ResearchPad - biochemistry-and-chemical-biology https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Heterogeneity of proteome dynamics between connective tissue phases of adult tendon]]> https://www.researchpad.co/article/elastic_article_7267 Muscles are anchored to bones through specialized tissues called tendons. Made of bundles of fibers (or fascicles) linked together by an ‘interfascicular’ matrix, healthy tendons are required for organisms to move properly. Yet, these structures are constantly exposed to damage: the interfascicular matrix, in particular, is highly susceptible to injury as it allows the fascicles to slide on each other.

One way to avoid damage could be for the body to continually replace proteins in tendons before they become too impaired. However, the way proteins are renewed in these structures is currently not well understood – indeed, it has long been assumed that almost no protein turnover occurs in tendons. In particular, it is unknown whether proteins in the interfascicular matrix have a higher turn over than those in the fascicles.

To investigate, Choi, Simpson et al. fed rats on water carrying a molecular label that becomes integrated into new proteins. Analysis of individual proteins from the rats’ tendons showed great variation in protein turnover, with some replaced every few days and others only over several years. This suggests that protein turnover is actually an important part of tendon health. In particular, the results show that turnover is higher in the interfascicular matrix, where damage is expected to be more likely.

Protein turnover also plays a part in conditions such as cancer, heart disease and kidney disease. Using approaches like the one developed by Choi, Simpson et al. could help to understand how individual proteins are renewed in a range of diseases, and how to design new treatments.

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<![CDATA[Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation]]> https://www.researchpad.co/article/elastic_article_6357 By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions. We identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome.

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<![CDATA[Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9]]> https://www.researchpad.co/article/elastic_article_6353 A single protein sometimes does multiple jobs. For instance, our immune system uses a small number of multipurpose proteins to respond quickly to a large number of threats. One example is the protein S100A9. It acts as an antimicrobial by preventing microbes from getting the nutrients they need, while also stimulating inflammation by inducing the release of molecules that recruit white blood cells.

S100A9, like all proteins, is made up of a chain of small building blocks. These building blocks interact with each other and with other molecules in the environment. The sequence of the building blocks thus determines what jobs the protein can do. Therefore, a single change to the sequence of building blocks can have a dramatic effect: one change might render the protein faulty, while another change might allow it to do a new job.

Proteins face similar challenges humans do when trying to do several things at once. A person driving a car while using their phone will not do either task well. Likewise, a protein that does two jobs faces challenges a single-purpose protein does not.

Harman et al. were interested in how S100A9 was able to evolve and maintain its dual functionality, despite this potential problem. They started by asking when S100A9 acquired its two purposes. They measured the antimicrobial and inflammatory activity of S100A9 proteins from humans, mice and opossums. The activities of S100A9 in these species was similar, suggesting that S100A9 acquired its different jobs in the ancestor of mammals, some 160 million years ago.

Next, Harman et al. computationally reconstructed ancestral forms of S100A9 by comparing hundreds of similar proteins and building an evolutionary tree. They then measured the antimicrobial and inflammatory activity of these ancestral proteins. By comparing the last ancestor that did not have these activities to the first ancestor that did, they identified the sequence changes that gave S100A9 its dual activity. Importantly, these changes are located in separate regions of the protein, meaning they could occur independently, without affecting each other. Further, the same sequence change that converted S100A9 into an inflammatory signal also introduced a mechanism to regulate this activity.

The results suggest that a small number of sequence changes – or even a single change – can make a protein more versatile. This means that evolving multipurpose proteins may not be as difficult as is often thought.

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<![CDATA[The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling]]> https://www.researchpad.co/article/elastic_article_6343 Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

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<![CDATA[A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter]]> https://www.researchpad.co/article/N31301349-16ac-43e0-9228-476ce24b03ef ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

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<![CDATA[Cooperative interactions facilitate stimulation of Rad51 by the Swi5-Sfr1 auxiliary factor complex]]> https://www.researchpad.co/article/Nbfc8d5d1-da72-4c2a-9797-ce3b29738b07

Although Rad51 is the key protein in homologous recombination (HR), a major DNA double-strand break repair pathway, several auxiliary factors interact with Rad51 to promote productive HR. We present an interdisciplinary characterization of the interaction between Rad51 and Swi5-Sfr1, a conserved auxiliary factor. Two distinct sites within the intrinsically disordered N-terminus of Sfr1 (Sfr1N) were found to cooperatively bind Rad51. Deletion of this domain impaired Rad51 stimulation in vitro and rendered cells sensitive to DNA damage. By contrast, amino acid-substitution mutants, which had comparable biochemical defects, could promote DNA repair, suggesting that Sfr1N has another role in addition to Rad51 binding. Unexpectedly, the DNA repair observed in these mutants was dependent on Rad55-Rad57, another auxiliary factor complex hitherto thought to function independently of Swi5-Sfr1. When combined with the finding that they form a higher-order complex, our results imply that Swi5-Sfr1 and Rad55-Rad57 can collaboratively stimulate Rad51 in Schizosaccharomyces pombe.

