bluuebunny/crossref_metadata_2025_split huggingface dataset:
BNG 3‐36 as a slow‐binding, competitive inhibitor against HDAC4 (768.19)
Authors: Mueller Dustin , Yu Junru , Omlid Joseph , Singh Raushan , Leedahl Travis , Balasubranian Narayanganesh , Cook Gregory , Srivastava D.K. 
Published: June 2021

Histone deacetylase 4 (HDAC4) is a high priority drug target for the treatment of various human diseases including Huntington’s disease and various cancers. Pan‐HDAC inhibitors, such as Suberoylanilide Hydroxamic Acid (SAHA) have already been screened and approved by the FDA as a treatment for some forms of cancer. BNG 3‐36 is a novel, thiol based compound that binds to HDAC4 utilizing a unique slow‐binding feature, inhibiting the catalytic mechanism of the enzyme. When added to HDAC8, however, this particular inhibitor shows normal competitive features. When the inhibitor is added to HDAC8, a member of another class of HDACs, the slow‐binding feature is not seen and instead is a normal competitive. We show that the inhibitor has a slow‐binding competitive feature when used with HDAC4 and has a normal competitive feature when used with HDAC8.Grant Funding Source: Supported by the NIH
On the forces stabilizing supertertiary structure in multi‐domain proteins (770.2)
Authors: Lemmon Eric , Shanbhogue Prajna , McCann James , Bowen Mark 
Published: June 2021

Many proteins are modular in that they contain a series of independently‐folded domains. Often, these sub‐domains have no affinity for each other in solution, but can maintain a stable supertertiary structure when connected by polypeptide linkers. Little is known about the ultraweak interactions that dictate the stability of supertertiary structure. It is our hypothesis that surface features select for a specific structure, while the linker length determines the effective concentration thereby setting the interaction energy. To test this we attempted to redesign the multi‐domain scaffold protein PSD‐95. Structural studies have observed a stable supertertiary structure for the two N‐terminal PDZ domains from PSD‐95, but differing structural models were unable to identify interdomain interactions. We added a new interdomain salt bridge and linker extensions of varying rigidity. These manipulations rearranged the supertertiary structure but did not lead to isotropic motion of domains. This suggests that the forces stabilizing the structure are sufficient to withstand a 1.5‐fold decrease in effective domain concentration.Grant Funding Source: Supported by NIMH Grant MH081923
Role of substrate loop residues in catalytic loop motion and substrate binding in human glutathione synthetase (773.2)
Authors: Ingle Brandall , Shrestha Bisesh , Anderson Mary , Cundari Thomas 
Published: June 2021

Human glutathione synthetase (hGS) catalyzes the second step in the synthesis of the key antioxidant glutathione. The homodimeric enzyme displays negative cooperativity towards the ɣ‐glutamylcysteine substrate. The presented work probes the significance of residues within the substrate binding loop of hGS responsible for loop motion throughout the catalytic cycle and provides insight into the precise mechanism of communication between active sites. Molecular dynamics simulations and docking studies elucidate the interactions that mediate binding. Experimental point mutations, coupled activity assays, kinetic studies, and differential scanning calorimetry provide information on the role of these residues in activity, cooperativity and thermal stability. A deeper understanding of the residues responsible for loop motion within the substrate binding loop of hGS shows that interactions at the active site have significant impact on the structure and function of the enzyme.Grant Funding Source: Supported by NIH R15GM086833, Research Enhancement Program Grant (TWU), UNT Faculty Research Grant
Role of human glutathione synthetase H‐loop residues in substrate binding and activity (773.4)
Authors: Feyen Jessica , Ingle Brandall , Berthold Meghan , Davis Myra , Sandoval Esmeralda , Hinojos Caleb , Crutchfield Amanda , Cundari Thomas , Anderson Mary 
Published: June 2021

Glutathione (GSH) is an antioxidant essential for mammalian cell survival and is found in every cell of almost all organisms. GSH cannot be taken up by the cell, so understanding the intercellular synthesis of this important antioxidant is crucial. GSH is synthesized in a two‐step process. In the second step, human glutathione synthetase (hGS) binds γ‐glutamylcysteine and glycine and then uses the energy of ATP to make GSH. hGS is a homodimer that displays negative cooperativity, toward the γ‐glutamylcysteine substrate, a phenomenon which contributes to metabolic regulation but is not yet fully understood. The H‐loop (147‐TISASF‐152) of hGS is highly conserved and located near the binding site of γ‐glutamylcysteine. The role of H‐loop residues was examined using site‐directed mutagenesis of H‐loop residues, followed by purification and kinetic characterization of H‐loop mutant hGS enzymes. Our results show that the H‐loop mutant hGS enzymes have altered binding affinity (Km) for the negatively cooperative substrate, γ‐glutamylcysteine.Grant Funding Source: Supported by NIH R15GM086833 (MEA), REP Grant (TWU, MEA), Welch Chem Dept Grant (TWU)
The biochemical characterization of the PDB entry 4EZI by in silico and in vitro quantitative techniques (777.2)
Authors: Hart Kaitlin , Dodge Greg , Horn Alison , Craig Paul , Bernstein Herbert 
Published: June 2021

