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Results for “Biochemistry”

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bioRxiv2020

Investigating peripubertal stress and exogenous corticosterone as endogenous stimulators of brain metabolism

Substantial research on the association between early-life stress and its long-lasting impact on lifetime mental health has been performed revealing that early-life environmental adversity strongly regulates brain function. Alterations of gene expression and behavior in the off-springs of paternally stressed rats were also revealed. However, the precise mechanisms underlying these changes remain poorly understood. Here, an improved characterization of these processes from investigations of the functional metabolism of animal models exposed to peripubertal stress (PS) is proposed. The ultimate goal of this study was to bring forward functional Magnetic Resonance Spectroscopy (fMRS) as a technique of interest for a better understanding of brain areas by endogenous stimulators such as stress. The present study evaluated, compared and classified effects of individual PS (iPS) and paternal PS (pPS) under corticosterone (CORT) challenge in the septal areas of adult rats. Acute stress was simulated by injection of CORT and metabolic concentration changes were analyzed as a function of time. Evaluation of Glucose and Lactate concentration changes allowed the classification of groups of rats using a Glc to Lac index. Moreover, metabolic responses of control rats (CC) and of pPS x iPS rats (SS) were similar while responses in pPS (SC) and iPS (CS) differed, revealing differential adaption of energetic metabolism and of glutamatergic neurotransmission. Findings have crucial interest for understanding the metabolic mechanisms underlying altered functional connectivity and neuronal plasticity in septal areas inducing increased aggressivity in early-life stressed rats.

biochemistry↗

bioRxiv2020

Binding of guide piRNA triggers methylation of the unstructured N-terminal region of Aub leading to assembly of the piRNA amplification complex

Piwi proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two Piwi proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimp uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of Piwi proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing. HighlightsO_LIsDMA modification of Aub is essential for ping-pong cycle, transposon silencing and fertility. C_LIO_LIpiRNA loading triggers Aub sDMA modification by exposing its unstructured N terminal region to the methylosome complex. C_LIO_LIA single molecule of Krimp simultaneously binds piRNA-bound Aub and empty Ago3 to assemble ping-pong complex. C_LIO_LIThe distinct structure of two Tudor domains of Krimp ensures binding of Ago3 and Aub in opposite modification and piRNA-loading states. C_LI

biochemistry↗

bioRxiv2020

PFN2 and NAA80 cooperate to efficiently acetylate the N-terminus of actin

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. Here, we define a specific role for PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80. PFN2 binding increases the intrinsic catalytic activity of NAA80. Furthermore, binding of PFN2 to NAA80 via its proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. The majority of NAA80 is stably bound to PFN2, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation. Data are available via ProteomeXchange with identifier PXD020188.

biochemistry↗

bioRxiv2020

Spatio-temporal control of phenylpropanoid biosynthesis by inducible complementation of a cinnamate 4-hydroxylase mutant

Cinnamate 4-hydroxylase (C4H) is a cytochrome P450-dependent monooxygenase that catalyzes the second step of the general phenylpropanoid pathway. Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, which carry hypomorphic mutations in C4H, exhibit global alterations in phenylpropanoid biosynthesis and have developmental abnormalities including dwarfing. Here we report the characterization of a conditional Arabidopsis C4H line (ref3-2pOpC4H), in which wild-type C4H is expressed in the ref3-2 background. Expression of C4H in plants with well-developed primary inflorescence stems resulted in restoration of fertility and the production of substantial amounts of lignin, revealing that the developmental window for lignification is remarkably plastic. Following induction of C4H expression in ref3-2pOpC4H, we observed rapid and significant reductions in the levels of numerous metabolites, including several benzoyl and cinnamoyl esters and amino acid conjugates. These atypical conjugates were quickly replaced with their sinapoylated equivalents, suggesting that phenolic esters are subjected to substantial amounts of turnover in wild-type plants. Furthermore, using localized application of dexamethasone to ref3-2pOpC4H, we show that phenylpropanoids are not transported appreciably from their site of synthesis. Finally, we identified a defective Casparian strip diffusion barrier in the ref3-2 mutant root endodermis, which is restored by induction of C4H expression. HighlightThe work presented this paper provides evidence of metabolite turnover, plasticity of the developmental window for lignification, and the impact of reduced and restored cinnamate-4-hydroxylase (C4H) expression on the Casparian strip.

