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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

Calcium-independent phospholipase A2{beta} (iPLA2{beta}) regulates several physiological processes including inflammation, calcium homeostasis and apoptosis. It is linked genetically to neurodegenerative disorders including Parkinsons disease. Despite its known enzymatic activity, the mechanisms underlying pathologic phenotypes remain unknown. Here, we present the first crystal structure of iPLA2{beta} that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. The ankyrin repeat domains wrap around the catalytic domains in an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. A single calmodulin binds and allosterically inhibits both catalytic domains. These unique structural features identify the molecular interactions that can regulate iPLA2{beta} activity and its cellular localization, which can be targeted to identify novel inhibitors for therapeutic purposes. The structure provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of iPLA2{beta} in the brain.

biochemistry↗

bioRxiv2017

Short-term stabilities of 21 amino acids in dried blood spots

BACKGROUNDDried blood spots (DBSs) have potential use in remote health applications for individual and population diagnosis, and can enable epidemiological surveillance for known and unknown diseases. The preparation and transportation of DBSs from remote settings often exposes these cards to extreme environmental stress that may impact the quality of the diagnostic data. Given these risks, it is essential to investigate the individual stabilities of biomarkers in DBSs. This paper details the stability of routinely-analyzed amino acids (AAs) on DBSs under environmental conditions that simulate a global health workflow.\n\nMETHODSThe extractions of 21 AAs from three sets of DBSs prepared on cellulose and cotton filter paper were optimized for quantitation by dansylation-UPLC/MRM-MS. The effects of sunlight exposure, temperature, humidity, and storage time were studied.\n\nRESULTSThe AAs were stable in DBSs after 4-hour sunlight exposure, and after storage at -20 and 4 {degrees}C for 30 days. At 25 and 40 {degrees}C, only 7 AAs showed significant concentration decreases over time, while 2 showed concentration increases. The changes were accelerated by high humidity. Histidine was the least stable AA under the conditions tested.\n\nCONCLUSIONSThis study provides quantitative data on the short-term stabilities of 21 AAs in DBSs on cellulose and cotton-based filter paper, under environmental conditions that simulate a global-health workflow. These results highlight the importance of assessing the stability of clinically-relevant biomarkers in DBSs. Based on the measured stabilities, we recommend that higher-temperature and high-humidity storage of DBS samples be avoided for AA analysis in remote health applications.

biochemistry↗

bioRxiv2017

A combinatorial approach for the synthesis of multi-phosphorylated peptides: new tool for studying phosphorylation patterns in proteins

Phosphorylation of proteins at multiple sites creates different phosphorylation patterns that are essential for their biological activity. For example, such patterns contribute to the redirection of signalling to alternative pathways. Multi-phosphorylated peptides are excellent tools to systematically study the impact of unique phosphorylation patterns on signalling, but their synthesis is extremely difficult. Here we present an efficient and general method for the synthesis of multi-phosphorylated peptides, using a combination of different tailor-made coupling conditions. The method was demonstrated for the synthesis of a library of Rhodopsin C terminal peptides with distinct phosphorylation patterns containing up to seven phosphorylated Ser (pSer) and Thr (pThr) residues in close proximity to one another. Our method can be used to synthesize peptides incorporating multiple phosphorylated amino acids at high efficiency. It does not require any special expertise and can be performed in any standard peptide laboratory. This approach opens the way for quantitative mechanistic studies of phosphorylation patterns and their biological roles.

biochemistry↗

bioRxiv2017

Membrane Proteins Significantly Restrict Exosome Mobility

Exosomes are membrane nanovesicles that intermediate cell-to-cell signaling through the transfer of their molecular cargo. The exosomes small size facilitates rapid migration through the extracellular matrix and into and out of circulation. Here we report that the mobility of the exosomes is much lower than would be expected from the size of their membrane vesicles. The difference is broadly distributed and caused by surface proteins, which significantly impede exosome migration. The observed wide range in the mobility implies that a subpopulation of hydrodynamically small exosomes is more likely to participate in signaling. The extracellular environment amplifies the size-dependent hindrance to the exosomes migration. The significant contribution of surface proteins to the transport resistance make the exosome mobility a dynamic property that changes with the extracellular environment which affects the membrane protein conformation, glycosylation, specific, and non-specific surface adsorption.

biochemistry↗

bioRxiv2017

Neuropathic mutations in MORC2 perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the HUSH complex. Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies.

