bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic interactions of TATA‐Box binding protein with promoters is regulated by GlcNAcylation
Authors: hardiville stephan , Han Guanghui , Ma Junfeng , Hu Ping , Banerjee Partha , Hart Gerald
Published: June 2021

O‐GlcNAcylation is a dynamic posttranslational modification tightly correlated to the concentration of UDP‐GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP), which varies according nutritional factors. Through HBP activity and production of UDP‐GlcNAc, OGT senses nutrient availability and forward the signal to downstream effectors by transferring a GlcNAc moiety to the targeted proteins. In return, the O‐GlcNAcase resets the signal. Interestingly, literature points out a regulation of the basal transcription machinery by O‐GlcNAc. TATA‐box binding protein (TBP) is a key player in regulating transcription since it is required for the activity of all three RNA polymerases. DNA‐bound TBP acts as a dock for recruitment of basal transcription factors and the RNA polymerase II to formation of a functional pre‐initiation complex (PIC). DNA‐bound TBP can also be recognized by NC2 and BTAF1. NC2 stabilizes the chromatin interaction of TBP, whereas the association of TBP with BTAF1 promotes its release. Here, we showed that TBP is O‐GlcNAcylated on its N‐terminus domain at T114, T126 and S158. O‐GlcNAcylated TBP is bound to DNA and the sugar inhibits the interaction with BTAF1. TBP site‐specific O‐GlcNAc mutants have been used to point out the T114 as the main regulator of its interaction. Using STZ‐treated rat models, we showed that TBP/BTAF1 association is decreased in diabetes. Owing to the fact that BTAF1 dynamically regulates the chromatin interaction of TBP, we investigated the dynamic behavior of TBP and TBP O‐GlcNAc by Strip‐FRAP and ChIP assays. Collectively, our results indicate that O‐GlcNAcylation of TBP at T114 regulates TBP/BTAF1 dynamics on gene promoters.
Regulatation of hepatic lipid metabolism by glutaredoxin‐1 and protein glutathione‐adducts
Authors: Shao Di , Han Jingyan , Hou Xiuyun , Fry Jessica , Behring Jessica , Yao Chunxiang , Weng Xiang , Pimentel David , Long Michelle , Roy Hemant , Seta Francesca , Cohen Richard , Matsui Reiko , Bachschmid Markus
Published: June 2021

Objective

Oxidants can contribute to cellular signaling via glutathione‐(GSH) adducts, which are reversible post‐translational modifications of proteins that alter their function. Glutaredoxin‐1 (Glrx) is an enzyme that specifically removes these GSH‐adducts and modulates enzyme activity. We aimed to study the physiological role of Glrx and protein GSH‐adducts in vivo

Methods

Whole body Glrx knockout mice (Glrx‐/‐) were created in a C57BL/6J strain and metabolic alterations were monitored over eight months

Results

Chow fed Glrx‐/‐ mice developed obesity, hepatic steatosis and hyperlipidemia. The key enzyme in hepatic lipid biosynthesis, fatty acid synthase (FASN), was upregulated by 50% in Glrx‐/‐ mice. Liver‐specific adenoviral Glrx reconstitution of Glrx‐/‐ mice down‐regulated hepatic FASN and normalized hepatic and plasma lipid levels. The central metabolic regulator sirtuin‐1 (SirT1), regulates FASN expression. Here, we found that elevated GSH‐adducts on SirT1 in Glrx‐/‐ mice inhibited SirT1 activity leading to increased FASN transcription.

Conclusions

Our data suggest that Glrx regulates SirT1 activity in vivo through GSH‐adducts thereby modulating hepatic and plasma lipids.
A lysosomal targeting defect of V0a1 suppresses V‐ATPase activity elevating lysosomal pH in Ppt1‐/‐ mice: amelioration by NtBuHA
Authors: Bagh Maria , Chandra Goutam , Peng Shiyong , Zhang Zhongjian , Mukherjee Anil
Published: June 2021

Lysosomal pH regulation is impaired in virtually all lysosomal storage disorders (LSDs). However, the mechanism of this defect remains unexplained. The vacuolar H+‐transporting ATPase (v‐ATPase), a multi‐subunit protein‐complex, regulates endosomal/lysosomal pH. The reversible assembly of its membrane‐bound V0 sector and cytosolic V1 sector regulates acidification of the lysosomes. Infantile Neuronal Ceroid Lipofuscinoses (INCL) is a neurodegenerative LSD caused by mutations in the CLN1 gene encoding palmitoyl‐protein thioesterase‐1(PPT1), a depalmitoylating enzyme. Dynamic palmitoylation (palmitoylation‐depalmitoylation) is critical for regulating the functions of many proteins, which requires palmitoyl‐acyltransferases (PATs) for palmitoylation and palmitoyl‐protein thioesterases (PPTs) for depalmitoylation.

