bluuebunny/crossref_metadata_2025 huggingface dataset:
The Three Blind Men: How Adhesion Proteins Binding Affects the Retinas of People with Retinoschisis
Authors: Strandberg Stacey , Bauer E. , Cobb E. , DebBaruah C. , Fricker E. , Griffith‐Topps A. , Jessick M. , Kuehn C. , Kulju T. , Lois C. , Sargent C. , Schraufnagel L. , Strandberg S. , Strother C. , Thota A. , Urban S. , Fleischmann Scott
Published: June 2021

X‐linked retinoschisis, an early‐onset macular degenerative disease occurring primarily in young boys, can result in blindness. As many as two hundred different mutations on the X chromosome can lead to a malformation of retinoschisin, a structural protein which normally holds the photoreceptors and bipolar cells together. The photoreceptors detect light, and bipolar cells process these stimuli for interpretation by the brain. An improperly folded retinoschisin prevents the photoreceptors and bipolar cells from interacting and prevents information from being transmitted to the brain. Retinoschisin is composed of sixteen subunits arranged in a double octomer. Because retinoschisin relies on extensive interactions between subunits, many different mutations can lead to a dysfunctional protein. The C110 and C142 bonds stabilize the spikes protruding from the discoid fold—the part of the protein that allows the retinoschisin to bind to the retinal cells. Mutations in these residues lead to the inability of the protein binding to retinal cells. Since the retinoschisin cannot attach to the retinal cells, the retina begins to split and causes eventual blindness. In order to further understand the structure and function of this protein, the Divine Savior Holy Angels SMART (Students Modeling a Research Topic) team modeled retinoschisin using 3D printing technology. Although no cure exists, current research is focusing on the structure of retinoschisin, and how mutations prevent adhesion between retinal cells with an ultimate goal of developing a gene therapy. By correcting the mutated X chromosome segment, retinoschisis could be prevented.

Support or Funding Information

Munzenmaier, Dr. Diane

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human RGS14 binds NHERF1 and regulates NPT2A‐mediated phosphate uptake: Disruption by genetic variants
Authors: Hepler John R. , Mamonova Tanya , Sneddon W. Bruce , Emlet David , Harbin Nicholas H. , Squires Katherine E. , Friedman Peter A.
Published: June 2021

RGS14 is a multifunctional scaffolding protein that integrates G protein, MAPK and Ca++/CaM signaling pathways. Most of what we know about RGS14 comes from studies in rodent brain where it acts as a natural suppressor of synaptic plasticity and hippocampal‐based learning, though other studies implicate a role for RGS14 in myocardial remodeling. Recent genome‐wide association studies have implicated RGS14 in disordered phosphate metabolism, an effect of chronic kidney disease. The RGS14 gene is adjacent to SLC34A1 on human chromosome 5 that encodes the NPT2A sodium‐phosphate cotransporter, which mediates hormone‐sensitive phosphate resorption in renal proximal tubules. Therefore, the goal of these studies was to define a possible role for RGS14 in hormone‐sensitive phosphate transport. Human/primate RGS14 differ from rodent proteins by possessing a carboxy‐terminal extension encoding a Type I PDZ ligand (DSAL). Of note, several rare human mutations have been reported in this region of RGS14 located at residues 563 and 565, corresponding to positions −1 (A565S, A565V) and −3 (D563N, D563G) of the PDZ ligand. The family of PDZ adapter proteins assemble a variety of membrane‐associated proteins, including transporters, receptors, and ion channels, and signaling molecules in short‐lived functional units. The PDZ adapter NHERF1 is expressed in renal proximal tubules, and contains two PDZ domains (PDZ1 and PDZ2). NHERF1 binds the PDZ ligand carboxy tail of NPT2A to promote its phosphate uptake, and this NPT2A:NHERF1 complex is inhibited by parathyroid hormone (PTH) acting at the PTH1 receptor. Here we report that RGS14 is expressed in proximal and distal renal tubules of humans, and that RGS14 binds to NHERF1 via the PDZ2 domain. PTH inhibits NPT2A uptake of phosphate, and RGS14 blocks this action. Of the RGS14 genetic variants tested, D563N disrupted binding to NHERF1 and RGS14 actions on PTH‐sensitive phosphate uptake, whereas D563G, A565S and A565V were innocuous. Computational analysis and molecular dynamics modeling of NHERF1 PDZ binding to the RGS14 C‐terminal PDZ ligand define the structural determinants of this interaction. Taken together, our findings are consistent with the idea that RGS14 contributes to PTH‐sensitive phosphate transport in humans, and that RGS14 coding variants may cause disordered phosphate metabolism.