biochemistry↗

bioRxiv2020

Modulate Siglec-7 Signaling via in situ Created High-affinity cis-Ligands

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan epitopes overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the mechanisms underlying these processes are just starting to be elucidated. We discover that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both transfer of sialylated glycans from NK cells to target tumor cells and accelerated de novo synthesis of sialosides on the tumor cell themselves. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating Siglec-7-regulated NK-activation. At high levels, the added Siglec-7 ligand suppresses NK-cytotoxicity through the recruitment of Siglec-7, whereas at low levels the same ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that glycan engineering of NK cells may provide a means to boost NK effector functions.

biochemistry↗

bioRxiv2020

AGC kinase members phosphorylate ubiquitin.

The posttranslational modification of proteins with ubiquitin controls most cellular processes, such as protein degradation or transport, cell signaling, or transcription1-5. Ubiquitin can be phosphorylated at multiple sites, which likely further modulates the function of protein ubiquitination6,7. However, except for PINK18-10, the kinases involved in ubiquitin phosphorylation remain unknown, which hampers our understanding of phospho-ubiquitin signaling. In this study, we performed genome-wide in vitro kinase screenings and discovered that AGC kinases phosphorylate ubiquitin. Ubiquitin phosphorylation by members of the PKA, PKC, PKG and RSK families as well as by less well-characterized kinases, such as SGK2, was not solely dependent on peptide specificity but required additional kinase recruitment to ubiquitin. The stabilization of the kinase interaction with ubiquitin resulted in phosphorylation of suboptimal kinase motifs on ubiquitin, suggesting that ubiquitin phosphorylation is dictated primarily through the recruitment of kinases to the ubiquitinated proteins. Hence, we identify AGC kinase members as enzymes that can phosphorylate ubiquitin in a mechanism regulated by protein interactions outside of the catalytic kinase domain and are only applicable to specific subsets of ubiquitinated proteins.

biochemistry↗

bioRxiv2020

Myeloid deletion and therapeutic activation of AMP-activated protein kinase (AMPK) do not alter atherosclerosis in male or female mice

ObjectiveThe dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Approach and ResultsWe aimed to clarify the role of myeloid-specific AMPK signaling by using LysM-Cre to drive the deletion of both the 1 and 2 catalytic subunits (MacKO), in male and female mice made acutely atherosclerotic by PCSK9-AAV and Western diet-feeding. After 6 weeks of Western diet feeding, half received daily injection of either the AMPK activator, A-769662 or a vehicle control for a further 6 weeks. After 12 weeks, myeloid cell populations were not different between genotype or sex. Similarly, aortic sinus plaque size, lipid staining and necrotic area were not different in male and female MacKO mice compared to their littermate floxed controls. Moreover, therapeutic intervention with A-769662 had no effect. There were no differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area or markers of autophagy showed no effect of either lacking AMPK signaling or systemic AMPK activation. ConclusionsOur data suggest that while defined roles for each catalytic AMPK subunit have been identified, global deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Moreover, we show that intervention with the first-generation AMPK activator, A-769662, was not able to stem the progression of atherosclerosis. Highlights- The deletion of both catalytic subunits of AMPK in myeloid cells has no significant effect on the progression of atherosclerosis in either male or female mice - Therapeutic delivery of a first-generation AMPK activator (A-769662) for the last 6 weeks of 12-week study had no beneficial effect in either male or female mice - Studying total AMPK deletion may mask specific effects of each isoform and highlights the need for targeted disruption of AMPK phosphorylation sites via knock-in mutations, rather than the traditional "sledgehammer" knockout approach

biochemistry↗

bioRxiv2020

An Antioxidant Enzyme Therapeutic for COVID-19

The COVID-19 pandemic has taken a significant toll on people worldwide, and there are currently no specific antivirus drugs or vaccines. We report herein a therapeutic based on catalase, an antioxidant enzyme that can effectively breakdown hydrogen peroxide and minimize the downstream reactive oxygen species, which are excessively produced resulting from the infection and inflammatory process. Catalase assists to regulate production of cytokines, protect oxidative injury, and repress replication of SARS-CoV-2, as demonstrated in human leukocytes and alveolar epithelial cells, and rhesus macaques, without noticeable toxicity. Such a therapeutic can be readily manufactured at low cost as a potential treatment for COVID-19.

