bluuebunny/crossref_metadata_2025 huggingface dataset:
Polar planets around highly eccentric binaries are the most stable
Authors: Chen Cheng , Lubow Stephen H , Martin Rebecca G
Published: April 2020

We study the orbital stability of a non-zero mass, close-in circular orbit planet around an eccentric orbit binary for various initial values of the binary eccentricity, binary mass fraction, planet mass, planet semimajor axis, and planet inclination by means of numerical simulations that cover 5 × 104 binary orbits. For small binary eccentricity, the stable orbits that extend closest to the binary (most stable orbits) are nearly retrograde and circulating. For high binary eccentricity, the most stable orbits are highly inclined and librate near the so-called generalized polar orbit which is a stationary orbit that is fixed in the frame of the binary orbit. For more extreme mass ratio binaries, there is a greater variation in the size of the stability region (defined by initial orbital radius and inclination) with planet mass and initial inclination, especially for low binary eccentricity. For low binary eccentricity, inclined planet orbits may be unstable even at large orbital radii (separation ${\gt}5 \, a_{\rm b}$). The escape time for an unstable planet is generally shorter around an equal mass binary compared with an unequal mass binary. Our results have implications for circumbinary planet formation and evolution and will be helpful for understanding future circumbinary planet observations.
Evolution of planar magnetic structure within the stream interaction region and its connection with a recurrent Forbush decrease
Authors: Shaikh Zubair I , Raghav Anil N , Vichare Geeta
Published: April 2020

In general, stream interaction region (SIR)-induced Forbush decreases are recurrent and low magnitude in nature. The diffusion–convection associated with the SIR plays an important role in their modulation. Here, we study the evolution of planar magnetic structure (PMS) within the SIR and its contribution to cosmic ray modulation. Interestingly, we found the presence of PMS structures within the SIR from the leading part of the SIR to the minimum of the cosmic ray intensity in two events. The PMS may have originated due to the high compression caused by the fast solar wind, which amplifies and aligns the pre-existing discontinuities in the ambient slow solar wind. The study also suggests that the existence of PMS, enhanced initial mass function (IMF) strength, and associated turbulent regions decreases the perpendicular diffusion coefficient and causes a decrease in the cosmic ray intensity observed on Earth. Moreover, a slow decrease in IMF magnitude concurs with the recovery phase of cosmic ray intensity.
PRMT7 regulates RNA-binding capacity and protein stability in Leishmania parasites
Authors: Ferreira Tiago R , Dowle Adam A , Parry Ewan , Alves-Ferreira Eliza V C , Hogg Karen , Kolokousi Foteini , Larson Tony R , Plevin Michael J , Cruz Angela K , Walrad Pegine B
Published: April 2020

RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.
Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence
Authors: Isaev Artem , Drobiazko Alena , Sierro Nicolas , Gordeeva Julia , Yosef Ido , Qimron Udi , Ivanov Nikolai V , Severinov Konstantin
Published: April 2020

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.
History and Constitutional Interpretation: Applying the ‘Cambridge School’ Approach to Interpreting Constitutions
Authors: Saunders Benjamin B , Kennedy Simon P
Published: April 2020

Constitutions are products of an alien culture. Constitutions are also ‘instruments of government’ and must be relevant to serve the needs of modern society. How ought we to reconcile the competing pressures that arise from the nature of a document that is at one and the same time a historical artefact and an instrument of contemporary government? We propose an answer modelled on the ‘Cambridge method’ of textual interpretation, whereby a constitution must first be considered as a historical text, on its own terms, without regard to today’s controversies. We argue that the best way of understanding a constitution is to take account of its historical context, and that meaning derived in this way ought to be the starting point when considering the provisions of the constitution. However, we consider that the historical context need not be determinative of the contemporary meaning of the constitution, and so departures from that meaning may be appropriate in some situations.
QEMSCAN as a Method of Semi-Automated Crystal Size Distribution Analysis: Insights from Apollo 15 Mare Basalts
Authors: Bell S K , Joy K H , Pernet-Fisher J F , Hartley M E
Published: April 2020

Crystal size distribution analysis is a non-destructive, quantitative method providing insights into the crystallization histories of magmas. Traditional crystal size distribution data collection requires the manual tracing of crystal boundaries within a sample from a digital image. Although this manual method requires minimal equipment to perform, the process is often time-intensive. In this study we investigate the feasibility of using the Quantitative Evaluation of Minerals by SCANing electron microscopy (QEMSCAN) software for semi-automated crystal size distribution analysis. Four Apollo 15 mare basalt thin sections were analysed using both manual and QEMSCAN crystal size distribution data collection methods. In most cases we observe an offset between the crystal size distribution plots produced by QEMSCAN methods compared with the manual data, leading to differences in calculated crystal residence times and nucleation densities. The source of the discrepancy is two-fold: (1) the touching particles processor in the QEMSCAN software is prone to segmenting overlapping elongate crystals into multiple smaller crystals; (2) this segmentation of elongate crystals causes estimates of true 3D crystal habit to vary between QEMSCAN and manual data. The reliability of the QESMCAN data appears to be a function of the crystal texture and average crystal shape, both of which influence the performance of the touching particles processor. Despite these limitations, QEMSCAN is able to produce broadly similar overall trends in crystal size distribution plots to the manual approach, in a considerably shorter time frame. If an accurate crystal size distribution is required to calculate crystal residence time or nucleation density, we recommend that QEMSCAN should only be used after careful consideration of the suitability of the sample texture and average crystal shape.
Measurement Properties of the Craniocervical Flexion Test: A Systematic Review
Authors: Araujo Francisco Xavier de , Ferreira Giovanni E , Scholl Schell Maurício , Castro Marcelo Peduzzi de , Ribeiro Daniel Cury , Silva Marcelo Faria
Published: April 2020