Support or Funding Information

National Institutes of Health (NIH) awards DK105811, DK111427 (PAF); NS037112, NS102652 (JRH).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of Telomere‐Rap1 aggravates cardiac aging by impairing fatty acid oxidation via PPARα signaling
Authors: Liu Hao , Cai Yin , Ying Fan , Liu Shiming , Xia Zhengyuan
Published: June 2021

The heart undergoes multiple functional and structural declining with aging, including impaired fatty acid metabolism, cardiac dysfunction and hypertrophy. Repressor activator protein 1 (Rap1) as a component of shelterin complex is essential for the maintenance of telomere function. Moreover, Rap1 also regulates gene transcription by binding to non‐telomeric sites such as the nuclear hormone receptor ‐ peroxisome proliferator‐activated receptor α (PPARα) which plays a central role in the cardiac fatty acid metabolism and mitochondrial function. However, it's still not clear whether or not the heart of Rap1−/− mice also suffers more aging associated disorders. Thus, the present study aims at the role of Rap1 in cardiac aging and its possible mechanism. Echocardiography showed enlarged left ventricular internal dimension (LVID) and LV mass, which suggested dilated hypertrophy, and elevated myocardial performance index (MPI) in Rap1−/− mice at age of 85weeks, indicating systolic and diastolic dysfunction (all P<0.05 vs. Rap1+/+). The greater adverse cardiac remodeling in aged Rap1−/− mice was paralleled by elevated cardiomyocyte area showed by Wheat germ agglutinin (WGA) staining (P<0.05 vs. Rap1+/+). p53 as a senescence marker was also increased indicating more severe cellular senescence in cardiomyocyte of Rap1−/− mice (P<0.05 vs. Rap1+/+). From apex cordis of Rap1 wildtype and knockout mice, electron microscopy showed more enlarged pleomorphic mitochondria containing cristae fragmentation, vacuolization and disrupted external membranes in aged Rap1 knockout mice compared to age‐matched wildtype counterparts. Decreased Acetyl‐CoA carboxylase (ACC) (catalyzes the production of malonyl‐CoA) and Elevated Carnitine palmitoyl transferase I (CPT‐1) (transports fatty acyl‐CoA into mitochondria) and Acyl‐CoA dehydrogenase long chain (ACADL, catalyzes most of fatty acid β‐oxidation) were detected in aged Rap1 knockout mice(all P<0.05 vs. Rap1+/+), indicating Rap1 deficiency may lead to reduced fatty acid oxidation during aging. In the heart of aged Rap1−/− mice, PPARα was reduced compared with age‐matched wildtype counterparts (P<0.05 vs. Rap1+/+), suggesting that with aging, Rap1 may down‐regulate PPARα signaling in the heart and control fatty acid metabolism resulting in cardiac dysfunction and hypertrophy. In conclusion, Rap1 deficiency may impair myocardial fatty acid oxidation via PPARα signaling, leading to more severe cardiac aging as cardiac dysfunction and hypertrophy. These findings may identify a new extratelomeric role of Rap1 in cardiac metabolism during aging.

Support or Funding Information

The authors' work was supported by the Health and Medical Research Fund (04151816, Hong Kong) and General Research Fund (17121315M, 17123915M and 17117217M, Research Grants Council of Hong Kong).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A model of steady state ultrafiltration through an endothelial glycocalyx bi‐layer formed by a porous outer layer and a more selective membrane‐associated inner layer
Authors: Curry FitzRoy E , Michel Charles
Published: June 2021

Ultrastructural investigations of the endothelial glycocalyx reveal a layer adjacent to the cell surface with a structure consistent with the primary ultrafilter of vascular walls. Theory predicts this inner layer can be no greater than 200–300 nm thick, a result to be reconciled with observations that red cells and large macromolecules are excluded from a region up to 1 micron or more from the cell membrane. We tested whether this apparent inconsistency might be accounted for by a model of steady state water and protein transport through a glycocalyx bi‐layer. Expressions for coupled water and albumin fluxes through the two layers were used to describe steady state ultrafiltration. The model demonstrated that albumin may accumulate at the interface between the porous layer and the selective inner layer. The osmotic pressure of accumulated albumin would significantly modify the observed permeability properties of the microvessel wall by an effective unstirred layer effect. Our results place constraints on the outer layer permeability properties. For example, with an perfusate albumin concentration of 50 mg/ml, Adamson et al ( Am. J. Physiol. 557: 889–907,2004) reported that the hydraulic conductivity measured in rat mesenteric microvessels under conditions of steady state ultrafiltration were similar to that measured before steady ultrafiltration was established. The only properties of the outer layer consistent with these observations are albumin permeability coefficients and hydraulic conductivities close to an order of magnitude larger than that of the inner layer. A high filtration coefficient for the outer layer is also consistent with estimates of the hydraulic conductance of the outer layer needed to account for red cell movement over the endothelial surface up to 1 micron thick.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Uncoupling protein (UCP)1 and exercise induced ‘browning’ of white adipose tissue is absent at thermoneutrality
Authors: Aldiss Peter , Lewis Jo , Ebling Fran , Bookcock David , Budge Helen , Symonds Michael
Published: June 2021

Background and aim

The induction of thermogenic genes in white adipose tissue (WAT) of exercise‐trained rodents is commonly seen in animals housed at standard temperatures (e.g. ~20°C) despite not being seen in exercise‐trained humans1,2. Here, we hypothesised that raising and exercise training rodents at thermoneutrality (e.g. ~28°C) thus mimicking human physiology would negate the induction of thermogenic genes typically seen at standard housing temperatures.

Methods

Twelve weanling (i.e. 3 weeks old) Sprague‐Dawley male rats were housed at thermoneutrality (28°C) and fed a high‐fat diet (HFD) to induce weight gain with (Ex) or without (C) exercise training. At 12 weeks of age Ex was commenced by swim‐training (i.e. 1 hour/day, 5 days per week) for 4 weeks. Metabolic assessment was undertaken in metabolic cages during the last 48h to assess energy intake, expenditure and physical activity in addition to the acute response to administration of a β3‐agonist (Mirabegron, 0.75mg/kg/d). Then, at 16 weeks of age following an overnight fast, tissues were taken for analysis of thermogenic genes and quantitative proteomics.