biochemistry↗

bioRxiv2020

Mode of Targeting to the Proteasome Determines GFP Fate

The Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasomes ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.

biochemistry↗

bioRxiv2020

Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

Nucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are attracting antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by RdRp. We found that the incorporation efficiency of Remdesivir-TP is higher than ATP, and we did not observe chain termination or delayed chain termination caused by single Remdesivir-TP incorporation, while multiple incorporations of Remdesivir-TP caused chain termination in our assay condition. The incorporation efficiency of Ribavirin-TP and Favipiravir-TP is very low either as ATP or GTP analogs, which suggested that mutagenesis may not be the mechanism of action of those two drugs against SARS-CoV-2. Incorporation of Sofosbuvir-TP is also very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2-C-Methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2-C-Methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.

biochemistry↗

bioRxiv2020

Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

Advanced analytical strategies including top-down and middle-up HPLC-MS approaches have become powerful alternatives to classical bottom-up analysis for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up analysis of polyclonal IgGs posing additional challenges due to extensive sequence variability. The presented workflow is based on Fc/2 portions as conserved subunits of IgGs and enables global profiling of subclasses and their glycosylation patterns, both of which influence IgG effector functions. To obtain subunits of murine IgGs, we established digestion with the bacterial protease SpeB. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry allowing relative quantification of IgG subclasses and their N-glycosylation variants. In order to assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. This analysis simultaneously revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine as demonstrated for monoclonal IgGs, which may be implemented for quality control of functional antibodies.

biochemistry↗

bioRxiv2020

The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans

Osteocalcin (OCN) is an osteoblast-derived hormone with pleiotropic physiological functions. Like many peptide hormones, OCN is subjected to post-translational modifications (PTMs) which control its activity. Here, we uncover O-glycosylation as a novel PTM present on mouse OCN and occurring on a single serine (S8) independently of its carboxylation and endoproteolysis, two other PTMs regulating this hormone. We also show that O-glycosylation increases OCN half-life in plasma ex vivo and in the circulation in vivo. Remarkably, in human OCN (hOCN), the residue corresponding to S8 is a tyrosine (Y12), which is not O-glycosylated. Yet, the Y12S mutation is sufficient to O-glycosylate hOCN and to increase its half-life in plasma compared to wildtype hOCN. These findings reveal an important species difference in OCN regulation, which may explain why serum concentrations of OCN are higher in mouse than in human. The authors have nothing to disclose

biochemistry↗

bioRxiv2020

Simulations support the interaction of the SARS-CoV-2 spike protein with nicotinic acetylcholine receptors and suggest subtype specificity

Changeux et al. recently suggested that the SARS-CoV-2 spike (S) protein may interact with nicotinic acetylcholine receptors (nAChRs). Such interactions may be involved in pathology and infectivity. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the S protein, to investigate this nicotinic hypothesis. We examine the binding of the Y674-R685 loop of the S protein to three nAChRs, namely the human 4{beta}2 and 7 subtypes and the muscle-like {beta}{gamma}d receptor from Tetronarce californica. Our results indicate that Y674-R685 has affinity for nAChRs and the region responsible for binding contains the PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. In particular, R682 has a key role in the stabilisation of the complexes as it forms interactions with loops A, B and C in the receptors binding pocket. The conformational behaviour of the bound Y674-R685 region is highly dependent on the receptor subtype, adopting extended conformations in the 4{beta}2 and 7 complexes and more compact ones when bound to the muscle-like receptor. In the 4{beta}2 and {beta}{gamma}d complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation similar to other known nAChR antagonists. In contrast, in the 7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of the simulations of the full-length S protein show that the Y674-R685 region is accessible for binding, and suggest a potential binding orientation of the S protein with nAChRs.