Using Ppt1‐/‐ mice that mimic INCL, we uncovered that subunit a1 of V0‐ATPase (V0a1) requires palmitoylation on Cys‐25 for localization to endosomal/lysosomal membrane. V0a1 is transported via clathrin/AP2 pathway from plasma membrane to early endosomes where PPT1‐deficiency suppressed depalmitoylation impairing clathrin/AP2/V0a1 dissociation. This defect prevented interaction of V0a1 with AP3, essential for trafficking to lysosomes and clathrin/AP2/V0a1 was recycled back to the plasma membrane. Consequently, disruption of V0/V1 assembly on lysosomal membrane suppressed V‐ATPase activity, essential for pH regulation. Importantly, a PPT1‐mimetic, NtBuHA, ameliorated these defects. Our findings uncover a novel mechanism underlying increased lysosomal pH in INCL and suggest that varying factors impairing V‐ATPase activity may disrupt lysosomal pH regulation in other LSDs.
Quantifying the Two P6 Populations in Various Strains of Nontypable Haemophilus influenzae
Authors: Shaul Jeffrey , Reulbach Casey , Kisselstein Breanne , Pichichero Michael , Michel Lea
Published: June 2021

Nontypeable Haemophilus influenzae (NTHi) is a pathogenic bacterium that causes acute otitis media (ear infections) and nasopharyngeal infections. There is a commonly used vaccine against the type B form of Hi (Hib), but there is no vaccine to protect against NTHi in the United States. The lipoprotein P6 found on the outer membrane (OM) of NTHi is currently one of the leading protein vaccine candidates for protection against NTHi. However, research from our lab has shown that P6 can be inserted into the OM in two different orientations: attached to the inner leaflet of the OM and facing in toward the periplasm or attached to the outer leaflet of the OM and facing out toward the extracellular space. When P6 is in its external orientation, it can interact with antibodies, making it a suitable target for vaccines. In this study, we used a biotinylation labeling technique and immunoblotting to separate and quantify the external (surface exposed) P6 and internal P6. The results of our study suggest that in the clinical strains of NTHi that we tested, only a minor population of P6 is surface exposed. We consider the possible ramifications of minimal surface exposure on P6′s vaccine candidacy.
Development and characterization of synthetic human antibodies for sensing cellular metabolic states
Authors: Tomic Jelena , McLaughlin Megan , Geyer Ron , Sidhu Sachdev , Moffat Jason
Published: June 2021

Metabolic reprogramming of cancer is reflected in aberrant activity of the hexosamine biosynthetic pathway that produces UDP‐GlcNAc (a donor substrate used to form the O‐GlcNAc modification on proteins). Rather than acting as a switch that turns signaling pathways on or off, O‐GlcNAc should be considered as a “rheostat” that controls the intensity of intracellular signals according to the nutrient and stress conditions. There is evidence that some cell surface proteins contain O‐GlcNAc modification in their extracellular regions. However, there is a lack of understanding for the role of extracellular O‐GlcNAcylation in biological processes.

Tuning the level of O‐GlcNAcylation is one way that tumor cells can adapt to varying nutrient and environmental conditions. We hypothesize that cancer cells can “sense” stress through the O‐GlcNAc pathway and, consequently, reprogram the cell surface and adapt to tougher conditions. Neo‐epitopes on the cell surface that emerge from tuning O‐GlcNAc pathway activity may be prime candidates as readouts that reliably report the status of metabolic reprogramming. These nutrient‐dependent cell surface epitopes may have functional roles in defining cancer cell identity.

In order to advance understanding of metabolic reprogramming from the perspective of cell surface, we combined genetics and advanced protein engineering using phage‐displayed synthetic human antibody libraries to generate binders that respond to changes in the O‐GlcNAc rheostat. Our method for discovery of surface and nutrient‐associated protein sensors (SNAPS) has allowed us to identify multiple antibodies that can detect different cellular metabolic states.
Structural and functional characterization of a maize‐derived hepatitis B surface antigen (HBsAg) for the development of an oral vaccine
Authors: Shah Shweta , Hayden Celine , Howard John , Rao A
Published: June 2021

Hepatitis B is a life‐threatening infection of the human liver. A vaccine against hepatitis B has been available since 1982 but still more than 780,000 people die every year due to hepatitis B. The overall costs associated with vaccines make it difficult for its worldwide administration. In addition, there are certain groups of people who are poor responders to the available vaccines. Both these problems may be overcome by the use of oral vaccine. To this end, HBsAg was produced in maize and orally fed to mice as a boosting dose. The oral vaccine produced a robust immune response both systemically and mucosally, as evidenced by high antibody titers. In order to characterize the active ingredient of the oral vaccine, the maize‐derived HBsAg was purified using a two‐step process. Gel filtration experiments show that maize derived HBsAg exists mostly as a higher order oligomer. TEM analysis shows that the HBsAg oligomer forms virus‐like particles (VLPs). The far UV‐CD spectrum shows minima at 208 nm and 222nm, typical of alpha helical structure. Further, we demonstrate that removal of fat from the maize material by supercritical fluid extraction (SFE) shows higher immunological response compared to full fat (FF) and hexane extracted material. The VLPs obtained from SFE were more regular in shape and size and form particles of ~ 20 nm in size. In contrast, VLPs obtained from FF seed material are of varying size ranging from 20 nm to 100 nm. Further immunological studies are underway but the work presented here advances the path toward the development of a more efficacious oral vaccine.
Cucurbiturils as Inhibitors of Complex Formation between LL‐37 and DNA
Authors: Brooks Celine , Zachary R , Craig Maria
Published: June 2021

In autoimmune diseases such as psoriasis and lupus, the ability of the LL‐37 peptide to form complexes with DNA plays a significant role in disease onset and pathogenesis. Cucurbiturils have the potential to bind to LL‐37 and to disrupt LL‐37/DNA complexes or to serve as sensors of LL‐37, which may be useful in the treatment of these diseases. Cucurbiturils are macrocyclic molecules that have the ability to form host‐guest complexes with a variety of guest molecules, such as amino acids, drugs, and organic compounds. They bind particularly well to guest molecules that contain cationic, aromatic, and hydrophobic regions, and binding of phenylalanine residues inside the glycouril ring have been observed. At physiological pH, LL‐37 has a net positive charge of +6 and four aromatic phenylalanine residues that may allow LL‐37 to form complexes with cucurbiturils. Electrophoretic mobility shift assays (EMSA) and western blotting were used to study the effect of cucurbit[7]uril on the binding affinity of LL‐37 for 24‐bp DNA fragments with CpG sequences and on LL‐37 self‐association.
Assessing Host Immune Response to Dengue Virus Infection at Single‐Cell Resolution
Authors: Sulistijo Endah , Miller‐Jensen Kathryn
Published: June 2021