biochemistry↗

bioRxiv2017

Structural Basis for Katanin Self-Assembly

The reorganization of microtubules in mitosis, meiosis and development requires the microtubule-severing activity of katanin. Katanin is composed of a AAA ATPase subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanins conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that wild-type katanin only forms heterodimers and heterotetramers. Heterododecamers were only observed for an ATP hydrolysis deficient mutant in the presence of ATP, suggesting an auto-inhibition mechanism that prevents oligomerization. X-ray structures of katanins AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states reveal conformational changes in AAA subdomains and N and C-terminal expansion segments that explain this auto-inhibition of assembly. These data lead to a model in which self-inhibited heterodimers bind to a microtubule, then transition into an assembly-competent conformation upon ATP binding. Microtubule-bound heterododecamers then promote tubulin extraction from the microtubule prior to oligomer dissociation.

biochemistry↗

bioRxiv2017

Temporal profiling of redox-dependent heterogeneity in single cells

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe roGFP. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach we defined redox-dependent heterogeneity of yeast cells, and characterized growth, as well as proteomic and transcriptomic profiles of subpopulations of cells that differ in their redox status, but are similar in age. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

biochemistry↗

bioRxiv2017

Control of Cardiac Mitochondrial Fuel Selection by Calcium

Calcium ion concentration modulates the function of pyruvate dehydrogenase, isocitrate dehydrogenase, and -ketoglutarate dehydrogenase. Previous studies have shown that despite its ability to affect the function of these dehydrogenases, [Ca2+] does not substantially alter mitochondrial ATP synthesis in vitro under physiological sub-strate conditions. We hypothesize that, rather than contributing to respiratory control, [Ca2+] governs fuel selection. Specifically, cardiac mitochondria are able to use different primary carbon substrates to synthesize ATP aerobically. To determine if and how [Ca2+] affects the relative use of carbohydrates versus fatty acids we measured oxygen consumption and tricarboxylic acid cycle intermediate concentrations in suspensions of cardiac mitochondria with different combinations of pyruvate and palmitoyl-L-carnitine in the media at various [Ca2+] and ADP infusion rates. Results reveal that when both fatty acid and carbohydrate substrates are available, fuel selection is sensitive to both calcium and ATP synthesis rate. When no Ca2+ is added under low ATP-demand conditions, {beta}-oxidation provides roughly half of acetyl-CoA for the citrate synthase reaction with the rest coming from the pyruvate dehydrogenase reaction. Under low demand conditions with increasing [Ca2+], the fuel utilization ratio shifts to increased fractional consumption of pyruvate, with 83{+/-}10% of acetyl-CoA derived from pyruvate at the highest [Ca2+] evaluated. With high ATP demand, the majority of acetyl-CoA is derived from pyruvate, regardless of the Ca2+ level. Our results suggest that changes in work rate alone are enough to effect a switch to carbohydrate use while in vivo the rate at which this switch happens may depend on mitochondrial calcium.\n\nKey PointsO_LIDespite its effects on activity of mitochondrial dehydrogenases, Ca2+ does not substantially alter mitochondrial ATP synthesis in vitro under physiological substrate conditions. Nor does is appear to play an important role in respiratory control in vivo in the myocardium.\nC_LIO_LIWe hypothesize that Ca2+ plays a role mediating the switch in fuel selection to increasing carbohydrate oxidation and decreasing fatty acid oxidation with increasing work rate.\nC_LIO_LITo determine if and how Ca2+ affects the relative use of carbohydrates versus fatty acids in vitro we measured oxygen consumption and TCA cycle intermediate concentrations in suspensions of purified rat ventricular mitochondria with carbohydrate, fatty acid, and mixed substrates at various [Ca2+] and ATP demand rates.\nC_LIO_LIOur results suggest that changes in work rate alone are enough to effect a switch to carbohydrate use in vitro while in vivo the rate at which this switch happens may depend on mitochondrial calcium.\nC_LI

biochemistry↗

bioRxiv2017

Development of a riboswitch-based platform for live cell imaging of RNAs in mammalian cells

RNAs directly regulate a vast array of critical cellular processes, emphasizing the need for robust approaches to fluorescently tag and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular Riboglow platform leverages different color fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescent turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small non-coding U RNA in live mammalian cells. A direct side-by-side comparison revealed that Riboglow outperformed the dye binding aptamer Broccoli and performed on par with the current gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR/small/199240_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@11b5e3borg.highwire.dtl.DTLVardef@1c78fc0org.highwire.dtl.DTLVardef@aeba74org.highwire.dtl.DTLVardef@1531e3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

Gum Arabic promotes oxidation and ester hydrolysis.