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<![CDATA[Correction: Endothelial PKA activity regulates angiogenesis by limiting autophagy through phosphorylation of ATG16L1]]> https://www.researchpad.co/article/N63d19ade-13da-4b37-a1de-55afc96d0697 ]]> <![CDATA[Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function]]> https://www.researchpad.co/article/Nc2fad685-f858-495d-8178-d7538f92930d

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand.

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<![CDATA[A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment]]> https://www.researchpad.co/article/Nbe9a7b76-8c01-489a-b41d-c82050dff1cb

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

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<![CDATA[The guide sRNA sequence determines the activity level of box C/D RNPs]]> https://www.researchpad.co/article/N32fdb9c1-8366-4e0a-92c1-866d17aeaba5

2’-O-rRNA methylation, which is essential in eukaryotes and archaea, is catalysed by the Box C/D RNP complex in an RNA-guided manner. Despite the conservation of the methylation sites, the abundance of site-specific modifications shows variability across species and tissues, suggesting that rRNA methylation may provide a means of controlling gene expression. As all Box C/D RNPs are thought to adopt a similar structure, it remains unclear how the methylation efficiency is regulated. Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate–guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.

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<![CDATA[Chemoptogenetic ablation of neuronal mitochondria in vivo with spatiotemporal precision and controllable severity]]> https://www.researchpad.co/article/N9f8a4863-a649-4758-a829-2914bd3c5856

Mitochondrial dysfunction is implicated in the pathogenesis of multiple neurological diseases, but elucidation of underlying mechanisms is limited experimentally by the inability to damage specific mitochondria in defined neuronal groups. We developed a precision chemoptogenetic approach to target neuronal mitochondria in the intact nervous system in vivo. MG2I, a chemical fluorogen, produces singlet oxygen when bound to the fluorogen-activating protein dL5** and exposed to far-red light. Transgenic zebrafish expressing dL5** within neuronal mitochondria showed dramatic MG2I- and light-dependent neurobehavioral deficits, caused by neuronal bioenergetic crisis and acute neuronal depolarization. These abnormalities resulted from loss of neuronal respiration, associated with mitochondrial fragmentation, swelling and elimination of cristae. Remaining cellular ultrastructure was preserved initially, but cellular pathology downstream of mitochondrial damage eventually culminated in neuronal death. Our work provides powerful new chemoptogenetic tools for investigating mitochondrial homeostasis and pathophysiology and shows a direct relationship between mitochondrial function, neuronal biogenetics and whole-animal behavior.

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<![CDATA[Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme]]> https://www.researchpad.co/article/N98045833-8cd0-44a3-b20d-435809bf7fc2

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

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<![CDATA[Mitochondrial ClpX activates an essential biosynthetic enzyme through partial unfolding]]> https://www.researchpad.co/article/N3fec9f84-a006-4f25-8be8-1f684b7c85e6

Mitochondria control the activity, quality, and lifetime of their proteins with an autonomous system of chaperones, but the signals that direct substrate-chaperone interactions and outcomes are poorly understood. We previously discovered that the mitochondrial AAA+ protein unfoldase ClpX (mtClpX) activates the initiating enzyme for heme biosynthesis, 5-aminolevulinic acid synthase (ALAS), by promoting cofactor incorporation. Here, we ask how mtClpX accomplishes this activation. Using S. cerevisiae proteins, we identified sequence and structural features within ALAS that position mtClpX and provide it with a grip for acting on ALAS. Observation of ALAS undergoing remodeling by mtClpX revealed that unfolding is limited to a region extending from the mtClpX-binding site to the active site. Unfolding along this path is required for mtClpX to gate cofactor binding to ALAS. This targeted unfolding contrasts with the global unfolding canonically executed by ClpX homologs and provides insight into how substrate-chaperone interactions direct the outcome of remodeling.

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<![CDATA[RNA-guided retargeting of Sleeping Beauty transposition in human cells]]> https://www.researchpad.co/article/Nd081b2f7-794f-4db4-bde8-115a5e22b2d2

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, and occurred in an asymmetric pattern with a bias towards sites in a relatively narrow, 300 bp window downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

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<![CDATA[Structure-based discovery of potent and selective melatonin receptor agonists]]> https://www.researchpad.co/article/N7d8f55b0-fe17-4881-99f8-4635a1c63635

Melatonin receptors MT1 and MT2 are involved in synchronizing circadian rhythms and are important targets for treating sleep and mood disorders, type-2 diabetes and cancer. Here, we performed large scale structure-based virtual screening for new ligand chemotypes using recently solved high-resolution 3D crystal structures of agonist-bound MT receptors. Experimental testing of 62 screening candidates yielded the discovery of 10 new agonist chemotypes with sub-micromolar potency at MT receptors, with compound 21 reaching EC50 of 0.36 nM. Six of these molecules displayed selectivity for MT2 over MT1. Moreover, two most potent agonists, including 21 and a close derivative of melatonin, 28, had dramatically reduced arrestin recruitment at MT2, while compound 37 was devoid of Gi signaling at MT1, implying biased signaling. This study validates the suitability of the agonist-bound orthosteric pocket in the MT receptor structures for the structure-based discovery of selective agonists.