Objective

Patients with neck pain commonly have altered activity of the neck muscles. The craniocervical flexion test (CCFT) is used to assess the function of the deep neck flexor muscles in patients with musculoskeletal neck disorders. Systematic reviews summarizing the measurement properties of the CCFT are outdated. The objective of this study was to systematically review the measurement properties of the CCFT for assessing the deep neck flexor muscles.

Methods

The data sources MEDLINE, EMBASE, Physiotherapy Evidence Database, Cochrane Central Register of Controlled Trials, Scopus, and Science Direct were searched in April 2019. Studies of any design that reported at least 1 measurement property of the CCFT for assessing the deep neck flexor muscles were selected. Two reviewers independently extracted data and rated the risk of bias of individual studies using the COnsensus-based Standards for the selection of health Measurement INstruments (COSMIN) risk-of-bias checklist. The overall rating for each measurement property was classified as “positive,” “indeterminate,” or “negative.” The overall rating was accompanied with a level of evidence.

Results

Fourteen studies were included in the data synthesis. The ratings were positive, and the level of evidence was moderate for interrater and intrarater reliability and convergent validity. There was conflicting rating and level of evidence for discriminative validity. Measurement error was indeterminate, with an unknown level of evidence. Responsiveness was negative, with a limited level of evidence. A limitation of this study was that only papers published in English were included.

Conclusions

The CCFT is a valid and reliable test that can be used in clinical practice as an assessment test. Because of the conflicting and low-quality evidence, caution is advised when using the CCFT as a discriminative test and as an outcome measure. Future better-designed studies are warranted.
Muscle biopsy in anti-neutrophil cytoplasmic antibody–associated vasculitis: diagnostic yield depends on anti-neutrophil cytoplasmic antibody type, sex and neutrophil count
Authors: Lacou Mathieu , Leroy Maxime , Le Lan Nowenn , Toquet Claire , Espitia-Thibault Alexandra , Graveleau Julie , Masseau Agathe , Agard Christian , Volteau Christelle , Mussini Jean-Marie , Hamidou Mohamed , Néel Antoine
Published: April 2020

Objectives

This study aimed to examine the sensitivity of muscle biopsy (MB) in ANCA-associated vasculitis (AAV), identify factors predicting MB positivity and assess the prognostic value of a positive MB.

Methods

We conducted a single-centre retrospective study of AAV with an MB performed at diagnosis. AAV classification [granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA)] followed the European Medicines Agency algorithm. A logistic regression model was used to identify the factors associated with MB positivity. Survival curves were generated using the Kaplan–Meier method.

Results

Among 276 AAV patients (1995–2018), 101 had an MB. Seventy-eight patients were included: 33 with GPA, 25 with MPA and 20 with EGPA. MB samples were positive in 45 cases (58%): 17 GPA, 16 MPA and 12 EGPA. Univariate analysis focussed on GPA and MPA, revealed that the MB yield was higher in females [22/31 (71%) vs 11/27 (41%); P = 0.02] and in anti-MPO patients [25/37 (68%) vs 6/19 (32%) for anti-PR3; P = 0.01]. By multivariate analysis, three factors predicted MB positivity: anti-MPO ANCA [odds ratio (OR) 10.67 (CI 2.09, 81.68)], female sex [OR 5.3 (CI 1.16, 32.35)] and neutrophil count [OR 1.33 (CI 1.07, 1.8)]. MB positivity had no impact on relapse, death or end-stage renal disease–free survival.

Conclusions

MB is a safe and efficient diagnostic tool for AAV. Predictors of MB yield include ANCA type, sex and neutrophil count. MB cannot substitute for kidney biopsy when indicated, but should be considered in other cases.
Mechanistic Dissection of Spatially Encoded GPCR Signaling Through High‐throughput CRISPR Screening
Authors: Tsvetanova Nikoleta
Published: April 2020