This study is part of the SBEVSL project, the purpose of which is to propose and confirm the function of uncharacterized protein structures, in the Protein Data Bank, by use of in silico and in vitro quantitative techniques. For over 3000 structures in the PDB, no function has been assigned. These structures were first screened with the PyMOL molecular graphics system, along with ProMOL plugin, which compares query structures against a library of enzyme active sites. The most promising alignments were further refined by sequence and structure comparison using BLAST and Dali. A structure of considerable interest is presented here: 4EZI. When 4EZI was queried with PyMOL/ ProMOL, the best alignment was with the dipeptidyl peptidase IV, 1ORV. After analysis with BLAST and Dali, the alignments revealed 4EZI matched closely to the esterase family, specifically 3H2H. This match was confirmed experimentally. The protein was purified using methods of transformation and column chromatography. Then, the predicted molecular weight was confirmed using SDS‐PAGE. Protein concentrations were determined by a BCA assay and enzymatic activity was confirmed using p‐nitrophenol esters of short‐chain carboxylic acids. In conclusion, 4EZI has been characterized as an esterase based both on in silico and in vitro quantitative methods. Funding was provided by RIT, Dowling College, and NIGMS 2R15GM078077‐02 &amp; 3R15GM078077‐02S1.Grant Funding Source: Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1
Stop activity and preserve the molecular integrity of tissue samples by heat stabilization (779.1)
Authors: Göransson Charlotta , Söderquist Marcus , Borén Mats , Sköld Karl 
Published: June 2021

The action of proteolytic and other protein‐modifying enzymes rapidly change the composition of the proteome and post translational modifications (PTM) after sampling. Subsequent analytical results reflect a mix of the in vivo molecular status and degradation products and display increased inter‐sample variation. Effective enzyme inactivation and standardization of sample handling eliminate this problem.A heat‐stabilization system has been used to generate rapid, homogenous thermal denaturation of enzymes to stop degradation in tissues. Comparisons were made to snap‐freezing and inhibitors, and in time study manner, compared with different post‐mortem intervals. Using mass spectrometry, Western blot, RPPA and activity assays, the protein and peptide content, including PTM’s were examined.The results show rapid changes in phospho‐states on a variety of different proteins detected only minutes after excision whereas after heat‐stabilization, phospho‐levels remain unchanged during 2 hours in room temperature. In three minutes post‐mortem both proteins and endogenous peptides/neuropeptides, including PTM’s, are subjected to substantial degradation. On the contrary, amounts and identities of the detected proteins/peptides in heat‐stabilized samples show maintained integrity. Similarily, levels of pCREB, pGSK3β and pERK1/2 were unchanged for 2 hours, whereas snap‐frozen samples showed a dramatic decrease in levels after 10 min in room temperature.Post‐mortem changes may distort our view of in vivo protein expression. Adequate suppression of both phosphatases and kinases is important for phospho‐state analysis. Heat‐stabilization stops activity thereby enables sample analysis to reflect the in vivo status as closely as possible. This approach may be of great help in research on neurodegenerative disease such as Alzheimer and Parkinson to differentiate true biomarkers from those proteins found in any situation where cells are under stress.
LYCAT, an acyltransferase required for 1‐stearoyl esterification of phosphoinositides, plays a critical role in receptor trafficking dynamics (782.1)
Authors: Botelho Roberto , Bone Leslie , Antonescu Costin 
Published: June 2021