We studied the interactions between gum Arabic and select catecholic compounds like caffeic acid, chlorogenic acid and catechol. We observed that GA is capable of promoting the auto-oxidation of the above-mentioned compounds into darkly colored pigments without the addition of redox-sensitive cations. Gum Arabic appeared to be unique among polysaccharide-based materials as many other types of polysaccharides promote the oxidation of the above-mentioned compounds only in the presence of redox cations like Fe2+ or Cu2+. RP-HPLC and SEC chromatographic techniques were employed to monitor the reactions and to observe the formation of high molecular mass, pigmented materials from gum Arabic and all three catecholic compounds. FT-IR spectroscopic analysis revealed that, despite their darkly colored appearances, the gum Arabic/pigment materials synthesized contain mostly gum Arabic and very little pigment. As chlorogenic acid is an ester of caffeic acid, we studied the capacity of gum Arabic to promote ester hydrolysis using acetylsalicylic acid as the model compound. We observed that gum Arabic did promote the hydrolysis of acetylsalicylic acid into salicylic acid. However, in all our experiments involving the pigment formation between gum Arabic and chlorogenic acid, we did not observe any evidence that chlorogenic acid was hydrolyzed leading to the release of caffeic acid during these reactions. In addition, we observed that heat treatment of gum Arabic did not affect its pro-oxidizing capability, but it did negatively affect its capability to hydrolyze salicylic acid. Thus, these two types of chemical reactivity present in the gum Arabic material may be associated with different components of the gum Arabic material.

biochemistry↗

bioRxiv2017

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes a significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution interactions are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results we suggest a model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.

biochemistry↗

bioRxiv2017

Sequence, Structure and Context Preferences of Human RNA Binding Proteins

Production of functional cellular RNAs involves multiple processing and regulatory steps principally mediated by RNA binding proteins (RBPs). Here we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of an RBP in vitro from deep sequencing of bound RNAs. Analyses of these data revealed several interesting patterns, including unexpectedly low diversity of RNA motifs, implying frequent convergent evolution of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, we observed extensive preferences for contextual features outside of core RNA motifs, including spaced \"bipartite\" motifs, biased flanking nucleotide context, and bias away from or towards RNA structure. These contextual features are likely to enable targeting of distinct subsets of transcripts by different RBPs that recognize the same core motif. Our results enable construction of \"RNA maps\" of RBP activity without requiring crosslinking-based assays, and provide unprecedented depth of information on the interaction of RBPs with RNA.

biochemistry↗

bioRxiv2017

Base-resolution mapping reveals distinct m1A methylome in nuclear- and mitochondrial-encoded transcripts

Gene expression can be post-transcriptionally regulated via dynamic and reversible RNA modifications. N1-methyladenosine (m1A) is a recently identified mRNA modification; however, little is known about its precise location, regulation and function. Here, we develop a base-resolution m1A profiling method, based on m1A-induced misincorporation during reverse transcription, and report distinct classes of m1A methylome in the human transcriptome. m1A in 5-UTR, particularly those at the first nucleotide of mRNA, associate with increased translation efficiency. A different subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome and are dependent on the methyltransferase TRMT6/61A. Additionally, we show for the first time that m1A is prevalent in the mitochondrial-encoded transcripts. Manipulation of m1A level via TRMT61B, a mitochondria-localizing m1A methyltransferase, demonstrates that m1A in mitochondrial mRNA interferes with translation. Collectively, our approaches reveal distinct classes of m1A methylome and provide a resource for functional studies of m1A-mediated epitranscriptomic regulation.

biochemistry↗

bioRxiv2017

Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) that regulates all aspects of life. To date, investigation of human cell signalling has focussed on canonical phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that phosphorylation of histidine also plays a central role in regulating cell biology. Phosphoproteomics workflows rely on acidic conditions for phosphopeptide enrichment, which are incompatible with the analysis of acid-labile phosphorylation such as histidine. Consequently, the extent of non-canonical phosphorylation is likely to be under-estimated.\n\nWe report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), which permits enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive and positional phosphorylation patterns on histidine, arginine, lysine, aspartate and glutamate in human cell extracts, including 310 phosphohistidine and >1000 phospholysine sites of protein modification. Remarkably, the extent of phosphorylation on individual non-canonical residues vastly exceeds that of basal phosphotyrosine. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring roles of acid-labile phosphorylation in any proteome using mass spectrometry.