Our recent work has overturned the prevailing textbook model for GPCR signaling, which held that GPCR activation at the plasma membrane is responsible for all downstream consequences of signaling. We have demonstrated that receptor/second messenger signaling on internal compartments but not plasma membrane drives all receptor‐dependent transcriptional responses, and the site of receptor activation can dictate how cells “interpret” different ligands, including medically relevant drugs. This phenomenon of functional specialization of signaling based on receptor localization has been gaining significant support in the last few years from us and other groups, yet not much is known about its underlying mechanisms and functional consequences. To begin to address these open questions, we set up a functional genomic screen aimed at identifying factors regulating ß2‐adrenergic receptor (ß2‐AR)‐dependent transcription‐ a location‐biased GPCR signaling response. We combined pooled CRISPR interference libraries targeting all annotated transcripts in the human genome and a novel fluorescent transcriptional reporter for GPCR/cAMP signaling that we have developed with fluorescence‐activated cell sorting and deep sequencing. Through this comprehensive screen, we have identified both known and novel mediators of the GPRC pathway. We are currently defining the biochemical mechanisms and specificity of the new regulators and their contributions to location‐biased receptor signaling, and have intriguing data linking a highly‐penetrant genetic risk factor for psychosis to these pathways.

Support or Funding Information

National Institute of Mental Health [Grant MH10963301]
Biochemical and Toxicological Evaluations of Aqueous Extract of Parquetina nigrescens (Afzel.) leaves on Mifepristone‐induced Polycystic Ovarian Syndrome in Rats
Authors: Femi-olabisi Fehintoluwa Joy , Faokunla Opeyemi O
Published: April 2020

The biochemical and toxicological effects of aqueous extract of Parquetina nigrescens leaves (AEPNL) at the doses of 50, 100, and 200 mg/kg body weight on mifepristone‐induced in Polycystic Ovarian Syndrome (PCOS) was investigated in female Wistar rats. Fifty female Wistar rats (190.00 ± 13.00 g) were assigned into 5 groups (A – E) of ten each: animals in group A received 0.5 ml of distilled water orally on daily basis for 30 days while the mifepristonized rats in groups B, C, D and E also received orally 0.5 ml of distilled water and same volume of the extract corresponding to 50, 100, and 200 mg/kg body weight of AEPNL respectively after which levels of some biochemical and toxicological indices were determined. AEPNL aggravated mifepristone‐treatment related increases in albumin, total protein and liver aspartate aminotransferase activity and mitigated the increases in globulin, total bilirubin, urea, creatinine, liver and serum alkaline phosphatase and alanine aminotransferase activities, and no treatment‐related histoarchitectural changes occurred in the liver, kidney and uterus of the female rats. Therefore, the aqueous extract of Parquetina nigrescens leaves attenuated and also aggravated some biochemical parameters in the serum, liver and kidney but with no histological changes in the liver, kidney and uterus of the mifepristonized female Wistar rats.
The role of sympathetic activation and carbon dioxide tension in human neurovascular coupling
Authors: Rosentreter Ryan E , Mirzadzare Niloofar , Cho Woojin , Potemkin Michael , Scott Berkeley A , Soriano Jan Elaine , Fisher James P , Phillips Aaron A
Published: April 2020

Neurovascular coupling describes a series of processes that serve to match blood supply to neuronal metabolism within the central nervous system. We know that the sympathetic nervous system plays a role in several cerebrovascular control pathways, and preclinical non‐human work demonstrates that the sympathetic nervous system may play a role in neurovascular coupling. These preclinical models utilize isolated cell preparations lacking key inputs to the neurovascular unit such as that of the autonomic nervous system, or utilize anesthetics which blunt cerebrovascular regulatory pathways.

Here, our objective was to provide preliminary insight into the role of sympathetic nervous system activation in neurovascular coupling by using our non‐anesthetized, non‐stressed, human, truly in vivo model. We utilized moderate lower body negative pressure (−40 mmHg) to elevate sympathetic activity without affecting mean arterial pressure. Beat‐by‐beat blood pressure was recorded via finger photoplethysmography, while cerebral blood velocity in the middle and posterior cerebral arteries was measured via transcranial Doppler. Neurovascular coupling was elicited using our standardized visual stimulus protocol and data was analyzed using our custom software. As lower body negative pressure leads to sympatho‐excitation and hyperventilation, in a subset of individuals we maintained end‐tidal carbon dioxide levels identical to baseline during lower body negative pressure.

Absolute cerebral blood velocity was influenced by arterial carbon dioxide levels. Neurovascular coupling was preserved during lower body negative pressure induced sympatho‐excitation and reduced arterial carbon dioxide levels.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada, Libin Cardiovascular Institute, Hotchkiss Brain Institute, Compute Canada
Static Magnetic Field accelerates diabetic wound healing by facilitating resolution of inflammation
Authors: Ren Huiwen , Shang Wenlong , Yu Ying
Published: April 2020

Impaired wound healing is commonly encountered in patients with diabetes mellitus, which may lead to severe outcomes such as amputation, if untreated timely. Macrophage plays a critical role in the healing process including the resolution phase. Although magnetic therapy is known to improve microcirculation, its effect on wound healing remains uncertain.

In the present study, the model of diabetic mice was used to prove the effect of static magnetic field (SMF) in wound healing process. The positive role of SMF in macrophage polarization was observed in wound areas of db/db mice by the immunofluorescence staining and flow cytometric analysis. In order to examine the molecular mechanism of SMF in the wound healing, peritoneal macrophages were isolated from db/db mice and treated by LPS or IL‐4.