Phosphoinositides (PtdInsPs) are important signaling lipids that regulate many cellular processes, including membrane trafficking and cell proliferation. There are seven interconvertible species of PtdInsPs based on the inositol headgroup phosphorylation. However, there are additional molecular species of PtdInsPs that differ by their acyl chain type, though the predominant species consists of 1‐stearoyl‐2‐arachidonoyl (18:0/20:4). To date, very little is known about the functional relevance of the various acyl‐dependent PtdInsP species. LYCAT is an acyltransferase that appears responsible for inserting stearoyl at sn‐1 of PtdInsPs. Using siRNA‐dependent gene silencing of LYCAT in cultured cells, we have begun to elucidate the role of LYCAT in membrane trafficking and signaling. We present evidence that LYCAT silencing profoundly alters the trafficking of transferrin and its receptor. Specifically, LYCAT‐silenced cells exhibit reduced transferrin uptake, abated cell surface levels of transferrin receptor and dispersal of the receptor away from a perinuclear distribution. Our data suggest that the above defects are due to a fraction of the transferrin receptor population becoming trapped in a non‐cycling compartment. Thus, LYCAT seems essential for proper receptor sorting and trafficking, likely by controlling the levels of 1‐stearoyl‐containing PtdInsPs.Grant Funding Source: Supported by Canadian Institutes for Health Research
microRNA define the glycocode: identification of critical glycogenes that shape the cell (788.6)
Authors: Kurcon Tomasz , Agrawal Praveen , Koppolu Sujeethraj , Pilobello Kanoelani , Mahal Lara 
Published: June 2021

Cell surface glycans create an intricate source of biological information which is dynamically altered by cells in developmental processes and used by pathogens during host invasion. Mapping of the glycocode and identification of critical glycan altering enzymes in biological processes is confounded by the complexity and redundancy of glycan biosynthetic machinery. Recently, we integrated microRNA (miRNA) expression data with our glycomics analysis for the NCI‐60 cancer cell line panel, and showed that miRNA act as a major regulator of the human glycome by targeting critical nodes within the glycan biosynthesis pathway. Among others, our analysis revealed a strong association of fucose signature and miRNA‐200 family (miR‐200f), known regulators of epithelial to mesenchymal transition (EMT) a process during which miR‐200f is downregulated. Within the NCI‐60, expression pattern of miR‐200f maps onto fucose signature: both are high in epithelial and low in mesenchymal cells, indicating that fucose might be a marker of epithelial/mesenchymal cell state. In line with our findings we utilize miRNA target prediction software and identify a secreted α‐fucosidase (FUCA2) and O‐linked fucose β‐1,3‐glucosyltransferase (B3GALTL) as most common predicted targets. Both enzymes have the potential to reduce availability of terminal fucose and are downregulated by miRNA‐200f. We show that B3GALTL has a direct role in EMT as B3GALTL knockout induced a change in morphology from mesenchymal to epithelial in mesenchymal breast (MB‐231) and melanoma (M14) cell lines. Taken together our data validates miRNA based mapping of the glycome and identifies B3GALTL as an important regulator of EMT.Grant Funding Source: Sponsored by NIH
Molecular basis for import cargo discrimination by importin α isoforms (793.2)
Authors: Pumroy Ruth , Cingolani Gino 
Published: June 2021

The human genome encodes seven isoforms of importin α grouped into three subfamilies, α1, α2 and α3. All isoforms have an N‐terminal auto‐inhibitory Importin β binding (IBB)‐domain and a C‐terminal helical core that associates with cargos bearing a Nuclear Localization Signal (NLS‐cargos). Although all importin α isoforms can associate with classical NLS‐cargos, certain cargos are selectively imported by specific isoforms. This specificity is not seen in vitro, as NLS‐cargos bind different ΔIBB isoforms with comparable affinity. To identify rules for importin α selection, we have determined high‐resolution crystal structures of representative isoforms from each family of importin α in complex with a model NLS‐cargo. In parallel, structure‐based mutagenesis and an in vitro binding assay were used to examine the degree of IBB‐dependent auto‐inhibition among different isoforms. This work led us to three findings: (i) all isoforms share an identical NLS‐binding groove across the inner surface of ARM 2‐8. (ii) Although the NLS‐binding groove is invariant, importin α isoforms present different curvature, which positions NLS‐cargos with subtly different binding energetics. (iii) Importin α isoforms are differentially auto‐inhibited by their IBB‐domains. We propose that these three factors determine NLS‐cargo discrimination and explain the specificity for importin α isoforms observed in living cells.Grant Funding Source: Supported by T32 GM 100836‐2
Yeast phospholipase C is required for normal glucose metabolism, acetyl‐CoA homeostasis, and global histone acetylation (800.1)
Authors: Zhang Tiantian , Galdieri Luciano , Vancura Ales 
Published: June 2021