Results

Ex attenuated weight gain, but did not affect food intake, energy expenditure, final body weight, fat mass, plasma insulin or glucose or HOMA‐IR. There was also no increase in energy expenditure in response to administration of Mirabegron with UCP1 mRNA in IWAT remaining undetectable in EX despite an upregulation of genes considered to be classical ‘browning’ inducers i.e. PGC1a, ADRB3 and DIO2. We found 189 proteins were differentially regulated in IWAT following exercise. Among the most significant were those regulating nuclear factor kappa B signalling (LRRFIP2), the mitochondrial electron transport chain (NDUFS6) and oxidative stress (SOD3). Gene ontology analysis demonstrated these enriched 51 groups based on their biological process including ‘regulation of alternative mRNA splicing, via spliceosome’, ‘DNA damage checkpoint’ and ‘regulation of type 2 immune response’ whilst only 13 groups were enriched based on their molecular function including ‘metallodipeptidase activity’, ‘ATPase regulator activity’ and ‘pre‐mRNA binding’.

Conclusion

Unlike at standard housing temperatures, exercise induced ‘browning’ of IWAT was absent in animals raised at thermoneutrality corroborating human data. Proteomic analysis further highlights alternate novel pathways in adipose tissue regulated by exercise training that could have functional relevance.

Support or Funding Information

British Heart Foundation [grant number FS/15/4/31184/]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sonic hedgehog binds to Patched molecules on sensory stem cells to initiate smell and taste function
Authors: Henkin Robert
Published: June 2021

Smell and taste function are tripartite systems involving sensory receptors, nerves and brain. Pathological changes in these systems depend primarily on impaired receptor function. Receptors in these systems are unique in that they contain neither blood vessels nor lymphatics and do not exhibit mitosis. For their growth and development, sensory receptors depend upon stem cell stimulation with growth/transcription factors secreted into saliva for taste and nasal mucus for smell. There are many of these growth/transcription factors but one of the major ones is sonic hedgehog (SHH) which causes receptor growth and development. Many pathological entities inhibit smell and taste function and do so by inhibition of these growth/transcription factors including SHH. Treatment with phosphodiesterase inhibitors activates SHH secretion which corrects these various pathologies. However, the molecular mechanism by which SHH acts on these stem cells to activate them is unclear. Recent studies have demonstrated that the efficacy of SHH at a molecular level depends upon its binding to two Patched molecules at a distinct site on the cell membrane of these sensory stem cells. This binding yields a signaling competent complex which, in the taste and smell systems, initiate growth and perpetuation of olfactory and gustatory receptors and results in their growth and development. This binding initiates the olfactory and gustatory signals which initiate smell and taste function.

Support or Funding Information

This institution did not receive any funding from agencies in the public, commercial, or not‐for‐profit sectors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPK ablation aggravates colorectal tumorigenesis via epigenetic suppression of antioncogenes
Authors: Zhu Meijun , Sun Xiaofei , Du Min
Published: June 2021

Background

The incidence of colorectal cancer (CRC) is closely related to metabolic diseases. AMP‐activated protein kinase (AMPK) is known as a key metabolic regulator. The objective of study is to assess the regulatory role of AMPK in metabolic alteration, epigenetic modification and colorectal tumorigenesis.

Methods

AMPKα1‐floxed mice was cross‐bred with Lgr5Cre mice to specifically knockout AMPK α1 gene in intestinal stem cells and their derived epithelial cells. The wild‐type and AMPK knockout mice were subjected to azoxymethane (AOM)/DSS induced‐CRC induction.

Results

AMPK stem cell specific knockout caused colonic hyperproliferation and pathological features in mice without CRC induction; augmented adenocarcinoma number and size as well as dysperplasia in AOM/DSS‐indiced mice model. The aggravated colorectal tumorigenesis induced by AMPK ablation likely resulted from alteration of metabolites, as indicated by reduced production of acetyl‐CoA, a substrate required for histone acetylation, and α‐ketoglutarate, an obligate substrate for ten‐eleven translocation hydroxylases (TETs) mediated DNA demethylation, in colonic tissues. Consistently, α‐ketoglutarate producing isocitrate dehydrogenase 1 (IDH1) content and the activity TET1 were markably decreased in AMPK deficient mice, correlated with reduced metastasis inhibitors, Adamts1 (A disintegrin and metalloproteinase with thrombospondin motif 1) and Mal (myelin and lymphocyte), as well as hypermethylation of Mal and Mgmt (O‐6‐methylguanine‐DNA methyltransferase) promotor regions.

Conclusion

Data suggested critical roles of AMPK in integrating metabolism with epigenetic modifications and colorectal tumorigenesis.