We found 0.6 T SMF significantly accelerated wound closure rate by 50%. Histological analyses revealed significantly shorter distances by 26.7% between the epithelial tips of punched wound and distances by 33.3% between the edges of the panniculus carnosus in SMF group at day 7 postoperatively. SMF promoted M2 macrophage recruitment by 87.5% and reduced M1 macrophages accumulation by 24.3%, which was observed by immunofluorescence staining in wound areas of db/db mice. Flow cytometry analysis also revealed that M2 macrophages (F4/80+CD206+) were increased and M1 macrophages (F4/80+CD206−) were significantly decreased, indicating SMF facilitates M2 polarization in inflamed tissues. The expression levels of pro‐inflammatory markers (iNOS, IL‐6, IL‐1β, CCR7) in injured tissues were paradoxically downregulated in the SMF group. Meanwhile, SMF significantly raised the expression levels of reparative genes (CD206, Fizz1, Arg‐1, IL‐10). The pro‐inflammatory iNOS expression was decreased and anti‐inflammatory Arg‐1 was increased in SMF/LPS/IL‐4‐treated macrophages. Furthermore, SMF inhibited LPS‐induced STAT1 phosphorylation and augmented IL‐4‐induced STAT6 phosphorylation in the macrophages.

In summary, our study showed for the first time the effect of 0.6 T SMF on wound healing in diabetic mice. These results indicate that SMF accelerates diabetic wound healing by promoting macrophage polarization and resolution of inflammation through modulation of the JAK‐STAT pathway. Therefore, focusing on SMF in therapeutic interventions might be useful for treating diabetic wound by re‐normalizing the healing process.

Support or Funding Information

This work was supported by the National Natural Science Foundation of China (grants 81703591).
Fuzzy Interactions Between 14‐3‐3ζ And Tau Regulate Tau Aggregation
Authors: Huang Fan , Shu Qianhui , Qin Zhaojie , Tian Jianglin , Gao Meng , Huang Yongqi
Published: April 2020

The microtubule associated protein tau is vital for elongation and stability of microtubules in the central nervous system. However, aberrant accumulation of tau amyloid fibrils in neurons is a character of a neurodegenerative pathology called Alzheimer’s disease. Extensive studies have revealed that several factors promote aggregation of tau, including mutations, polyanions, phosphorylation, and interactions with other proteins. Previous studies have shown that the conserved scaffolding protein family 14‐3‐3 proteins interact with tau in a phosphorylation dependent manner and 14‐3‐3 proteins promote tau aggregation. Crystal structure of 14‐3‐3/tau complex revealed that phosphorylated tau segments bind to the ligand binding grooves of 14‐3‐3 dimer. Consequently, it is promising that 14‐3‐3 specific binding ligands will reduce/inhibit tau aggregation. Indeed, rational designed inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. Although thermodynamics characterization showed that non‐phosphorylated tau segments bind to 14‐3‐3 much weaker than phosphorylated tau segments, 14‐3‐3ζ also promotes non‐phosphorylated tau aggregation. In this work, we investigated the interactions between tau K18 construct and 14‐3‐3ζ. We found that non‐phosphorylated and PKA‐phosphorylated tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Replacing Ser324 with Ala had little effect of tau aggregation mediated by 14‐3‐3ζ protein. We further saturated the 14‐3‐3ζ ligand binding grooves by a high affinity peptide R18 and found that the presence of R18 could not reduce tau aggregation. Molecular dynamics simulations showed that tau K18 interact with 14‐3‐3ζ via extensive fuzzy interactions. Our results suggest that besides binding to the canonical ligand binding groove, tau also interacts with 14‐3‐3ζ via nonspecific interactions which are responsible for tau aggregation and cannot be inhibited by 14‐3‐3ζ specific inhibitors.

Support or Funding Information

This work was supported by Hubei University of Technology
Mechanistic Roles of Electrostatic Interactions in the Molecular Recognition of Intrinsically Disordered Proteins
Authors: Gao Meng , Huang Yongqi
Published: April 2020

Electrostatic interactions could play important roles in the molecular recognition process of intrinsically disordered proteins (IDPs) as many IDPs and the recognition elements contain charged residues. Previous studies have demonstrated that electrostatic forces could accelerate the encounter process via electrostatic steering effect and increase the efficiency of binding upon encounter for several IDPs. To further investigate the universal role of electrostatic interactions in IDPs molecular recognition, we collected the experimentally determined binding kinetics of IDPs. For many studied IDPs, the association rate constants are reduced for about ten folds when the salt concentration increases from low (~50 mM) to high (~500 mM), indicating the presence of favorable electrostatic interactions. We further performed molecular dynamics simulations using structure‐based coarse‐grained models. Simulations predicted that long‐range electrostatic interactions accelerate the binding rate in a range consistent with experimental results. Simulations also revealed that electrostatic forces generally enhance the binding kinetics by increasing the encounter rate and by enhancing the evolving efficiency. Correlating the charge density with binding kinetics revealed that the overall charge density is relate with enhancement of the capture rate constant and the charge distribution affects both the evolution rate constant and escape rate constant. Analysis of the transition path times showed that electrostatic interactions stabilize the intermediate state along the binding process. Besides affecting the binding kinetics, electrostatic interactions also redistributed the flux among multiple parallel binding transition paths. As a summary, our results suggest that favorable electrostatic interactions are present in many IDPs recognition processes. Under physiological salt concentrations, electrostatic interactions are expected to enhance the binding rate constant by several folds.