Dengue virus (DENV) is a mosquito‐borne pathogen, commonly found in tropical and subtropical climates, that results in a broad range of disease pathologies ranging from asymptomatic to dengue fever (DF) to the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Despite reports characterizing elevated levels of inflammatory cytokines in serum and correlating the manifestation of the severe disease symptoms to increased immune responses, attempts to delineate the signaling profile have been hindered by the low‐efficiency, dose‐dependent DENV infection of cultured cells. Understanding the signaling profile may reveal the hallmarks of disease progression and dictate the manifested severity of the infection. In this study, we eliminate the complexity of interpreting averaged signaling profile from heterogeneous population by isolating infected cells in a single‐cell barcode chip (SCBC), a device that enable simultaneous detection of multiple secreted cytokines at the single‐cell level. This strategy will allow us to effectively differentiate DENV‐infected from non‐infected cells, profile the types and levels of cytokines and chemokines secreted by these infected cells, and examine how various immune cells response to DENV infection. This single‐cell signaling profile will provide insight into how DENV infection may perturb the host immune system signaling networks and aid in the identification of therapeutic targets that can modulate DENV infection and suppress progression into DHF/DSS.

Supported by NIH grant U19‐AI089992
Circadian rhythms orchestrate lymph node homing and egress
Authors: Druzd David , de Juan Alba , Kraus Kerstin , Scheiermann Christoph
Published: June 2021

Adaptive immunity is dependent on the immunosurveillance of tissues by lymphocytes and their recirculation between blood, lymph nodes and lymph. Thus far, various molecules have been implicated in this migratory process including chemokines and adhesion molecules that play a critical role in the homing and egress events occurring within lymph nodes. However, how this fundamental process of immunity is being regulated at the organismal level at different times is unknown. Here, we present data demonstrating that robust circadian oscillations, exhibiting a period length of ~24h, occur in cellular numbers in murine lymph nodes, showing a ~2‐fold difference with a trough at the onset of the behavioral rest phase (light) and a peak at the onset of the active period (dark). Oscillations were also observed in the capacity of lymphocytes to home to lymph nodes, peaking at the onset of the activity phase. These oscillations corresponded to rhythmic expression of the adhesion molecules ICAM‐1 and VCAM‐1 as well as the chemokine CCL21 within high endothelial cell venules. We detected similar oscillations in leukocyte counts within efferent lymph, exhibiting a ~3‐fold difference between peak and trough values, with egress peaking at the onset of behavioral activity. Egress could be modulated acutely in a rhythmic manner using the functional sphingosine‐1‐phosphate receptor antagonists FTY720 and SEW2871 indicating circadian sensitivity to these drugs. Taken together, our data provide new insights into the temporal control of adaptive immunity by orchestrating the homing of lymphocytes into lymph nodes and their egress from these sites in a rhythmic manner.

Funded by the Emmy‐Noether‐Program of the German Research Foundation (DFG)
Structural Determinants in Hinge‐Bend Motions of Neurolysin
Authors: Bibel Brianna , Sigman Jeffrey
Published: June 2021

Neurolysin (EC 3.4.24.16; NLN) is a zinc (II) metallopeptidase, in the M3 family of enzymes, along with close relatives such as thimet oligopeptidase (TOP). NLN is believed to play a role in diverse physiological processes through cleavage of neuropeptides, including neurotensin and bradykinin. NLN and similar enzymes are thought to operate via a hinge‐clasp mechanism whereby a substrate enters the active site of the open, inactive form and conformational changes in the hinge region lead the enzyme to adopt a closed, active form. These conformational changes likely provide a mechanism for limiting the length of peptides processed by NLN and TOP. This research will use site‐directed mutagenesis followed by spectroscopic and kinetic assays of the engineered enzymes to examine the importance of residues in the clasp region on the mechanism of substrate selectivity and hydrolysis. To this end we have optimized the expression and purification of the cytosolic isoform of mouse NLN – subcloned into a pET28b(+) vector, overexpressed in E. coli, and purified by Ni‐affinity chromatography. We have created homology models of the open and closed conformations of mouse neurolysin; these models suggest that alpha helices 3 and 6, located on either side of the hinge region, play a role in modulating the stability of the closed, active form of the enzyme. Using our homology models, we have analyzed this region and identified several potential stabilizing interactions, including a salt bridge between GLU135 and LYS189, residues highly conserved in NLN and similar enzymes. Analysis of the effect of mutations in the clasp region on enzyme stability and activity are currently underway.

Grant Funding Source

This research was funded by the Robert W. and Beverly J. Summers Scholarship through Saint Mary's College of California's Summer Research Program.
Bioengineered Styrene Monooxygenase: Elucidating the Impact of the N‐Terminus in the Efficiency and Regulation of the Flavin Transfer Reaction
Authors: Okonkwo Nonye , Gassner Geroge
Published: June 2021

Metabolism of toxic styrene by Pseudomonas putida (S12) bacteria is initiated by a two‐component flavoenzyme system, which consists of the FAD‐specific styrene monooxygenase A (SMOA) and an NADH‐specific styrene monooxygenase B (SMOB), a reductase. Wild‐type SMOB catalyzes the reduction of FAD using a BiBi sequential mechanism and subsequently transfers FAD to the active site of SMOA, which catalyzes the epoxidation of styrene to (S)‐styrene oxide. However, during steady‐state catalysis, we recently discovered that the N‐terminally tagged version, N‐SMOB, employs a double‐displacement mechanism (Kantz, 2005). The efficient delivery of reduced FAD to the SMOA presents a challenge for most two‐component SMO systems and this research aims to further understand the regulation of N‐terminally engineered flavoenzymes.