Phospholipase C (Plc1p) is required for the initial step of inositol polyphosphates (InsPs) synthesis. Plc1p is also required for normal level of histone acetylation; plc1∆ cells that do not synthesize any InsPs display decreased acetylation of bulk histones, global hypoacetylation of chromatin histones, and altered transcriptional regulation. In accordance with the role of Plc1p in supporting histone acetylation, plc1∆ mutation is synthetically lethal with mutations in several subunits of SAGA and NuA4 histone acetyltransferase (HAT) complexes. Conversely, the growth rate, sensitivity to multiple stresses, and the transcriptional defects of plc1∆ cells are partially suppressed by deletion of histone deacetylase HDA1. The histone hypoacetylation in plc1Δ cells is due to the defect in Grr1p/proteasome‐dependent degradation of repressor Mth1p, and consequently lower expression of the HXT genes and reduced conversion of glucose to acetyl‐CoA, a substrate for HATs. Our current experiments test three possible mechanisms whereby InsPs affect Mth1p degradation: (i) InsPs are required for Mth1p phosphorylation prior to degradation, (ii) InsPs are required for Grr1p activity, and (iii) InsPs are required for the activity of the proteasome.Grant Funding Source: Supported by NIH
Molecular basis of the activation of the Rabex‐5 GEF activity by Rabaptin‐5 in endocytosis (802.27)
Authors: Zhang Zhe , Zhang Tianlong , Yu Fang , Chen Jiangye , Ding Jianping 
Published: June 2021

Rabex‐5 and Rabaptin‐5 function together to activate small GTPase Rab5 and further promote early endosomal fusion in endocytosis. The Rabex‐5 GEF activity is autoinhibited by the Rabex‐5 CC domain (Rabex‐5CC) and activated by the Rabaptin‐5 C2‐1 domain (Rabaptin‐5C21) with yet unknown mechanism. We report here the crystal structures of Rabex‐5CC, the Rabex‐5CC‐Rabaptin‐5C212 complex, and the Rabex‐5Δ‐Rabaptin‐5C212 complex. Our structural and biological data together show that Rabex‐5CC assumes an amphipathic α‐helix which may bind weakly to a nonpolar surface groove of the GEF domain to occlude the substrate‐binding site, resulting in autoinhibition. The linker between the GEF and CC domains plays a role in the autoinhibition probably by modulating the relative conformation of the two domains. Binding of Rabaptin‐5C212 to Rabex‐5 displaces Rabex‐5CC, yielding a largely exposed substrate‐binding site and thus releasing the GEF activity. These results reveal the molecular mechanism of the regulation of the Rabex‐5 GEF activity.Grant Funding Source: Supported by grants from the National Natural Science Foundation of China (31230017 and 31000327)
Cdk1‐dependent phosphorylation of leukemia‐associated RhoGEF during mitosis (802.30)
Authors: Helms Michelle , Grabocka Elda , Martz Matthew , Suzuki Nobuchika , Wedegaertner Philip 
Published: June 2021

Rho GTPases are crucial players in the regulation of actin cytoskeleton‐dependent processes such as mitosis. Our laboratory has discovered a novel role and localization of the leukemia‐associated Rho guanine‐nucleotide exchange factor (LARG), a regulator of G protein signaling (RGS)‐RhoGEF, during mitosis. Rho and LARG have been implicated in diseases such as cancer and heart disease, thus highlighting the importance for further research. The purpose of this study is to determine the molecular mechanism and role of mitotic‐dependent phosphorylation of LARG. Using mitotic‐shift assays and kinase inhibitors, we report that LARG undergoes a mitotic‐dependent and Cyclin‐dependent kinase 1 (Cdk1) inhibitor‐sensitive phosphorylation. Cell synchronization followed by Western blot also identified the temporal feasibility of Cdk1 as a potential kinase. Furthermore, using in vitro kinase assays, we show that LARG can be directly phosphorylated by Cdk1. Using both N‐ and C‐terminal deletion and multi‐site phosphorylation mutants, we demonstrate that the mitotic‐dependent shift of LARG relies on phosphorylation occurring in both termini. Using custom phosphospecific antibodies, we confirm that two sites that are phosphorylated during mitosis and also in a Cdk1‐dependent manner. We conclude that Cdk1 phosphorylates LARG during mitosis, and phosphorylation of LARG may play a role in LARG activity and function.Grant Funding Source: Supported by NIH GM062884
Proteomic analysis of chronic morphine treated SH‐SY5Y human neuroblastoma cells (803.10)
Authors: Randell Kesa , Taka Equar , Goodman Carl 
Published: June 2021