biochemistry↗

bioRxiv2017

Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin

Histone deacetylase 6 (HDAC6) is a cytoplasmic HDAC isoform able to remove acetyl groups from cellular substrates such as -tubulin. In addition to the two deacetylase domains, HDAC6 has a C-terminal zinc-finger ubiquitin (Ub)-binding domain (ZnF-UBP) able to recognize free Ub. HDAC6-Ub interaction is thought to function in regulating the elimination of misfolded proteins during stress response through the aggresome pathway. Small molecules inhibiting deacetylation by HDAC6 were shown to reduce aggresomes, but the interplay between HDAC6 catalytic activity and Ub-binding function is not fully understood. Here we describe two methods to measure the HDAC6-Ub interaction in vitro using full-length HDAC6. Both methods were effective for screening inhibitors of the HDAC6-Ub protein-protein interaction independently of the catalytic activity. Our results suggest a potential role for the HDAC6 deacetylase domains in modulating HDAC6-Ub interaction. This new aspect of HDAC6 regulation can be targeted to address the roles of HDAC6-Ub interaction in normal and disease conditions.

biochemistry↗

bioRxiv2017

SIRT2 and lysine fatty acylation regulate the oncogenic activity of K-Ras4a

Ras proteins play vital roles in numerous biological processes and Ras mutations are found in many human tumors. Understanding how Ras proteins are regulated is important for elucidating cell signaling pathways and identifying new targets for treating human diseases. Here we report that one of the K-Ras splice variants, K-Ras4a, is subject to lysine fatty acylation, a previously under-studied protein post-translational modification. Sirtuin 2 (SIRT2), one of the mammalian nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, catalyzes the removal of fatty acylation from K-Ras4a. We further demonstrate that SIRT2-mediated lysine defatty-acylation promotes endomembrane localization of K-Ras4a, enhances its interaction with A-Raf, and thus promotes cellular transformation. Our study identifies lysine fatty acylation as a previously unknown regulatory mechanism for the Ras family of GTPases that is distinct from cysteine fatty acylation. These findings highlight the biological significance of lysine fatty acylation and sirtuin-catalyzed protein lysine defatty-acylation.

biochemistry↗

bioRxiv2017

Early urinary candidate biomarkers in a rat model of experimental autoimmune encephalomyelitis

Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system and is difficult to diagnose in early stages. Without homeostatic control, urine was reported to have the ability to accumulate early changes in the body. We expect that urinary proteome can reflect early changes in the nervous system. In this study, the early urinary proteome changes in a most employed multiple sclerosis rat model (experimental autoimmune encephalomyelitis (EAE)) were analyzed to explore early urinary candidate biomarkers, and early treatment of methylprednisolone were used to evaluate the therapeutic effect. Compare with controls, twenty-five urinary proteins were altered at day 7 when there were no clinical symptoms and no obvious histological changes. Among them, twenty-three have human homologs and fourteen were reported to be differently expressed in the serum/cerebrospinal fluid/brain tissues of multiple sclerosis patients or animal models. Functional analysis showed that the dysregulated proteins were associated with asparagine degradation, neuroinflammation and lipid metabolism. After the early treatment of methylprednisolone, the incidence of encephalomyelitis in the intervention group was only 1/13. This study demonstrates that urine may be a good source of biomarkers for the early detection of multiple sclerosis and early treatment can significantly delay disease progression. These findings may provide important information for early diagnosis and intervention of multiple sclerosis in the future.

biochemistry↗

bioRxiv2017

DNA unwinding is the primary determinant of CRISPR-Cas9 activity

Bacterial adaptive immunity utilizes RNA-guided surveillance complexes composed of CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs with the target strand of dsDNA, displacing the non-target strand to create an R-loop, where the HNH and RuvC nuclease domains can cleave opposing strands. Cas9 has been repurposed for a variety of important genome engineering applications. While many structural and biochemical studies have shed light on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. Our detailed kinetic characterization of the enzyme reveals that DNA binding is reversible, R-loop formation is rate-limiting, occurring in two steps, one for each of the nuclease domains. Although the HNH nuclease activity is stimulated by Mg2+ with a single measureable Kd, the RuvC activity requires two distinct Mg2+ binding events. The specificity constant for cleavage is determined through an induced-fit mechanism as the product of the equilibrium binding affinity for DNA and the rate of R-loop formation.

biochemistry↗