Here we evaluate the impact of an N‐terminal tag on the catalytic mechanisms of SMO. Fluorescence monitored titrations at 4°C determined the Kd value to be ~ 50 nM, an order of magnitude greater than the wild‐type reductase. The increased FAD binding affinity directly affects the steady‐state kinetics at 30°C, as the double‐displacement mechanism with NADH as the leading substrate becomes the preferred method of FAD reduction. Due to these significant changes in flavin binding affinity and catalytic mechanism, we investigated the pre‐steady state and FAD‐transfer kinetics using stopped‐flow fluorescence and absorbance studies. These findings will be presented together with their implications for the engineering of coupled N‐terminally tagged enzymes and N‐terminally linked fusion proteins as potential biocatalysts for the production of essential chiral epoxides.
Structure and Mechanism of 6‐Hydroxynicotinate 3‐Monooxygenase (NicC)
Authors: Gerwig Tyler , Yuen Meigan , Zhen Weifeng , Anderson Mark , Hicks Katherine , Snider Mark
Published: June 2021

NicC catalyzes the decarboxylation and oxidation of 6‐hydroxynicotinate (6HNA) to 2,5‐dihydroxypyridine (DHP) in the aerobic degradation of niacin to fumarate in both soil and pathogenic bacteria. The crystal structure of the Pseudomonas putida NicC apoenzyme has been solved to 2.2 Å resolution using SAD phasing. Steady‐state kinetic analysis of NicC activity fit the sequential ternary kinetic model, with inhibition profiles by NAD and DHP supporting a random bi bi mechanism. Dependence of kcat and kcat/KM on pH show critical roles of both general base and acid catalysis, with pKa values of 6.3 and 8.0, respectively. NicC catalytic turnover drops 60% upon incubation with either iodoacetamide or MMTS, suggesting a role for cysteine that will be examined further by analyzing the kinetic consequences of a NicC C201A mutation. The high catalytic efficiencies of NicC with 3‐chloro‐4‐hydroxybenzoic acid (kcat/KM = 1.5 x 105 M‐1s‐1), and 5‐chloro‐6‐hydroxynicotinate (1.4 x 105 M‐1s‐1), compared to that with 6HNA (kcat/KM = 5.5 x 104 M‐1s‐1), negates mechanistic proposals requiring tautomerization at C‐6. The 13V/K kinetic isotope effect is inverse (0.9986 ± 0.0002), supporting a change in hybridization at C‐3 from sp2 to sp3 prior to decarboxylation. The observed lack of irreversible inhibition by 6‐hydroxynicotinaldehyde negates mechanistic proposals requiring the covalent addition of cysteine at C‐3, suggesting a mechanism in which the –OH group from flavin hydroperoxide is added prior to decarboxylation.
Engineering New Substrate Specificity into the Active Site of Styrene Monooxygenases
Authors: Truong Phu , Gassner George
Published: June 2021

Styrene Monooxygenases catalyze the FAD‐dependent epoxidation of styrene to (S)‐styrene oxide in the first step of the styrene catabolic and detoxification pathways employed by a range of microorganisms. The robust tolerance of the styrene‐binding pocket to mutagenesis and the tunability of the active site chemistry of SMOs has identified these enzymes a valuable new target for development as biocatalysts with applications in chiral synthesis and bioremediation. In the present study, we systematically evaluate the impact of changing hydrophobic volume in the styrene‐binding pocket of an N‐terminally histidine‐tagged epoxidase (NSMOA) by using the active site‐directed mutants, V211A, V303A, V211I, and V303I. We find the equilibrium midpoint potential of FAD bound to these mutants to be indistinguishable from the wild‐type protein, indicating that the electronic environment of the FAD is not significantly changed by the amino acid substitutions. The kinetics of the epoxidation reaction of a library of substrate analogs were monitored by single‐turnover studies by stopped‐flow fluorescence spectroscopy. Evaluation of the kinetic results by sequential exponential fitting indicates that the substrate specificity of NSMOA (V303A) shifts to favor epoxidation of the sterically‐bulky substrate 4‐vinylbiphenyl over styrene by 1.5 fold. We conclude that active site directed mutations that change the hydrophobic volume in the active site of NSMOA provide a direct means of tuning the substrate specificity without perturbing the electronic properties of the FAD.
Evidence for the Sacrificial Role of the Auxiliary [4Fe‐4S] Cluster of Lipoyl Synthase
Authors: Lanz Nicholas , Pandelia Maria‐Eirini , McLaughlin Martin , Rectenwald Justin , Silakov Alexey , Drennan Catherine , Krebs Carsten , Booker Squire
Published: June 2021

Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, the insertion of two sulfur atoms at C6 and C8 of an n‐octanoyl chain on a lipoyl carrier protein (LCP). LipA contains two [4Fe‐4S] clusters, one of which is required to reductively cleave S‐adenosylmethionine to generate two 5′‐deoxyadenosyl 5′‐radical intermediates that abstract hydrogen atoms from C6 and C8 of the octanoyl chain, while the second cluster has been hypothesized to donate the inserted sulfur atoms. Thus, a single turnover inactivates LipA due to destruction of its second [4Fe‐4S] cluster. Herein, we provide compelling evidence for this controversial role for a [4Fe‐4S] cluster in enzymatic catalysis. Reactions conducted under conditions wherein only one sulfur atom is inserted into the organic substrate results in the LCP substrate becoming cross‐linked to LipA through the Fe/S cluster. Extensive characterization of the cross‐linked species by analytical techniques, Mössbauer spectroscopy, and X‐ray crystallography shows that it contains 3 iron and 3 sulfide ions in a cubane‐like structure that is connected to the LCP by a bridging monothiolated octanoyl linker. HYSCORE spectroscopy was used to demonstrate a direct interaction between a substrate radical, generated using a conjugated substrate analogue, and an iron atom of an Fe/S cluster of LipA.
Production of Bacterial Cellulose, Cloning and Characterization of Novel β‐glucosidases from Gluconacetobacter xylinum
Authors: Auta Richard , Loh Joshua , Radecka Iza , Hooley Paul
Published: June 2021

Bacterial cellulose (BC) is produced by bacteria has a unique structural and mechanical properties and is highly pure as compared to plant cellulose. In this study, BC was produced using Hestrin‐Schramm (HS) media. Two forms of cellulose were observed; a gel‐like membrane and a leathery membrane. Fourier Transform infrared spectroscopy (FTIR) investigation revealed both forms to be cellulose and indicated the presence of CH2 and OH group at the absorption wavelength of 3300 cm‐1, 1650 cm‐1 and 1025 cm‐1. Scanning Electron Microscopy (SEM) results showed fine morphology of the cellulose matrix and fibre; and XRD indicated the majority of the cellulose to be type‐1 cellulose (crystalline in nature). Plasmid DNA sequence analysis revealed an open reading frame of 678 bp. The significance of this work is discussed.
Elucidating the Nicotinic Acid Degradation Pathway in Bacillus niacini
Authors: Daniel Abigail , Snider Mark
Published: June 2021

As byproducts of oil, tar and wood processing, and common elements of herbicides, pesticides and pharmaceuticals, N‐heteroaromatic compounds are pervasive in the environment and in many cases pose toxicological threats. Bacteria have evolved to degrade this class of compound and the pathway for nicotinic acid degradation serves as a model for better understanding the mechanistic principles involved. Recently a novel gene cluster hypothesized to be responsible for the aerobic catabolism of nicotinic acid by the common soil bacterium, Bacillus niacini, was predicted. The focus of this current study is to experimentally examine that hypothesis. Of the first seven genes in cluster, six have been cloned, and the resulting protein products have been expressed and purified to homogeneity. Further, kinetic analyses of the resulting enzymes will be performed using HPLC and tandem LC‐MS/MS to determine the chemical structures of the degradation intermediates and to properly annotate the function of the genes in the cluster. This work is funded by Clare Boothe Luce and Henry J. Copeland funds for student research and Independent Study at the College of Wooster.
Identification of an Intermediate Methyl Carrier and the Stereochemical Outcomes of H‐atom Abstraction and Methylthiolation by the Radical SAM Enzyme RimO
Authors: Landgraf Bradley , Lee Kyung‐Hoon , Booker Squire
Published: June 2021

RimO is a radical S‐adenosylmethionine (RS) enzyme that catalyzes the insertion of a methylthio group (‐SCH3) at C3 of a conserved aspartyl residue of protein S12, a component of the 30S subunit of the bacterial ribosome. RimO contains a [4Fe‐4S] cluster to which S‐adenosylmethionine (SAM) binds, allowing for its reductive cleavage to methionine and a 5′‐deoxyadenosyl 5′‐radical. Unlike most RS enzymes, methylthiotransferases (MTTases) contain an additional [4Fe–4S] cluster that is believed to be the source of the sulfur atom of the inserted –SCH3 group. It was believed that the sequence of MTTase reactions involved initial sulfur insertion into the substrate followed by capping with a SAM‐derived methyl group. We show that RimO from Thermotoga maritima catalyzes the synthesis and subsequent insertion of a ‐SCH3 group by mediating methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of its substrate and the requisite reductant that triggers radical chemistry. Consistent with the assignment of the acceptor as an Fe‐S cluster (presumably the auxiliary [4Fe–4S] cluster), denaturation of the SAM‐treated protein with acid results in production of methanethiol (CH3SH). We show chemical competence of the methylated intermediate by using unlabeled and [methyl‐d3] SAM and demonstrate that RimO catalyzes multiple turnovers when CH3SH is supplied exogenously. Using deuterated isotopologs at C3 of aspartate and 1H NOESY NMR, we determined the stereospecificity of H‐atom abstraction and the absolute configuration of the chiral center created upon ‐SCH3 insertion by RimO.
The Reduction Effect of Low Molecular Weight Chitosan Oligosaccharide (GO2KA1) on Postprandial Blood Glucose Levels in Healthy Individuals
Authors: Kang Yu‐Ri , Jo Sung‐Hoon , Lee Se‐Woon , Kim Seong‐Cheol , Apostolidis Emmanouil , Kim Young‐Cheul , Min Hye‐Sun , Lee Mee‐Sook , Kwon Young‐In
Published: June 2021

We investigated the effects of chitosan oligosaccharide (GO2KA1, a‐glucosidase inhibitor) on the level of postprandial blood glucose in adult subjects with normal blood glucose level. Postprandial blood glucose levels were tested for 2 hr after sucrose administration with or without 500 mg of GO2KA1. GO2KA1 significantly lowered the mean, maximum, and minimum level of postprandial blood glucose at 30 and 90 min after meal. GO2KA1 treatment reduced the blood glucose‐time curve (AUC) and the blood glucose peak (Cmax) value while the time of peak plasma concentration of glucose (Tmax) value was significantly increased (p<0.05). Our findings suggest that GO2KA1 reduces postprandial blood glucose rise due to slow absorption of glucose in the small intestine, via carbohydrate hydrolyzing enzymes inhibition.
Size Separation of Large Proteins and Protein Complexes
Authors: Liljedahl Pia , Salomonsson Inger , Graner Therese
Published: June 2021

The elucidation of the biological function of protein complexes is an area of intense research efforts but isolation and analysis of the complexes is challenging as they can be very large and unstable. Superose™ 6 Increase is a new agarose‐based SEC (size exclusion chromatography) medium with a wide separation range, Mr 5000 to 5 000 000 for globular proteins. The wide separation range allows separation of very large proteins, complexes and membrane proteins both for preparative and analytical purposes under mild, non‐dissociative conditions.