The development of opioid tolerance is a barrier to its clinical use. Continuous exposure to opioids, like morphine, induces several molecular and cellular changes at the receptor and signal transduction pathway. Chronic opioid treatment of differentiated SH‐SY5Y human neuroblastoma cells has been shown to be an excellent in vitro model for studying cellular changes observed after opioid exposure. The present study was conducted to gain a better understanding of alterations in protein expression induced after opioid treatment, using mass spectrometry based proteomics, which in recent years has had a major impact on investigating the molecular and cellular mechanism of protein‐protein interactions. Cells were treated with morphine (10 μM), naloxone (10 μM), or 1 hr naloxone (10 μM) pre‐treatment followed by morphine (10 μM) for 24 hours, harvested, and separated by 2‐D gel electrophoresis. Analysis of these gels revealed alterations in protein expression induced by morphine treatment. Proteins with the most significant changes following drug treatment were identified using MALDI‐TOF. Calmodulin, alpha‐enolase, stathmin, nucleoside diphosphate kinase A, and nucleophosmin were identified. These results form the basis for the analysis of differential protein expression in SH‐SY5Y cells. The proteins detected will further be investigated to determine their role in morphine tolerance.Grant Funding Source: NIH‐NIMHD G12MD007582
Regulator of G protein signaling 6 promotes anxiety and depression by attenuating serotonin‐mediated activation of the 5‐HT1A receptor‐adenylyl cyclase axis (803.12)
Authors: Stewart Adele , Maity Biswanath , Wunsch Amanda , Meng Fantao , Wu Qi , Wemmie John , Fisher Rory 
Published: June 2021

The limited efficacy, delayed onset of action, and side effects restrict the clinical utility of selective serotonin (5‐HT) reuptake inhibitors (SSRIs), which remain first line therapies in the treatment of mood disorders. Regulator of G protein Signaling (RGS) proteins have been implicated as key regulators of 5‐HT1ARs, believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS protein involved remains unknown. Here, we identify RGS6 as the critical negative regulator of 5‐HT1AR‐dependent anxiolytic and antidepressant actions. RGS6‐/‐ mice exhibit spontaneous anxiolytic and antidepressant behavior, a phenotype reversible by 5‐HT1AR blockade. Effects of the SSRI fluvoxamine and 5‐HT1AR agonist 8‐OH‐DPAT were also potentiated in RGS6+/‐ mice. The phenotype of RGS6‐/‐ mice was associated with decreased phospho‐CREB and reduced PKA activity implicating enhanced Gαi‐dependent adenylyl cyclase (AC) inhibition as a causative factor in RGS6‐/‐ animal behavior. In fact, direct activation of AC with forskolin reversed the antidepressant phenotype of RGS6‐/‐ mice. Thus, by inhibiting 5‐HT activation of postsynaptic 5‐HT1ARs, RGS6 exerts powerful anxiogenic and pro‐depressant actions indicating that RGS6 inhibition may represent a viable means to enhance the efficacy of serotonergic drugs through selective potentiation of 5‐HT1AR‐mediated inhibition of AC.Grant Funding Source: Supported by NIH CA161882, 1R01MH085724‐01, 5 R01 HL113863‐01
Multiple palmitoyl acyltransferases modify DAT palmitoylation (803.5)
Authors: Rastedt Danielle , Foster James , Vaughan Roxanne 
Published: June 2021

The dopamine transporter (DAT) mediates the reuptake of synaptic dopamine, thus regulating dopaminergic neurotransmission. DAT undergoes palmitoylation in which a saturated 16‐carbon palmitate group is linked to cysteine via a thioester bond. Palmitoylation regulates diverse aspects of neuronal protein trafficking and function, but its role in DAT function is poorly understood. Palmitoylation is a reversible process in which the protein undergoes cycles of palmitoylation and depalmitoylation catalyzed by palmitoyl acyltransferases (PATs) and palmitoyl‐protein thioesterases (PPTs). The 23 member family of DHHC (Asp‐His‐His‐Cys) proteins, identified as PATs, remains poorly characterized and the identities of the PATs and PPTs involved in the regulation of DAT palmitoylation are unknown. To address this question, we co‐expressed each PAT individually with DAT and assessed DAT palmitoylation, total expression, and transport activity. Preliminary results indicate that specific neuronal‐linked enzymes DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17 increase DAT palmitoylation, while several other DHHC enzymes have no effect. Furthermore, DHHC enzymes found to increase DAT palmitoylation also increase total DAT levels, while treatment with the PAT inhibitor, 2‐bromopalmitate, led to DAT degradation. These findings suggest that palmitoylation enhances total DAT levels, opposing its degradation. Grant Funding Source: Supported by DA 13147, DA 031991, EPS 0184442