Support or Funding Information

USDA‐NIFA#2018‐67017‐27517 and WSU ERI competitive grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caspases from Scleractinian coral show unique regulatory features
Authors: Clark Clay , Shrestha Suman , Tung Jussica , Swartz Paul , Grinshpon Robert , Mydlarz Laura
Published: June 2021

Coral bleaching is an immune response that rejects compromised symbionts. Porites astreoides and Orbicella faveolata are coral species that exhibit different responses to stress. Disease‐resistant P. astreoides employs adaptive autophagic mechanisms when immunocompromised, while disease‐sensitive O. faveolata activates caspase‐mediated apoptosis. We characterized homologs of caspases from O. faveolata (O.fav‐3a and O.fav‐3b) and P. astreoides (P.ast‐7 and P.ast‐3). O.fav‐3a and P.ast‐7 contain a human initiator/inflammatory CARD‐like domain despite high sequence similarity to effector caspases. Experimental results from substrate‐phage display show that O.fav‐3a and P.ast‐7 prefer aspartate in the P1 position, which is corroborated by activity against canonical substrate, DEVD‐AFC. In contrast, substrate‐phage display studies showed that O.fav‐3b and P.ast‐3 prefer valine at the P1 position, but the enzymes are largely inactive on the canonical substrate VEID‐AFC, suggesting additional requirements for the active enzyme. We determined the structure of P.ast‐7 bound with DEVD‐FMK inhibitor to 1.57Å resolution by X‐ray crystallography. The data reveal a hydrogen‐bonding network with a unique insertion (RYP) in one active site loop and suggest that the sequence may be autoinhibitory when substrate is not bound. In addition, the structure reveals an N‐terminal peptide bound near the active site that may serve as a regulatory exosite.

Support or Funding Information

UT Arlington

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification and enzymatic assay of BmaI1, a quorum sensing signal synthase
Authors: Kellner‐Rogers Julian , Ricciardi Meghan , Ulrich Scott Michael
Published: June 2021

Quorum sensing is a bacterial cell‐cell communication system that functions through the synthesis, secretion, and detection of signaling molecules called autoinducers. Quorum sensing enables bacteria to assess their cell density and coordinate group behaviors that are advantageous at high cell density. Many bacteria that cause acute and chronic infections in humans use quorum sensing to control expression of virulence factors and formation of biofilms, so inhibitors of quorum sensing hold significant promise as novel antibacterial agents. By targeting bacterial virulence as opposed to growth and survival, quorum sensing inhibitors avoid the selective pressure for drug resistance that is inherent to traditional antibiotics.

The main quorum sensing system in Gram‐negative bacteria is based on the synthesis and detection of acyl homoserine lactone (AHL) autoinducers. Efforts to inhibit AHL‐mediated quorum sensing have focused on developing synthetic AHL analogs that act as antagonists of AHL receptors. These compounds have been shown to block virulence controlled by quorum sensing, validating quorum sensing as an antibacterial drug target.

The overall goal of the project is to develop inhibitors of AHL synthase enzymes, an alternative quorum sensing drug target that remains under‐explored. Towards this goal, we cloned and purified BmaI1, an AHL synthase from Burkholderia mallei. AHL synthases use S‐adenosyl methionine and acylated ACP protein as substrates. Generating Acyl‐ACP requires purification of ACP, and several sequential enzymatic modifications to make it suitable as an AHL synthase substrate. We have developed a small, synthetic substitute for acyl ACP that is utilized by BmaI1, avoiding the difficult and time‐consuming production of acyl‐ACP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single particle cryo‐EM studies of membrane proteins
Authors: Cheng Yifan
Published: June 2021

In the last few years, major technological advancements, particularly the development of new direct electron detection cameras and associated technologies, have enabled single particle electron cryo‐microscopy (cryo‐EM) to become the technique of choice for high‐resolution structure determination of many challenging biological macromolecules. We pioneered some of the technologies now being routinely used, and applied them to determine atomic structures of a number of integral membrane proteins, such as some major members of transient receptor potential (TRP) channel superfamily. Some of our studies paved the way of applying this method to a wide range of integral membrane proteins. Nowadays, atomic structures of membrane proteins that are refractory to crystallization are being determined by this method in a rapid pace.

Technologically, we further aimed to enable high‐resolution structural studies of membrane proteins in near native lipid bilayer environment. To this end, we pioneered a number of approaches, including reconstituting membrane protein into lipid nanodisc or into saposin based lipid nanoparticles, and developed methodology of using conformational specific monoclonal Fab or other designed scaffoldings to facilitate image alignment of very small integral membrane proteins reconstituted into lipid nanoparticles. In a number of recent studies, we observed specific lipid‐protein interactions that play critical role in regulating the channel functions. In TRPV1 ion channel, as an example, certain specific lipid‐protein interactions enhance binding of a spider toxin to the channel through formation of a toxin‐lipid‐TRPV1 tripartite complex, and an endogenous phosphatidylinositol lipid occupies the capsaicin‐binding site of the channel. These observations provide important clues about physiological mechanisms of channel regulation.

Support or Funding Information

This work is funded by NIH grants: R01GM098672, S10OD020054 and S10OD021741.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chaperone interactions of human α‐crystallin homo‐oligomers with their physiological substrate γD‐crystallin
Authors: Acosta‐Sampson Ligia Isabel , King Jonathan
Published: June 2021