Separation of a protein involved in structural coloration on Superdex 200 10/300 resulted in co‐elution of two differently sized protein populations. The wider separation range of Superose 6 Increase 10/300 enabled separation of the two populations. Based on elution position, the size of the separated material was estimated to Mr > 700 000

Purification of membrane proteins often require addition of expensive detergents in the buffer to keep the proteins stable and thus low consumption of buffer is important. Purification of a membrane protein complex on Superose 6 Increase 5/150 (3 ml bed volume) resulted in good separation from aggregated material and degradation products.
Substrate Specifity and Quaternary Structure of a Novel Class of Tryptophan Synthases
Authors: Busch Florian , Sterner Reinhard
Published: June 2021

The analysis of numerous genome sequences uncovered that the TrpB enzyme family IPR023026 is divided into two subfamilies named TrpB1 and TrpB2. The widely distributed TrpB1 enzymes are part of the canonical abba tryptophan synthase complex. They synthesize tryptophan by the condensation of L‐serine with indole, which is provided by the associated TrpA enzyme. TrpB1 and TrpA form a permanent complex. We characterized several TrpB2 enzymes and found that they differ in several respects from TrpB1. First, they synthesize tryptophan by the condensation of O‐phospho‐L‐serine with indole. Second, complex formation between TrpB2 and TrpA is transient and ligand‐dependent. Third, the quaternary structure of TrpB2‐TrpA strikingly differs from those of TrpB1‐TrpA complexes. In summary, our results unravel significant differences between two closely related enzyme subfamilies regarding substrate recognition/specificity and hetero‐oligomerization.

Ehrmann, A., Richter, K., Busch, F., Reimann, J., Albers, S.‐V. & Sterner, R. (2010). Ligand‐induced formation of a transient tryptophan synthase complex with αββ subunit stoichiometry. Biochemistry 49, 10842‐10853

Busch, F., Rajendran, C., Mayans, O. Löffler, P., Merkl, R. & Sterner, R. (2014). TrpB2 enzymes are O‐phospho‐L‐serine dependent tryptophan synthases. Biochemistry53, 6078‐6083.
Structural‐Function Mechanism of Vibrio cholerae Cytolysin: a b‐barrel Pore‐Forming Toxin
Authors: Rai Anand , Chattopadhyay Kausik
Published: June 2021

b‐barrel pore‐forming toxins (b‐PFTs) represent an important class of proteins that disrupt membrane permeability of the target cells by forming transmembrane pores. Vibrio cholerae cytolysin (VCC) is a prominent b‐PFT that forms heptameric transmembrane pores in the target eukaryotic cells. Our studies have shown that the membrane binding and pore formation by VCC is governed by multiple regulatory mechanisms. Interaction of VCC with the cell surface glycan(s) appears to modulate the efficacy of the process. Physicochemical conditions like pH also appear to regulate interactions of the toxin with the membranes. It is also evident that VCC may employ distinct structural motifs to mediate interactions with the membrane lipid components. Our study has also explored the sequence of events for the functional pore formation by VCC. We have identified the key amino acid residue(s) in VCC that are crucial to initiate membrane oligomerization. Modification of such residues traps the protein in its membrane‐bound monomeric state that further abrogates oligomerization, membrane insertion and functional transmembrane pore formation. Overall our study provides novel insight regarding the mechanistic aspects of VCC pore‐formation mechanism and also extends our knowledge regarding the structure‐function mechanism of the b‐PFTs family proteins.

This work was supported by The Department of Biotechnology, India (grant no. BT/PR13350/BRB/10/751/2009), and by the Indian Institute of Science Education and Research Mohali, India.
Acute Stimulation of GLUT1 Cell Surface Localization in Blood‐Brain Barrier Endothelial Cells
Authors: Ojelabi Ogooluwa , Carruthers Anthony
Published: June 2021

The human brain relies on constant uptake of glucose from the blood stream for its energy needs. However, the blood‐brain barrier (BBB) prevents simple diffusion of glucose and other substrates from circulation into the brain interstitial fluid. Thus, glucose must be transported across the BBB endothelial cells (ECs) by the glucose transporter, GLUT1. Misregulation of BBB GLUT1 expression or function results in severe cognitive and developmental disorders. Until recently, BBB GLUT1 was believed to be predominantly at the cell surface, and that increased GLUT1‐mediated glucose transport was simply achieved by incorporation of newly synthesized GLUT1 into the plasma membrane. However, recent data from our lab suggest that AMP kinase (AMPK) controls GLUT1 localization between intracellular and cell surface membranes. To understand how AMPK regulate GLUT1 trafficking in BBB ECs, we are using a reversible cell surface biotinylation technique to study GLUT1 kinetics in the presence or absence of AMPK activation. From our preliminary data in basal, murine brain endothelial (bEnd.3) cells we obtain a first order rate constant of exocytosis (kex) and endocytosis (ken) of 0.091min‐1 and 0.436min‐1 respectively for GLUT1 trafficking. Next, we will determine if AMPK stimulates GLUT1 kex or inhibits GLUT1 ken. This will aid in identifying downstream signaling partners in AMPK‐regulated GLUT1 trafficking. Finally, we will test whether signals that couple neuronal activation to increased BBB glucose transport, act via the AMPK pathway. Findings from this study will provide fundamental insights into cerebral glucose transporter misregulation in diseases and may reveal mechanisms for pharmacologic manipulation of the BBB permeability.
Structure of an H+‐coupled, Substrate‐bound MATE Transporter Yields Mechanistic Insights into Multidrug Extrusion
Authors: Lu Min , Radchenko Martha , Symersky Jindrich , Nie Rongxin , Guo Yi
Published: June 2021