Support or Funding Information

This work was supported by Hubei University of Technology
Effect of knock out of ASIC3 on cardiovascular reflexes arising from hindlimb muscle in ligated rats
Authors: Kim Joyce S. , Ducrocq Guillaume P. , Kaufman Marc P.
Published: April 2020

The exercise pressor reflex (EPR) arises from contracting the skeletal muscle and is manifested by increases in arterial pressure, heart rate and blood flow to working muscles. Group III and IV afferents comprise the sensory arm of the exercise pressor reflex. The exercise pressor reflex is exaggerated in patients with peripheral artery disease, which is an atherosclerotic process that occludes blood flow to working muscles. The exaggeration of the exercise pressor reflex is believed to be caused by a metabolite whose concentration is increased when the working muscles are inadequately perfused. Previous work has shown that pharmacological blockade of acid‐sensing ion channel 3, which is found on the endings of group III and IV afferents, prevents the exaggeration of the exercise pressor reflex in rats with simulated peripheral artery disease. In the present experiments, we compared the magnitude of the exercise pressor reflex in decerebrated rats with a functional knockout of the ASIC3 (KO) with the magnitude of the reflex in their decerebrated wild type counterparts (WT). We found that the exercise pressor reflex in ASIC3 KO rats was significantly smaller than the exercise pressor reflex in their WT counterparts (p<0.05). In addition, ASIC 3 KO rats demonstrated significantly smaller pressor responses to injection of diprotonated phosphate (86mM; pH 6.0), lactic acid (12mM; pH 2.85), and capsaicin (0.2μg; pH 7.2) into the arterial supply of the hindlimb (p< 0.05). In contrast, both ligated WT and ASIC3 KO rats displayed similar pressor responses to calcaneal tendon stretch (p>0.05). We conclude that ASIC3 plays an important role in evoking the exaggerated exercise pressor reflex seen in rats with peripheral artery disease.

Support or Funding Information

NIH Grants R01‐AR‐059397 and P01‐HL‐134609
Structural Disorder and Induced Folding of the WWC Family Proteins
Authors: Rodriguez Diego Javier , Kwok Ethiene , Chatterjee Tilottama , Baker Kasie , Nyarko Afua
Published: April 2020

The WWC family consists of three proteins‐ KIBRA/WWC1, WWC2, and WWC3. Each protein contains two WW domains which recognize proline‐rich sequences of the type PPxY (P is proline, Y is tyrosine, x is any amino acid). WW domains are named for two conserved tryptophans spaced 23 residues apart and adopt a three‐stranded antiparallel beta sheet fold. The WWC proteins are “atypical” in that, the second WW domain (WW2) contains an isoleucine rather than a conserved tryptophan. Recent studies of the KIBRA WW domains shows that this substitution leads to unfolding of the WW2 domains. However, the domain undergoes binding‐induced folding when bound to some partners but remains largely unfolded when bound to others. High sequence similarity of the WW domains in the WWC family suggests that unfolding and binding induced folding of the WW2 domain may be conserved in this family, but it is unclear why some partners induce folding of the WW2 domain while others do not. Our working hypothesis is that partners which contain PPxY motifs in close proximity will bind the WWC proteins with relatively high affinity and induce folding of the WW2 domain. In this study, we test this hypothesis with two binding partners, Large Tumor Suppressor 1 (LATS1), a kinase and tumor suppressor, and Synaptopodin (SYNPO), a synaptic and podocyte protein. We demonstrate by isothermal titration calorimetry that wildtype LATS1 and SYNPO which have two PPxY motifs each, separated by 194 and 15 residues respectively, bound KIBRA and WWC3 with modest micromolar binding affinities. However, mutant LATS1 (LATS1‐VA) and SYNPO (SYNPO_AA) constructs in which the PPxY motifs are separated by two amino acids, bound KIBRA and WWC3 with 20 to 180‐fold enhanced affinity. Using solution magnetic resonance spectroscopy, we demonstrated that the WW2 domain of KIBRA and WWC3 remain unfolded when bound to wildtype LATS1 and SYNPO, while the enhanced binding of the mutants LATS1_VA and SYNPO_AA is accompanied by folding of the WW2 domain. The structural heterogeneity of the WW2 domain may be an important for modulating binding to diverse PPxY motif partners.