The chaperone α‐crystallin, an ATP‐independent small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses, along with the γ and β family of Greek key crystallins. αA‐ and αB‐crystallin (αA‐ and αB‐crys) subunits (~ 20 kDa) form an hetero‐oligomeric, polydisperse complex of ~ 800 kDa in the lens. The chaperone function involves suppressing aggregation by binding aggregation‐prone species. Aggregates isolated from mature‐onset cataracts, the major cause of sight loss worldwide, contain damaged and misfolded forms of β γ‐crystallins. Human γD‐crystallin (γD‐crys) is a stable and long‐lived major structural crystallin localized in the lens nucleus. Human γD‐crys refolds in vitro through population of a partially folded intermediate with the C‐terminal domain folded and the N‐terminal domain disordered. This species partitions between productive refolding and competing aggregation pathways. The γD‐crys intermediates were populated by dilution out of GuHCl at 37°C, and subsequently incubated with α‐crys. Turbidity assays show that a 1:20 or 1:10 (monomer/monomer) ratio of γD‐crys to αA‐ or αB‐crys, respectively, suppressed aggregation by 90%. α‐Crys homo‐oligomers presumably suppress γD‐crys aggregation by binding γD‐crys partially folded intermediates formed during refolding. The α‐crys/γD‐crys complex were stable for many hours after initial incubation. It is not clear if the long‐lived nature of the complex reflects internalization of the substrate into the lumen of the α‐crys oligomer, or whether partially unfolded crystallin species are tightly bound to the chaperone surface.

Supported by NIH Grant GM 17980 and NEI grant EY 015834 (to J.K.)
Effects of methylglyoxal on amyloid‐beta peptide
Authors: Michelson Randal Cerf , Seidler Norbert W
Published: June 2021

Diabetics exhibit elevated levels of carbohydrates and their oxidized byproducts such as methylglyoxal (MGO). With the excess production and accumulation of the amyloid‐beta peptide (ABP) in AD, the linkage between diabetes and AD may involve ABP and MGO. We studied the effects of MGO‐induced ABP modification on conformational properties. Human isoform ABP fragment 1–42 was used in our study. Fluorescence and absorbance spectroscopy assessed MGO‐induced production of advanced glycation endproducts (AGEs), size exclusion chromatography monitored conformational changes, and thioflavin T fluorescence tracked fibrillation. MGO decreased the amount of high molecular weight (HMW) ABP aggregate and increased the low molecular weight (LMW) species relative to the control. Products obtained from incubation of MGO with free arginine, which absorbed in the 220nm range, also appear in HMW ABP when incubated with MGO. The LMW ABP species contained an MGO‐derived adduct that absorbed in the 280nm range, suggesting that conformational differences in the structures may promote reactivity to different amino acid residues. MGO affected the rates of ABP aggregation as compared with control, indicating that AGEs may influence ABP packing densities. These observations suggest a plausible molecular explanation for the relationship between diabetes and AD, providing novel avenues for therapeutic intervention.
The sHSP and molecular chaperone, aB crystallin, regulates microtubule assembly
Authors: Clark John I , Houck Scott A. , Ghosh Joy G.
Published: June 2021

Purpose:

The small heat‐shock protein (sHSP) αB crystallin, which protects against unfolding and aggregation of proteins has a regulatory effect on microtubule assembly. The sequences responsible for αB crystallin ‐ microtubule interactions were identified and tested.

Methods:

Five interactive sequences in αB crystallin were identified and studied for their effects on the assembly of microtubules using synthetic peptides and mutants of αB crystallin. Assembly of microtubules was measured using a fluorescence DAPI assay (PloS one 6:3498).

Results:

αB crystallin inhibited microtubule assembly at molar ratios >2:1 αB crystallin:tubulin and promoted microtubule assembly at molar ratios between 1:4 – 2:1. The synthetic peptide corresponding to the αB crystallin interactive sequence, 113FISREFHR120, partially inhibited microtubule assembly. In contrast, the peptides 131LTITSSLSSDGV142 and 156ERTIPITRE164promoted microtubule assembly. Mutations in these sequences altered the nature of the interactions with microtubules.

Conclusion:

Three interactive sequences on the surface of human αB crystallin modulate microtubule assembly through the dynamic subunit model of small heat shock proteins.
Mg2+‐Assisted Catalysis by B. stearothermophilus TrpRS is Promoted by Allosteric Effects
Authors: Carter Charles W. , Weinreb Violetta , Li Li , Campbell Cassandra L. , Kaguni Laurie S.
Published: June 2021

Mg2+ ion accelerates TrpRS‐catalyzed Trp activation ~105 fold, providing 35% of the reduction in ΔG‡. This catalytic contribution is achieved indirectly, by interactions from active‐site lysine residues to the non‐bridging phosphate oxygen atoms that coordinate the Mg2+, lengthening metal‐oxygen distances and weakening interaction with the triphosphate. This electrostatic compromise is coupled to an endergonic twist of the anticodon‐binding domain relative to the active site, whose unfavorable energetics arise from long‐range interactions, and which leads to (exergonic) untwisting during the catalytic step. We investigated these interactions quantitatively, determining mutant effects in multi‐variant thermodynamic cycles with [ATP]‐dependent Michaelis‐Menten kinetics and Mn2+ for Mg2+ substitution. These cycles confirm the coupling of lysines to metal coordination and rule out a proposed interaction between the metal and the nearly invariant aspartate, D146. The two lysine residues most strongly coupled to the metal bind the PPi leaving group; the third interacts with the Pα phosphate. Coupling was also observed for a remote mutation, F37I, which affects both kcat and the use of Mn2+. Tηεσειντεραχτιℴνσ αρε χℴνσιστεντ ωιτη στραιν αρισινγ φρℴμ αν υνφαϖℴραβλε χℴνφℴρματιℴν νεχεσσαρψ τℴ ασσεμβλε τηε αχτιϖε σιτε αρℴυνδδ A TΠ ανδ ωηιχη εσταβλισηεσ α δελιχατε βαλανχε ℴφ ελεχτρℴστατιχ ανδ μεχηανιχαλ φℴρχεσ τηατ στραιν τηε σχισσιλε Πα‐O‐Pβ linkage. Supported by NIGMS 48519 and 45295.
Site‐Specific Variations in RNA Folding Thermodynamics
Authors: Bharill Shashank , Gryczynski Zygmunt
Published: June 2021