Multidrug and toxic compound extrusion (MATE) proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar drugs to the influx of either Na+ or H+. The ~900 MATE transporters identified thus far can be classified into the NorM, DinF (DNA‐damage‐inducible protein F) and eukaryotic subfamilies based on amino‐acid sequence similarity. Structures of Na+‐coupled, extracellular‐facing NorM transporters had been determined, which revealed twelve membrane‐spanning segments that are related by a quasi‐twofold rotational symmetry as well as a multidrug‐binding cavity situated near the membrane‐periplasm interface. Here we report the crystal structures of an H+‐coupled MATE transporter from the DinF subfamily, with and without substrate, unveiling a surprisingly asymmetric arrangement of twelve transmembrane helices and a largely hydrophobic multidrug‐binding chamber located in the middle of the lipid bilayer. Combining structural and biochemical analyses, we confirmed the biological relevance of the substrate‐binding site and suggested a direct competition between H+ and substrate during DinF‐mediated drug transport. Our findings provided fundamental brushstrokes to the molecular picture depicting how a MATE transporter works and laid the groundwork for future experimental efforts aimed at overcoming multidrug resistance.
Determining The Topology of The Membrane Bound O‐acyltransferase Porcupine
Authors: Sanchez Luis , Fraley Gabriel , Bueno Raymund , Galli Lisa , Burrus Laura
Published: June 2021

Porcupine (PORCN), a membrane bound O‐acyltransferase (MBOAT) of the endoplasmic reticulum (ER), is essential for Wnt palmitoylation, transport, secretion, and activity. Because all human Wnts require PORCN for their activity, the inhibition of PORCN has become an attractive therapeutic target for the treatment of various cancers displaying an overexpression of Wnt ligands. In order to design and understand how potential PORCN inhibitors work, the topology of PORCN must be determined. Although various bioinformatics algorithms offer numerous predictions, no clear membrane topology of PORCN has emerged. Therefore, we aimed to experimentally establish the membrane topology of PORCN. Preliminary data orient the N and C terminus toward the ER lumen and cytoplasm, respectively. Additional experiments in which consensus glycosylation sites were introduced into the PORCN sequence suggest that the loop containing A134 is localized to the ER lumen. Therefore, we hypothesized that the A134 containing loop is entirely localized to the ER lumen. To test this hypothesis, we expressed two PORCN constructs with FLAG tags inserted at residues 118 and 145 respectively, and a myc tag at the C‐terminus, in COS‐7 cells. Contrary to our hypothesis, immunostaining of COS‐7 cells selectively permeabilized with Triton X‐100 or digitonin revealed that the FLAG tag on both variants was localized to the cytoplasm. These apparently contradictory data suggest that PORCN may exist in multiple conformations. Together, our findings provide structural information about PORCN that can help in the design and understanding of PORCN inhibitors that could be used for the treatment of Wnt driven cancers

This research was made possible by a grant received from the National Institute of Health (NIH).
Impact Various Pcp1p Mutations Have on Mitochondrial Genome Maintenance and Morphology
Authors: Both Andries , Huddleston Mary , Gordon Donna
Published: June 2021

Pcp1p is a transmembrane protease in the mitochondrial inner membrane of Saccharomyces cerevisiae. Mutations in PCP1 negatively impact cellular respiration due to defects in mitochondrial morphology and loss of mitochondrial DNA (mtDNA) that arise as a result of the improper processing of the Pcp1p substrate, Mgm1p. To understand the structural basis of Pcp1p activity, a mutagenesis screen was carried out and temperature sensitive PCP1 alleles selected based on their varied ability to utilize nonfermentable carbon (E/G). Defects in mitochondrial morphology for each of the mutants were assessed by fluorescence microscopy using mtGFP. In addition to an increase in ‘fragmented’ mitochondria, a number of the mutants had GFP targeted to the cytosol, suggesting defects in mitochondrial membrane potential. TEM will be carried out to better characterize the mitochondria ultrastructure for each of these mutants. Poor or absent growth on E/G suggests that these mutant pcp1 alleles are likely to have acquired mtDNA mutations or have lost their entire mitochondrial genome. To assay the extent of mtDNA loss, quantitative PCR was carried out to determine the relative mitochondria to nuclear genome ratio using ACT1, COX1, and COX3 primers. Strains that grew similarly to PCP1 had relative mitochondria to nuclear ratios of 0.8 to 1, while mutants that were unable to grow on E/G had normalized ratios that ranged from 0 to 0.3. A number of the pcp1 alleles had relative ratios that fell between 0.5 and 0.8, which correlates with biochemical data that suggests partial peptidase activity. Quantitative differences in mitochondrial genome content will be confirmed by DAPI. The data provides insight into the role different amino acids play in influencing Pcp1p peptidase activity.
Phosphatidylinositol 4,5‐bisphosphate Activates the Cell Adhesion Molecule CD44 and Assembles a CD44‐Ezrin Heterotetramer Complex as Revealed by Small Angle neutron Scattering
Authors: Bu Zimei
Published: June 2021