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<![CDATA[Fatal amyloid formation in a patient’s antibody light chain is caused by a single point mutation]]> https://www.researchpad.co/article/Nd0826784-b84e-4977-b846-f2c00b322855

In systemic light chain amyloidosis, an overexpressed antibody light chain (LC) forms fibrils which deposit in organs and cause their failure. While it is well-established that mutations in the LC’s VL domain are important prerequisites, the mechanisms which render a patient LC amyloidogenic are ill-defined. In this study, we performed an in-depth analysis of the factors and mutations responsible for the pathogenic transformation of a patient-derived λ LC, by recombinantly expressing variants in E. coli. We show that proteolytic cleavage of the patient LC resulting in an isolated VL domain is essential for fibril formation. Out of 11 mutations in the patient VL, only one, a leucine to valine mutation, is responsible for fibril formation. It disrupts a hydrophobic network rendering the C-terminal segment of VL more dynamic and decreasing domain stability. Thus, the combination of proteolytic cleavage and the destabilizing mutation trigger conformational changes that turn the LC pathogenic.

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<![CDATA[Regulation of mRNA translation by a photoriboswitch]]> https://www.researchpad.co/article/Nc2ffddfd-1feb-4795-aff8-54d3618d464d

Optogenetic tools have revolutionized the study of receptor-mediated processes, but such tools are lacking for RNA-controlled systems. In particular, light-activated regulatory RNAs are needed for spatiotemporal control of gene expression. To fill this gap, we used in vitro selection to isolate a novel riboswitch that selectively binds the trans isoform of a stiff-stilbene (amino-tSS)–a rapidly and reversibly photoisomerizing small molecule. Structural probing revealed that the RNA binds amino-tSS about 100-times stronger than the cis photoisoform (amino-cSS). In vitro and in vivo functional analysis showed that the riboswitch, termed Werewolf-1 (Were-1), inhibits translation of a downstream open reading frame when bound to amino-tSS. Photoisomerization of the ligand with a sub-millisecond pulse of light induced the protein expression. In contrast, amino-cSS supported protein expression, which was inhibited upon photoisomerization to amino-tSS. Reversible photoregulation of gene expression using a genetically encoded RNA will likely facilitate high-resolution spatiotemporal analysis of complex RNA processes.

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<![CDATA[Structural basis for the activation of PLC-γ isozymes by phosphorylation and cancer-associated mutations]]> https://www.researchpad.co/article/N1bb6834f-9ba5-4e2a-bf53-38581a0dc83e

Direct activation of the human phospholipase C-γ isozymes (PLC-γ1, -γ2) by tyrosine phosphorylation is fundamental to the control of diverse biological processes, including chemotaxis, platelet aggregation, and adaptive immunity. In turn, aberrant activation of PLC-γ1 and PLC-γ2 is implicated in inflammation, autoimmunity, and cancer. Although structures of isolated domains from PLC-γ isozymes are available, these structures are insufficient to define how release of basal autoinhibition is coupled to phosphorylation-dependent enzyme activation. Here, we describe the first high-resolution structure of a full-length PLC-γ isozyme and use it to underpin a detailed model of their membrane-dependent regulation. Notably, an interlinked set of regulatory domains integrates basal autoinhibition, tyrosine kinase engagement, and additional scaffolding functions with the phosphorylation-dependent, allosteric control of phospholipase activation. The model also explains why mutant forms of the PLC-γ isozymes found in several cancers have a wide spectrum of activities, and highlights how these activities are tuned during disease.

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<![CDATA[A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells]]> https://www.researchpad.co/article/N7a884d1b-4205-49f2-86ea-ef7b4bf351e3

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

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<![CDATA[A sex difference in the response of the rodent postsynaptic density to synGAP haploinsufficiency]]> https://www.researchpad.co/article/N9e1ec4da-c323-4f41-bb71-2a24053a1670

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of Syngap1 in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young Syngap1+/-mice. Here we show that only females and not males show a highly significant correlation between an increase in TARP and a decrease in synGAP in the PSDs of Syngap1+/-rodents. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP haploinsufficiency.

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