biochemistry↗

bioRxiv2020

Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is tailored by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminus threaded through the macrocycle. This unique lariat topology, which provides considerable stability towards heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of arginine deimination to yield citrulline. Although a citrulline-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for arginine deimination has remained unknown. Herein we describe the use of reactivity-based screening to discriminate bacteria that produce arginine-versus citrulline-bearing citrulassins, culminating in the discovery and characterization of 11 new lasso peptide variants. Phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the citrulline-containing variants. Absence of this gene correlated strongly with citrulassin variants only containing arginine (des-citrulassin). Heterologous expression of the PAD in a non-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in arginine deimination. The family of PADs were then bioinformatically surveyed for a deeper understanding of its genomic context and potential role in post-translational modification of RiPPs.

biochemistry↗

bioRxiv2020

Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA

RNA interfering is a eukaryote-specific gene silencing by 20[~]23 nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3' end-shortened variants of let-7a, miR-27a, and specific miR-17-92 families that make AGO3 an extremely competent slicer by an [~] 82-fold increase in target cleavage. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.

biochemistry↗

bioRxiv2020

The mechanism of selective kinesin inhibition by kinesin binding protein.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, crescent-shaped right-handed -solenoid that sequesters the tubulin-binding surface of the kinesin motor domain, structurally distorting the motor domain and sterically blocking MT attachment. KBP uses its -solenoid concave face and edge loops to bind the kinesin motor domain and selective mutation of this extended binding surface disrupts KBP inhibition of kinesin transport in cells. The KBP-interacting surface of the motor domain contains motifs exclusively conserved in KBP-interacting kinesins, providing a basis for kinesin selectivity.

biochemistry↗

bioRxiv2020

Structural basis for the inhibition of the papain-like protease of SARS-CoV-2 by small molecules

SARS-CoV-2 is the pathogen responsible for the COVID-19 pandemic. The SARS-CoV-2 papain-like cysteine protease has been implicated in virus maturation, dysregulation of host inflammation and antiviral immune responses. We showed that PLpro preferably cleaves the K48-ubiquitin linkage while also being capable of cleaving ISG15 modification. The multiple functions of PLpro render it a promising drug target. Therefore, we screened an FDA-approved drug library and also examined available inhibitors against PLpro. Inhibitor GRL0617 showed a promising IC50 of 2.1 M. The co-crystal structure of SARS-CoV-2 PLpro-C111S in complex with GRL0617 suggests that GRL0617 is a non-covalent inhibitor. NMR data indicate that GRL0617 blocks the binding of ISG15 to PLpro. The antiviral activity of GRL0617 reveal that PLpro is a promising drug target for therapeutically treating COVID-19. One Sentence SummaryCo-crystal structure of PLpro in complex with GRL0617 reveals the druggability of PLpro for SARS-CoV-2 treatment.

biochemistry↗

bioRxiv2020

Resveratrol shortens the chronological lifespan of Saccharomyces cerevisiae by a pro-oxidant mechanism

Resveratrol consumption has linked with normalization of the risk factors of some diseases such as colon cancer and type 2 diabetes. The antioxidant phenotype caused by resveratrol has recognized as a key piece in the health benefits exerted by this phytochemical. Although the antioxidant activity showed by resveratrol has attributed at the molecule per se, recent evidence indicates that the antioxidant effect occasioned by resveratrol could be associated with a pro-oxidant mechanism. The hypothesis that resveratrol inhibits complex III of the electron transport chain as its main target suggests that resveratrol increases reactive oxygen species (ROS) generation produces via reverse electron transport. This idea also explains that cells respond to the oxidative damage caused by resveratrol, inducing their antioxidant systems. The free radical theory of aging postulates that organisms age due to the accumulation of the harmful effects of ROS in cells. For these reasons, we hypothesize that resveratrol shortens the chronological life span (CLS) of Saccharomyces cerevisiae due to a pro-oxidant activity. Herein, we provide evidence that 100 M resveratrol supplementation at 5% glucose: 1) shorted the CLS of CTT1 and YAP1 genes deleted strains; 2) decreased the H2O2 release in the WT strain, and maintain unaltered the H2O2 release in the ctt1{Delta} strain; 3) lessened exponential growth of ctt1{Delta} strain, which was reverted with the adding of GSH; 4) increased catalase activity in the WT strain, a phenotype that was not observed in the ctt1{Delta} strain. Altogether, these results indicate that resveratrol decreases CLS by a pro-oxidant mechanism.

biochemistry↗