Support or Funding Information

This work is supported by start‐up funds from Oregon State University (OSU), OSU College of Science Disease Mechanism and Prevention Fund, and a New Investigator Award from the Medical Research Foundation of Oregon. NMR experiments were collected at the Oregon State University NMR Facility funded in part by the National Institutes of Health, HEI Grant 1S10OD018518, and by the M. J. Murdock Charitable Trust grant # 2014162
Functional Studies of the Non‐Ribosomal Peptide Synthetase Cyclization Domain in Epothilone Biosynthesis Using Site‐Directed Mutagenesis
Authors: Kim Christopher , Gonzalez Reyaz , Soule Jessica , Dieu Viet , MacRae Mark , Dowling Daniel Peter
Published: April 2020

Epothilones are anticancer natural products from Sorangium cellulosum that are comprised of a 16‐membered macrolactone ring with a thiazole side chain. This family of products is synthesized by a hybrid assembly line of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) proteins. PKS modules insert small acyl groups into natural products through C‐C bonds, whereas NRPS modules incorporate amino acids. Two proteins, EpoA and EpoB, form the hybrid PKS‐NRPS biosynthetic starter unit that generates 2‐methylthiazole, a chemical moiety that contributes to epothilone bioactivity. EpoB harbors a cyclization (Cy) domain that is responsible for the generating 2‐methylthiazoline, which is subsequently oxidized to the thiazole. Initial crystallographic studies of the Cy domain revealed a primary tunnel entrance for the upstream carrier protein to deliver its phosphopantetheine‐tethered substrate to the active site. Interestingly, Cy domain structures from other systems contain a different upstream tunnel location. To uncover the roles of the two possible upstream tunnels in EpoB Cy, site‐directed mutations were incorporated that contain larger, bulkier residues predicted to interfere with phosphopantheine arm access to the active site. A previously developed LC/MS assay for determining the 2‐methylthiazole product was adapted in order to monitor product formation among the mutants in comparison to the wild type. These results contribute to an understanding of the function of each tunnel and provide insight to how the protein modules of the PKS‐NRPS biosynthetic starter units work together to catalyze the formation of 2‐methylthiazole.

Support or Funding Information

Funding provided by NIGMS (Project number: 1R15GM123425‐01)
The Role of TIMP3 in Microvascular Endothelial Cell‐Extracellular Matrix Interaction and Regulation of Microvascular Barrier Function
Authors: Kulkarni Nidhi , Mehta Sanjay , Wang Lefeng , Pape Cynthia , Gill Sean
Published: April 2020

Background

During sepsis, disruption of the pulmonary microvascular endothelial cell (PMVEC) barrier is associated with lung dysfunction and death. PMVEC‐PMVEC interactions through adherens and tight junctions are crucial for maintaining barrier function, as disruption of these junctions causes protein leak. Multiple mechanisms support PMVEC junctional and barrier function, including PMVEC binding to the extracellular matrix (ECM). Data suggest that decreased PMVEC‐ECM interactions under pro‐inflammatory conditions, indicated by altered focal adhesion kinase (FAK) phosphorylation, may be associated with increased leak. Tissue inhibitor of metalloproteinases 3 (TIMP3) is shown to regulate barrier function as PMVEC from Timp3−/− mice have increased leak; however, the specific mechanisms are unknown. Interestingly, previous studies in the developing lung found that TIMP3 can mediate PMVEC‐ECM interactions. Based on this, I hypothesized that TIMP3 maintains PMVEC barrier function by promoting PMVEC‐ECM interactions.

Method

PMVEC from wild type (WT) and Timp3−/− mice were cultured on different matrices and stimulated with pro‐inflammatory cytokines (sepsis) vs control PBS. PMVEC‐ECM interactions were assessed using adhesion assays, immunocytochemistry, and the XPERT‐permeability assay.

Results

Examination of PMVEC adhesion to fibronectin, laminin, vitronectin, and collagen I indicated preferential adhesion of WT PMVEC to fibronectin with lowest adhesion to collagen. While Timp3−/− PMVEC appeared to also preferentially adhere to fibronectin, overall adhesion to the ECM vs. WT PMVEC was reduced. Assessment of phosphorylated FAK (pFAK) through immunocytochemistry revealed reduced abundance of pFAK in both cell types under cytomix‐treatment vs. PBS. Additionally, pFAK organisation and localised distribution around the periphery of cell borders appeared to be reduced in Timp3−/− vs. WT PMVEC. The XPERT‐permeability assay revealed differences in total inter‐PMVEC avidin fluorescence and specifically avidin leak diffusion distance between PMVEC and the ECM in WT vs. Timp3−/− cells, especially under cytomix‐treatment.

Significance

These findings demonstrate that sepsis disrupts the interaction of WT PMVEC with ECM, which is further enhanced in Timp3−/− cells. The results suggest that TIMP3 may play an important role in regulating PMVEC‐ECM interactions, and could help elucidate therapeutic targets to maintain or rescue PMVEC barrier integrity in sepsis.