The fluorescent base analogue 2‐aminopurine (2‐AP) is commonly used to study specific conformational and protein binding events involving nucleic acids. Here, combinations of steady‐state and time‐resolved fluorescence spectroscopy of 2‐AP were employed to monitor conformational transitions within a model hairpin RNA from diverse structural perspectives. RNA substrates adopting stable, unambiguous secondary structures were labeled with 2‐AP at an unpaired base, within the loop, or inside the base‐paired stem. Steady‐state fluorescence was monitored as the RNA hairpins made the transitions between folded and unfolded conformations using thermal denaturation, urea titration, and cation‐mediated folding. Unstructured control RNA substrates permitted the effects of higher‐order RNA structures on 2‐AP fluorescence to be distinguished from stimulus‐dependent changes in intrinsic 2‐AP photophysics and/or interactions with adjacent residues. Thermodynamic parameters describing local conformational changes were thus resolved from multiple perspectives within the model RNA hairpin. These data provided energetic bases for construction of folding mechanisms, which varied among different folding‐unfolding stimuli. Time‐resolved fluorescence studies further revealed that 2‐AP exhibits characteristic signatures of component fluorescence lifetimes and respective fractional contributions in different RNA structural contexts. Together, these studies demonstrate localized conformational events contributing to RNA folding and unfolding that could not be observed by approaches monitoring only global structural transitions.
Structural analysis of ColD: a unique PLP‐dependent sugar‐3‐dehydratase
Authors: Cook Paul D. , Holden Hazel M.
Published: June 2021

Colitose is a 3,6‐dideoxysugar found in the O‐antigen of some Gram‐negative bacteria. This unusual sugar is synthesized from mannose‐1‐phosphate via an intriguing four enzyme pathway. The focus of this investigation, GDP‐4‐keto‐6‐deoxymannose‐3‐dehydratase (ColD), is a unique pyridoxal‐5′‐phosphate (PLP)‐dependent enzyme that removes the hydroxyl from the sugar C‐3′ position. It is an interesting PLP‐dependent enzyme because it acts as both an aminotransferase and a dehydratase, but also because it lacks the conserved active site lysine residue typically observed in PLP‐dependent enzymes. We have solved wild‐type and mutant crystal structures of this enzyme in which various reaction intermediates have been trapped in the active site, including a PLP‐glutamate ketimine intermediate, a PLP‐glutamate geminal diamine, and a GDP‐linked sugar substrate analog. From these structures, it is clear that His 188 is the active site acid/base acting in both the aminotransferase and dehydratase reactions. Assays have revealed that H188K and H188N mutant ColD proteins are unable to catalyze the dehydration reaction. However, we demonstrate that the wild‐type ColD enzyme is capable of using GDP‐perosamine as a substrate to make its natural product. Taken together, these investigations have helped shed new light on the facinating manner in which ColD removes the hydroxyl group about the sugar C‐3′ position.
The Effects of Iron Overload and Deprivation on Iron Metabolic Proteins in Anopheles gambiae 4a3b Cells
Authors: Elliott Jamie L. , Geiser Dawn L. , Mayo Jonathan J. , Winzerling Joy J.
Published: June 2021

Female mosquitoes are vectors for malaria, dengue fever, yellow fever and West Nile encephalitis. Mosquitoes must blood feed to complete oogenesis. Consequently, manipulating the iron metabolic pathway has the potential to interfere with mosquito fecundity, thereby decreasing vector numbers and disease transmission rates. The blood meal supplies high levels of heme and non‐heme iron which has the potential to create free radicals; however the animal appears to experience little ill effect. One possible protection from oxidative stress is iron storage in ferritin. Ferritin is a 24 subunit protein composed of heavy and light chains. We have shown that Aedes aegypti (yellow fever mosquito) larval cells exposed to high doses of iron secrete iron‐loaded ferritin into the culture medium and concentrate iron in the membrane fraction. Anopheles gambiae (African malaria mosquito) 4a3b larval cells are a hemocyte‐like cell line that is involved in immunity. We are interested in the interaction of iron metabolism and infection in these animals. We exposed 4a3b cells to high levels of iron. Similar to A. aegypti CCL‐125 cells, iron localizes in the membrane fraction. However, transcripts for the ferritin subunits, iron regulatory protein 1 and a putative DMT1 responded differently. These iron dose experiments in 4a3b cells lay the ground work to examine the relationship of iron metabolism and immunity in mosquitoes. This work was supported by funds from the National Institutes of Health (GM056812), the Agricultural Experiment Station, the College of Agriculture and Life Sciences and the BIO5 Institute for Collaborative Bioresearch at The University of Arizona.
Measurement of glutathione turnover in human plasma by labeling from 2H‐enriched water and LC‐MS/MS
Authors: SUBRAMANIAN RAJAN KOMBU , Abbas Rime , David France , Sanabria Juan R. , Anderson Vernon E. , Brunengraber Henri
Published: June 2021

