity of intracellular transducers, prominently heterotrimeric G proteins, but also G protein-receptor kinases (GRKs) and arrestins. The recent surge in the number of structures of GPCR-G protein complexes has expanded our understanding of G protein recognition and GPCR-mediated signal transduction. However, many aspects of these mechanisms, including the existence of transient interactions with transducers, have remained elusive. Here, we present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. In contrast to all reported structures, our density map reveals the receptor C-terminal tail bound to the G{beta} subunit of the G protein heterotrimer. This observation provides a structural foundation for the role of the C-terminal tail in GPCR signaling, and of G{beta} as scaffold for recruiting G subunits and GRKs. By comparing all available complex structures, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

biochemistry↗

bioRxiv2019

Direct Visualization of Live Zebrafish Glycan via Single-step Metabolic Labeling with Fluorophore-tagged Nucleotide Sugars

Dynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. The metabolic glycan labeling coupled with bioorthogonal chemistry has paved the way for visulizing glycans in living organisms. However, a two-step labeling sequence is required, which is prone to tissue penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogs of nucleotide sugars directly. Injecting the fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables a systematic imaging of sialylation and fucosylation in live zebrafish embryos at various developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

biochemistry↗

bioRxiv2019

Dynamic membrane topology in an unassembled membrane protein

Helical membrane proteins constitute roughly a quarter of all proteomes and perform diverse biological functions. To avoid aggregation, they undergo cotranslational membrane insertion and are typically assumed to attain stable transmembrane topologies immediately upon insertion. To what extent post-translational changes in topology are possible in-vivo and how they may affect biogenesis is incompletely understood. Here, we show that monomeric forms of Small Multidrug Resistance (SMR) proteins display topological dynamics, where the N-terminal transmembrane helix equilibrates between membrane-inserted and non-inserted states. We characterize the kinetics of the process and show how the composition of the helix regulates the topological dynamics. We further show that topological dynamics is a property of the unassembled monomeric protein, as the N-terminal helix becomes fixed in a transmembrane disposition upon dimerization. Membrane protein topology can thus remain dynamic long after cotranslational membrane insertion, and can be regulated by later assembly processes.

biochemistry↗

bioRxiv2019

A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

DNA and RNA nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA nucleases. These nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.

biochemistry↗

bioRxiv2019

Activated thiol Sepharose-based proteomic approach to globally quantify protein oxidation

Reactive oxygen species (ROS) can act as second messengers for various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of redox signaling. We developed an Activated Thiol Sepharose (ATS)-based proteomic approach (ATSP) to globally quantify protein oxidation. ATSP can enrich for H2O2-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins, such as the M2 isoform of pyruvate kinase (PKM2). Treatment of HLRCC cells with dimethyl fumarate (DMF) or PKM2 activators alter PKM2 oxidation levels. This newly developed redox proteomics method can globally detect oxidation of reactive cysteines and can be employed to analyze multiple physiological and pathological conditions.

biochemistry↗

bioRxiv2019

An orthogonal c-Cbl recognition mode targets LynA for rapid degradation and builds specificity into the LynA checkpoint

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is critical factor regulating myeloid-cell activation. In a previous paper (Freedman et al., 2015) we showed in macrophages that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation, functioning as a rheostat regulating ITAM signaling. We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic and biochemical analysis, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via tyrosine 32 in its unique insert region. This orthogonal mode of c-Cbl recognition depresses the steady-state level of macrophage LynA. Mast cells, however, express little c-Cbl and have correspondingly high steady-state levels of LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression therefore builds cell specificity into the LynA checkpoint.

biochemistry↗

bioRxiv2019

Conservation of structure, function and inhibitor binding in UNC-51-like kinase 1 and 2 (ULK1/2)

Autophagy is essential for cellular homeostasis and when deregulated this survival mechanism has been associated with disease development. Inhibition of autophagy initiation by inhibiting the kinase ULK1 has been proposed as a potential cancer therapy. While inhibitors and crystal structures of ULK1 have been reported, little is known about the other closely related kinase ULK2. Here we present the crystal structure of ULK2 in complex with ATP competitive inhibitors. Surprisingly, the ULK2 structure revealed a dimeric assembly reminiscent of dimeric arrangements of auto-activating kinases suggesting a role for this association in ULK activation. Screening of a kinase focused library of pre-clinical and clinical compounds revealed several potent ULK1/2 inhibitors and good correlation of inhibitor binding behavior with both ULK kinases. Aurora A was identified as a major off-target of currently used ULK1 inhibitors. Autophagic flux assays demonstrated that this off-target activity by strongly inducing autophagy in different cellular systems conferred an additional layer of complexity in the interpretation of cellular data. The data presented here provides structural models and chemical starting points for the development of ULK1/2 dual inhibitors with improved selectivity for future exploitation of autophagy inhibition.

biochemistry↗

bioRxiv2019

Extracellular matrix mineralization in the mouse osteoblast-like cell line MC3T3-E1 is regulated by actin cytoskeleton reorganization and non-protein molecules secreted from the cells themselves

Bone tissue constantly undergoes turnover via bone formation by osteoblasts and bone resorption by osteoclasts. This process enables bone to maintain its overall shape while altering its local structure. However, the detailed mechanism of how osteoblast cell-signaling systems induce various structural changes in bone tissue have not yet been completely elucidated. In this study, we focused on the actin cytoskeleton as a regulatory system for bone formation and constructed an in vitro experimental system using the mouse osteoblast-like cell line MC3T3-E1. We found that, in MC3T3-E1 cells, the actin cytoskeleton had an important role in matrix mineralization via activation of specific developmental pathways and it was regulated by non-protein molecules secreted from MC3T3-E1 cells themselves. In MC3T3-E1 cells, we observed changes of actin cytoskeleton reorganization and accumulation of PIP2 related to actin filament convergences during cell differentiation, in the undifferentiated, early, middle and late stage. Actin cytoskeleton disruption with Cyto D, polymerization inhibitor of actin filament, in early and middle stage cells induced significant increase of osteocalcin mRNA expression normally expressed only in late stage, decrease of Alkaline phosphatase mRNA expression after 24h and abnormal matrix mineralization in MC3T3-E1 cells. Inhibition of Gi with PTX known to regulate actin cytoskeleton in middle stage induced changes in the actin cytoskeleton and PIP2 accumulation and suppression of matrix mineralization after 5 days. Furthermore, addition of non-protein molecules from culture medium of cells at various differentiation stage induced difference of PIP2 accumulation after 5 min, actin cytoskeleton in 20 min, and matrix mineralization after 5 days. These results not only provide new knowledge about the actin cytoskeleton function in bone-forming cells, but also suggest that cell signaling via non-protein molecules such as lipids plays important roles in bone formation.

biochemistry↗