Support or Funding Information

Heart and Stroke Foundation of Ontario, Ontario Thoracic Society Grant‐in‐Aid, NSERC Discovery Grant
Membrane Proteins — the Lipid Connection
Authors: Robinson Carol
Published: April 2020

We have used native MS of membrane proteins liberated form micelles and native membranes to study protein lipid interactions. Using different Orbitrap MS platforms developed for membrane proteins we show that it is possible to distinguish lipids, cofactors and drugs while in direct contact with membrane protein targets.

We also showed how drug binding mediates phosphorylation in a class A GPCR (P2Y1R) and that purification of three further class A GPCRs results in binding of specific lipids that mediate downstream coupling.

The ability to detect binding which has both structural and functional effects for example on lipid flippase activity or G‐protein coupling, is a powerful and important attribute of the study of membrane proteins by mass spectrometry.

Very recently we have added yet further insights by ejecting membrane protein directly from their native membrane environments. Recent results will be presented.

Support or Funding Information

Wellcome Trust Investigator AwardERC Advanced Grant MRC Programme Grant
Are Fibrillin‐Notch Interactions Important in Development and Disease?
Authors: Sakai Lynn Y. , Carlson Eric J.
Published: April 2020

In humans, mutations in the gene for fibrillin‐1 (FBN1) cause both the Marfan syndrome as well as several types of acromelic dysplasias. Musculoskeletal features of the Marfan syndrome (long bone overgrowth and arachnodactyly, joint hypermobility, and a muscle wasting phenotype) are the opposite of those in acromelic dysplasias (short stature and brachydactyly, joint stiffness, and a hypermuscular build). How mutations in the same gene can result in opposite phenotypes is poorly understood. Mutations in FBN2 cause congenital contractural arachnodactyly, which features a mixture of related musculoskeletal features (muscle hypoplasia, contractures of the large and small joints and arachnodactyly).

The fibrillins are multifunctional glycoproteins that polymerize into extracellular matrix microfibrils. Although we have associated fibrillin microfibril structure with the opposite musculoskeletal features of Marfan syndrome and acromelic dysplasias, we can only speculate that perturbations in growth factor signaling are involved. To begin to address this gap in our understanding, we hypothesize that direct interactions between fibrillin and cells coordinate growth factor signaling. Therefore, we searched for interactions between fibrillin and cellular receptors.

Fibrillins, like Notch receptors and Notch ligands, are composed of multiple tandemly repeated cbEGF‐like domains. Each fibrillin contains 43 cbEGF‐like domains. What if Notch receptors or ligands also bind to fibrillins in the extracellular matrix? Our biochemical data imply that fibrillin interactions with Notch receptors or ligands are just as likely to occur in vivo as interactions between Notch receptors and known Notch transmembrane ligands. Human genetic evidence support significant roles for Notch signaling components in vascular and musculoskeletal diseases, tissues primarily affected in the fibrillinopathies.

The current concept of Notch signaling is based strictly on cell‐cell communication. Our investigations have the potential to transform current understanding of context‐dependent Notch signaling by introducing the possibility that Notch signaling components also interact with fibrillin extracellular matrix.

Support or Funding Information

grants from the Shriners Hospitals for Children to Lynn Y. Sakai
Chromatin Regulation and Genome Maintenance by Mammalian SIRT6 and SIRT7
Authors: Chua Katrin F.
Published: April 2020

Members of the Sirtuin family of enzymes are important regulators of genomic stability, stress responses, and metabolic programs that impact on human physiology, aging, and age‐related disease processes. We previously showed that the mammalian Sirtuins SIRT6 and SIRT7 have high‐selectivity histone deacetylase activities at chromatin, and inactivation of SIRT6 or SIRT7 results in dysregulated histone acetylation states and gene expression programs, with pathological consequences at the cellular and whole organism levels. Recently, we have been exploring novel functions of SIRT6 and SIRT7 in silencing of heterochromatic regions of the genome, the deregulation of which has been linked to aging and cancer biology. We found that pericentric heterochromatin silencing by SIRT6 prevents acute cellular senescence that is triggered by pathologic pericentric transcripts. We also uncovered a second novel trigger of human cellular senescence, ribosomal DNA instability in nucleoli, and we showed that SIRT7 guards against senescence induced by this instability. In our studies, a long‐term focus has been identifying substrates of SIRT6 and SIRT7 and their roles in aging and disease pathways. In new work, we have identified a novel physiologic substrate of SIRT7 at chromatin, and we are characterizing the genomic landscape of this SIRT7‐dependent deacetylation target, and its downstream chromatin and nuclear signaling mechanisms. We will also discuss mechanistic insights into the functions of SIRT6 and SIRT7 from new proteomic, cellular, and mouse model studies.