We developed a LC‐MS/MS method for measuring the turnover of reduced and oxidized glutathione (GSH and GSSG) in human plasma. Because of differences in signal sensitivity and stability of GSH and GSSG, we assay both compounds as similar thioethers of GSH. The assay involves derivatization of GSH as carboxymethyl derivative, and GSSG as cyanomethyl derivative after reduction using dithiothreitol. Electrospray‐ionization mass spectrometry of thioethers was performed on a 4000 QTrap (Applied Biosystems) mass spectrometer coupled to an Agilent HPLC. We developed a multiple reaction monitoring (MRM) method based on the M0 and M1 fragmentation pattern. Fragmentation of the M1 peak results in a pair of product ions, i.e. 367.2 → (237.1, 238.1). To determine M1/M0 for the precursor ion, the intensity of both product ions must be summed for the numerator, while the denominator is determined solely by the intensity of the 366.2 → 237.1 fragment ion. The MRM difference of M1/M0 for the minimally labeled GSH and for natural abundance provides a precise quantitation of 2H incorporation. Using this method, we measured the turnover of plasma GSH/GSSG in human subjects whose body water was 0.5% enriched with 2H for 72 hr. The half‐lives of labeling of plasma GSH and GSSG, which are mostly of hepatic origin, are 4–5 hr. Supported by NIH grant 1R01ES013925.
REDOX PROPERTIES OF ATP‐SENSITIVE K+ CHANNELS IN BRAIN MITOCHONDRIA
Authors: Fornazari Maynara , de Paula Juliana , Castilho Roger Frigerio , Kowaltowski Alicia Juliana
Published: June 2021

Brain mitochondrial ATP‐sensitive K+ channel (mitoKATP) opening by diazoxide protects against ischemic damage and excitotoxic cell death. We studied here the redox properties of brain mitoKATP. MitoKATP activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mitoKATP activation in isolated brain mitochondria significantly prevented H2O2 release by these organelles, but did not change Ca2+ accumulation capacity. Interestingly, the activity of mitoKATP was highly dependent on redox state. Thiol reductant mercaptopropionylglycine prevented mitoKATP activity, while exogenous ROS activated the channel. In addition, the use of mitochondrial substrates that lead to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K+ transport activity through mitoKATP. Altogether, our results indicate that brain mitoKATP is a redox‐sensitive channel that controls mitochondrial ROS release.

Supported by FAPESP, CNPq, Redoxoma, Guggenheim Foundation and the NIH.
The calcium dependent cysteine protease μ‐calpain is located in the mitochondrial intermembrane space
Authors: Garcia Matthew , Badugu Rama Krishna , Bondada Vimala , Joshi Aashish , Geddes James W
Published: June 2021

The ubiquitous proteases, m‐ and μ‐calpain, are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. As a result, it is unclear how calpains become activated. We previously demonstrated that μ‐calpain was found enriched in mitochondrial fractions isolated from rat cerebral cortex and SH‐SY5Y neuroblastoma cells (Garcia, et. al. 2005. BBRC 338, 1241–1247), but the submitochondrial localization of μ‐calpain was not determined. In this study, we localized the large catalytic calpain 1 subunit along with the regulatory calpain small subunit 1, which together form μ‐calpain, to the mitochondrial intermembrane space. The results further demonstrate that calpain 1 is imported into mitochondria and contains a mitochondrial targeting sequence in its NH2‐terminus. This targeting sequence is not present in the large subunit of m‐calpain, which is not imported into mitochondria. The localization of μ‐calpain to the mitochondrial intermembrane space is consistent with a role for μ‐calpain in the cleavage of intermembrane space proteins including apoptosis inducing factor, providing a potential mechanism of mitochondrial μ‐calpain activation contributing to caspase independent cell death.

This work was supported by the NIH, USPHS grants; P01NS058484, P01AG010836, R01NS045726, T32DA022738 and by the Kentucky Spinal Cord and Head Injury Research Trust.
Functional Import of Exogenous Mature Cytochrome c into Mitochondria of Deficient Cells
Authors: Levy Richard J , Huang Zhishan , Edwards John G , Gao Qun , Wolin Michael S
Published: June 2021

Cytochrome c (cyt c), the electron carrier between Complexes III and IV, is required for oxidative phosphorylation. It is believed that only the immature form of the protein is imported into mitochondria. Here we test the hypothesis that exogenous mature cyt c can be functionally imported. Cyt c null cells (cyt c −/−) and control fibroblasts (NIH/3T3) were exposed to cyt c, acetylated cyt c, or buffer for 2 hours. Immunoblot analysis demonstrated markedly increased cyt c levels in mitochondria of cyt c exposed null and 3T3 cells. Following exposure to FITC‐conjugated cyt c, green punctate fluorescence was readily visualized in both cell types and co‐localized with MitoTracker Red. Using electron microscopy, gold particles were clearly seen within mitochondria and endosomes of null and 3T3 cells following exposure to either gold‐conjugated cyt c or acetylated cyt c. Cyt c‐dependent respiration was significantly decreased in null cells exposed to buffer or acetylated cyt c and increased following exposure to exogenous cyt c approaching 3T3 rates. The percentage of TUNEL positive nuclei was similar between groups. Exogenous cyt c readily entered the intact cell via endocytosis and was imported into mitochondria without initiating apoptosis. This import increased null cell cyt c‐dependent respiration. This may have important implications for the treatment of acquired and inherited mitochondrial disorders.
Integration of physiopathogenomics to identify progression of illness & stage‐appropriate therapeutic targets in a model of staphylococcal enterotoxininduced lethal shock.
Authors: Peyrefitte Christophe N , Hammamieh Rasha , Bi Shuguang , Jett Marti
Published: June 2021