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell‐cell and cell‐matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co‐localizes with the membrane‐cytoskeleton adapter protein Ezrin, which links the CD44 assembled receptor signaling complexes to the cytoskeletal actin and organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered and adopts an auto‐inhibited conformation, which prevents CD44ct from binding directly to activated Ezrin in solution. Binding to the signaling lipid phosphatidylinositol 4,5‐biphosphlate (PIP2) disrupts autoinhibition in CD44ct, and activates CD44ct to associate with Ezrin. Further, using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific hetero‐tetramer complex of CD44ct with Ezrin. This study reveals a novel auto‐regulation mechanism in the cytoplasmic tail of CD44 and the role of PIP2 in mediating the assembly of multimeric CD44ct‐Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of multimeric PIP2‐CD44‐Ezrin complexes.
Investigation of Cytoskeletal Proteins from Tetrahymena thermophila
Authors: Sterner Robert , Honts Jerry , Kilpatrick Adina
Published: June 2021

Over time, organisms have taken different paths to complexity. Although only a single eukaryotic cell, Tetrahymena thermophila derives complexity from its remarkably complex cell surface architecture. Proteomic analysis from our earlier experiments has led to the identification of nearly four dozen proteins isolated from the cortical cytoskeleton. A high molecular coiled coil structural protein (Epc1), an EF‐hand calcium binding protein (Tcb2), and a protein kinase (Epk1) have been investigated. These proteins are believed to have crucial regulatory and structural functions in addition to playing a role in calcium‐dependent regulation of ciliary motion. We are investigating the interactions of these proteins during cell division through confocal microcopy, fluorescence spectroscopy, and NMR solution studies. It was evident through the utilization of confocal microscopy that these proteins were found to localize to a narrow layer of the cortical cytoskeleton called the epiplasm, which was consistent with previous purification experiments. Fluorescence spectroscopy was used in order to monitor conformational changes in the C‐terminal domain of Tcb2, and we identified the presence of one high affinity calcium‐binding site with a low micromolar dissociation constant. The shift peaks observed during (1)H‐(15)N HSQC‐NMR monitored calcium titrations of the Tcb2 C‐terminal domain corroborated evidence of calcium‐dependent conformational changes in this domain. NMR spectra also illustrated that in both the presence and absence of calcium this domain is well folded and upon calcium addition undergoes dramatic conformational change. In the future, we will explore protein distributions relative to one another through FRET and double‐labeling experiments.
11β‐HSD1 Mediates Insulin Resistance and Type 2 Diabetes Mellitus through JNK Activation
Authors: Peng Kesong , Liang Guang
Published: June 2021

Background

Obesity‐induced insulin resistance is the major determinant of metabolic syndrome, which precedes the development of type 2 diabetes mellitus.11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is considered a potential therapeutic target in the treatment of type 2 diabetes mellitus, while the potential mechanism is not very clearly identified.

Methods

In vitro cultured 3T3‐L1 pre‐adipocytes, prednisone and over‐expressed 11β‐HSD1 were used to induce insulin resistance. Type 2 diabetes mellitus mouse models were treated with 11β‐HSD1 inhibitor PF00195715 and JNK inhibitor C66 by gavage, respectively.

Results

Stimulation of prednisone or over‐expressed 11β‐HSD1 significantly disturbed insulin signal pathway, decreased AKT phosphorylation and led to insulin resistance, while these effects were reversed by PF00195715 and C66. Either C66 or DN‐JNK significantly attenuated the 11β‐HSD1 over‐expression induced insulin resistance, indicating a mediated role of JNK. Oral administration with either PF00195715 or C66 in mouse models remarkably relieved insulin resistance and type 2 diabetes mellitus.

Conclusion

11β‐HSD1 mediates insulin resistance and type 2 diabetes mellitus through JNK activation.
Chronic Sweetener Intake and Oxidative Stress in The Central Nervous System
Authors: Rebolledo Zhenia , Contreras Irazu , Estrada Jose
Published: June 2021

Introduction

Diseases such as diabetes, obesity and metabolic syndrome are characterized by chronic low‐grade inflammation and increased oxidative stress, which causes changes damage to several tissues, including the central nervous system. Sweeteners are an alternative to sucrose for the treatment of these pathologies, since they provide flavor without the energy content; however, the use of these compounds has been extended to the general population, since they are included in a variety of products for human consumption. Despite being considered safe, there is evidence of significant metabolic alterations after chronic use of large quantities of these substances.

Objective

To determine the association between constant intake of commercial sucralose or stevia with an increased production of reactive oxygen species in the central nervous system of mice.

Method

4 groups of 6 female 8 week‐old BALB/c mice were supplemented with either sucralose, stevia or saccharose in their drinking water, or left unsupplemented (control), for 6 weeks. Subsequently, mice were sacrificed by anesthetic overdose and the nervous tissue was obtained. ROS production in the tissue was determined by the TBARS method.

Results

The highest production of ROS was observed in the control group (average concentration of MDA 186 mmol/mL). The sucrose (124.11 mmol/mL), sucralose (113 mmol/mL) and stevia (112.3 mmol/mL) samples all had lower ROS concentrations than the unsupplemented control.

Conclusions

In comparison with the sucrose‐supplemented and control groups, supplementation with either sucralose or stevia appear to promote decreased concentrations of ROS in the central nervous system, with the stevia group showing the lowest production.