Support or Funding Information

VA Merit Review NIH/NIA
tRNA: Splicing and Intron Turnover
Authors: Hopper Anita K. , Nostramo Regina T. , Bao Alicia , Peltier Lauren M.
Published: April 2020

In most eukaryotic genomes a subset of tRNA genes contains introns. Introns in tRNAs genes are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by a conserved heterotetrameric protein enzyme, tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast was surprisingly discovered to occur on the mitochondrial surface (Yoshihisa et al. 2003). We recently reported that the SEN complex also locates to the mitochondrial surface in fission yeast (Wan & Hopper 2018) and, therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for at least 500 million years. The mitochondrial outer membrane protein, Tom70, is required for efficient SEN subunit location to mitochondria in both budding and fission yeast (Wan & Hopper 2018). The freed introns are rarely detected in cells and until recently it was unknown how are they are turned over. We identified at least 5 separate, species‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate, possibly indicating their functions in stress response (Peltier, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNAs (Bao, unpublished), also indicative of their possible roles in gene regulation. Our initial experiments support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA (Nostramo, unpublished).

Support or Funding Information

NIH GMS 122884
Wiring the powerhouse: Systems‐to‐structure approaches for defining mitochondrial protein function
Authors: Pagliarini David
Published: April 2020

Mitochondria—epicenters of cellular metabolism and bioenergetics—are much less well understood than their iconic “powerhouse” moniker implies. Hundreds of mitochondrial proteins remain uncharacterized, classic pathways include many steps not yet assigned to specific enzyme functions, a large percentage of mitochondrial diseases remain unresolved, and there are no approved FDA drugs to combat mitochondrial dysfunction. The goal of my research program is to produce a more complete understanding of mitochondrial biology by systematically establishing the functions of orphan mitochondrial proteins and their roles within disease‐related processes. We begin by devising multi‐dimensional analyses designed to make new connections between these proteins and established pathways and processes. We then employ mechanistic and structural approaches to define the functions of select proteins at biochemical depth. This ‘systems biochemistry’ strategy is helping us address outstanding biological questions, including: Which orphan mitochondrial proteins fulfill missing steps in classic mitochondrial processes, such as the biosynthesis of coenzyme Q and the assembly of complex I, and how do they operate mechanistically? And, which resident signaling proteins direct the post‐translational regulation of mitochondrial activities? In answering these and other questions, we aim to help transform the mitochondrial proteome from a component list into a metabolic circuitry of connected functions, and to establish the biochemical underpinnings of mitochondrial dysfunction in human disease.

Support or Funding Information

NIH R01DK098672 NIH R35GM131795 NIH R01GM115591 NIH P41GM108538
Death by Lipids: role of noncoding RNAs in metabolic stress
Authors: Schaffer Jean E
Published: April 2020

Nutrient excess is linked to the pathogenesis of complications of obesity and type 2 diabetes. Fatty acid accumulation in non‐adipose tissues is initially well tolerated and leverages the nearly universal ability of cells to store fatty acids as triglycerides in lipid droplets. However, lipid overload eventually precipitates cell dysfunction through generation of toxic metabolites, activation of signaling cascades, endoplasmic reticulum stress, and oxidative stress. These pathways contribute to cell death, organ dysfunction and disease pathogenesis. Mechanisms that regulate the transition from adaptive lipid storage to initiation of lipotoxicity are not well understood. New insights have come from genetic screens in model cell culture systems. By leveraging the ability of retroviral promotor trap mutagenesis to disrupt genetic elements regardless of coding potential and using selection for resistance to palmitate‐induced cell death, our group discovered that disruption of specific small nucleolar RNAs (snoRNAs) protects against lipotoxic and oxidative stress in cultured cells. By extending our findings to in vivo models, we have demonstrated that knockdown or knockout of these snoRNAs rewires metabolism in ways that increase lipid metabolism, enhance oxidative stress tolerance, and confer resistance to lipotoxicity. Our studies highlight novel roles for snoRNAs as metabolic modulators and suggest that expression and function of this class of intronic non‐coding RNAs may have relevance to human health and disease.

Support or Funding Information

NIH R01 DK064989NIH R01 DK108357
Toolbox to evaluate biological function of histone deacetylases
Authors: Fierke Carol A.
Published: April 2020

Lysine acetylation is a post‐translational modification that is regulated by the opposing activities of histone acetyltransferases that catalyze protein lysine‐N‐acetylation, and histone deacetylases (HDACs) that catalyze removal of the ɛ‐N‐acetyl‐lysine modification. Acetylation regulates a plethora of cellular processes, and misregulation has been implicated in the development of myriad pathological states such as cancer, diabetes, aging, neurological disorders and genetic diseases. Inhibitors of metal‐dependent HDACs have been approved by the FDA for the treatment of T‐cell lymphoma and multiple myeloma. The full complement of in vivo substrates for the 11 metal‐dependent HDACs is unknown, and the functional basis for substrate selectivity is poorly defined. We are investigating the substrate sets for HDAC1, HDAC6 and HDAC8 using a number of methods, including: 1) identification of transient interactors using active‐site directed photocrosslinking covalent capture followed by mass spectrometric analysis, 2) measurement of HDAC activity with libraries of peptides and proteins coupled with the development of computational selectivity models, and 3) analysis of the role of phosphorylation and protein binding partners on substrate selectivity. These data are providing insight into the biological function of these important enzymes.

Support or Funding Information

National Institutes of Health R01‐GM‐040602