The onset of irreversible shock upon exposure to certain pathogens can progress rapidly from onset of flu‐like symptoms to near‐untreatable illness. Our approach has been to characterize host responses, an approach that offers other benefits that include assessment of “progression of illness”. This can take into account not only time post‐exposure, but also exposure level and susceptibility due to personal factors. Our studies have characterized host gene expression responses to biological threat pathogenic agents as well as to common influenza and other flu‐like pathogens in order to gain an understanding of host responses that lead to irreversible, critical illness. These studies integrate the clinical pathophysiological findings with host‐omics, from 1–72 h post‐exposure, developing algorithms to correlate these massive datasets with the progression of illness and identification of early indicators of impending vascular leakage and eventual collapse. Definition of the biosignatures at various stages during the progression to lethality have revealed stage‐appropriate therapeutic targets have the potentialo to be used for intervention strategies.
Enrichment of Phosphopeptides using Phos SpinTrap Fe, a new IMAC kit
Authors: Albenius Marianne , Risberg Gabriella , Salomonsson Inger , Hedlund Helena , Sjödahl Marika
Published: June 2021

There are many different types of post‐translational modifications (PTM) identified till now of which protein phosphorylation is one of the most important in eukaryotic cells and plays an essential role for regulating various cellular processes.

Immobilized Metal Affinity Chromatography (IMAC) is a common technique used for purification and enrichment of phosphopeptides and is based on the affinity of the metal ion for the phosphate group. Phos SpinTrap™ Fe is a new sample preparation kit based on Fe Sepharose™ 6 Fast Flow. The kit is designed for single use small‐scale phosphopeptide enrichment. A mixture of different phosphorylated proteins was spiked into a background of E. coli extract. The protein mixture was cleaved with trypsin and purified on a Phos SpinTrap Fe column. Several phosphopeptides were detected with masspectrometric methods after the enrichment. Conclusion: The Phos SpinTrap Fe kit shows high specificity for phosphopeptides and can be used for enrichment of phosphopeptides from complex samples.
A Fragment of Cartilage Collagen, Chondrostatin, Inhibits Migration of Breast Cancer Cells
Authors: Wang Zhepeng , Bryan Jennifer , Franz Carl , Siegel Adam , Wagenseil Jessica , Mecham Robert , Sandell Linda J
Published: June 2021

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. We have studied the interaction of procollagen type IIB N‐propeptide (chondrostatin) with several tumor cell lines. We found that chondrostatin binds to integrins αvβ3 and αvβ5 via RGD. In order to investigate downstream consequence of chondrostatin‐integrin interaction, we applied live cell imaging technology and phalloidin staining to investigate effects of chondrostatin on breast cancer cell migration and actin cytoskeleton reorganization.

MB231 breast cancer cells were incubated with or without chondrostatin, and visualized on a Leica DMI 6000B scope. Images of 12 pre‐selected microscopic fields were captured every 10 min using a Q Imaging Retiga Exi Fast 1394 camera. The cells were tracked using ImageJ software. The length of track over time was graphed, and the velocity of cell movement was calculated by linear regression analysis. The cell migrating velocity was reduced by 30%.

To begin to investigate the mechanism of the inhibition, the actin stress fiber was examined. Cells were treated with chondrostatin and actin stress fibers were stained with Alexa Fluor 546 Phalloidin and visualized by fluorescence microscopy. Actin fibers accumulated in cytoplasm when cells were incubated with chondrostatin.

These results show that chondrostatin can inhibit MB231 cell migration and suggest that rearrangement of the actin cytoskeleton may be involved. NIH R01 AR36994
The Potential Role of Hydrophobic Residues in Governing Putative Interactions Involving the TM Region of gp41
Authors: Hauser Sarah , Bell Ellis
Published: June 2021

There is a highly conserved sequence in gp41 which forms a transmembrane helix and is conserved in two putative co‐receptors, CD4 and Syndecan. This sequence is comprised of an X‐X‐X‐G‐G‐X‐X‐G‐X‐X‐X motif where X are hydrophobic residues. This region appears to play a critical role in viral entry and syncytium formation. The glycine motif provides a potential binding surface for interaction between gp41 and the similar region of either coreceptor. We hypothesize that the notch of the HIV1 gp41 interacts with equivalent notches in CD4 or Syndecan creating an interaction between the two molecules in the plane of the host cell membrane leading to effective infection by HIV. To determine whether hydrophobic residues surrounding the conserved glycine motif in gp41 CD4, and Syndecan play a role in directing such protein‐protein interactions we used bioinformatic and modeling techniques. Analysis of all known TM regions of gp41, CD4 and Syndecan reveal positional preferences for various hydrophobic residues. The effects of these amino acids and their location in the sequence and their possible effects on gp41‐CD4 or gp41‐Syndecan interactions were modeled through computational mutations and GRAMM docking. These approaches show how each mutation would cause a change in the interaction between gp41 and its receptors, and provided increased insight into understanding this interaction.