bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting the intranuclear functions of GAPDH: phosphoThr99‐GAPDH is translocated into nucleus of A549 cells after genotoxic stress (739.6)
Authors: Krynetskiy Evgeny , Krynetskaia Natalia , Phadke Manali , Mishra Anurag , Gothe Scott , Barrero Carlos , Merali Salim
Published: June 2021

GAPDH is a multifunctional cytosolic protein involved in various cellular processes including glycolysis, apoptosis, and accelerated senescence. Post‐translational modifications, e.g. phosphorylation and nitrosylation modulate functions of GAPDH in the cell. In this study, we analyzed the acidic isoforms of GAPDH which migrate in the pI range of 6.8 to 8.3. Using MALDI‐TOF analysis, we identified novel phosphorylation sites in GAPDH isoform (pI 8.3) at Y94, Ser98, and Thr99. We generated polyclonal antibody specific to phosphoThr99‐containing peptide within GAPDH, and demonstrated accumulation of T99‐phosphoGAPDH in the nuclear fractions of A549 cancer cells following the cytotoxic stress. Analysis of the extant crystal structures for human GAPDH and molecular modeling experiments indicate that T99 is adjacent to the NAD+‐binding site. Molecular mechanics and dynamics experiments suggest that mutation and / or phosphorylation of Thr99 may adversely affect the local geometry of the NAD+‐binding cleft. Site‐mutagenesis at positions Y94, S98, and T99 in the NAD+‐binding center reduced glycolytic activity of GAPDH by 50‐100% because of decreased affinity to NAD+ (Km=0.41±0.17 mM in T99I vs. 0.04±0.01 mM in wild type GAPDH). The studies on the functional consequences and a mechanism of Thr99 phosphorylation will open avenues to modulators of GAPDH activity, an important target in anticancer therapy.
Expanding the genetic code with pyroglutamate (751.1)
Authors: Benhaim Mark
Published: June 2021

Pyroglutamate forms via the cyclization of glutamine residues in proteins including amyloid β‐peptides associated with Alzheimer’s disease and oconase, an anti‐cancer agent. To study the role pyroglutamate plays in these proteins, we propose expanding the E. coli genetic code to include pyroglutamate by reassigning the amber stop codon to pyroglutamate. To include amino acid, we are modifying the archaeal RNA‐dependent Gln biosynthetic pathway to synthesize pyroglutamate on an amber suppressor tRNA. The relevant genes were cloned into a vector for expression in E. coli. To determine if the system can incorporate pyroglutamate into proteins, we are developing an enhanced yellow fluorescence protein reporter system. Once established, we will use the system to test the role of the pryoglutamate in proteins.

Grant Funding Source: Supported by Research Corporation
Choreography of bacterial protein biogenesis factors at the peptide exit of the ribosome (752.4)
Authors: Bornemann Thomas , Holtkamp Wolf , Wintermeyer Wolfgang
Published: June 2021

Nascent proteins emerging from translating ribosomes in bacteria are screened by ribosome‐associated protein biogenesis factors (RPBs), among them the signal recognition particle (SRP), trigger factor (TF), peptide deformylase (PDF), and methionine aminopeptidase (MAP). Whereas SRP and TF can bind to translating ribosomes simultaneously, the time of action for the modifying enzymes, PDF and MAP, is not clear. Here we examine the RPB choreography by monitoring FRET between labeled ribosomes and either TF or SRP. The competition binding analysis reveals how the RPB binding pattern changes depending on the state of the ribosome.

We observe that TF, like SRP, rapidly scans all ribosomes until it is stabilized on ribosomes presenting TF‐specific nascent chains. PDF and TF compete for binding to those complexes, whereas MAP can bind together with TF. The same competition pattern we observe with SRP and PDF or SRP and MAP on ribosomes exposing an SRP‐specific nascent chain. These results suggest that PDF and TF act sequentially in that PDF deformylates short nascent chains prior to stable TF binding to ribosomes carrying longer nascent chains. By contrast, competition between PDF and SRP, which is recruited to ribosomes carrying rather short nascent chains, may interfere with N‐terminal processing of nascent membrane proteins that enter the membrane co‐translationally in an SRP‐dependent fashion.
Chemical protein folding probes to quantify folded and functional cellular proteins to understand proteostasis network functions (753.1)
Authors: Zhang Xin , Liu Yu , Tan Yun Lei , Kelly Jeffery
Published: June 2021

While much is known about protein folding in buffers, current methodology is limited in probing how protein folding in vivo is influenced by the cellular proteostasis network quantitatively, particularly with regard to partitioning client proteins between folded and functional, soluble and misfolded, and aggregated conformational states in the face of genetic and environmental stresses. Herein we develop protein‐of‐interest folding probes that specifically recognize the functional and folded fraction of the protein‐of‐interest, , and serve to quantify the amount of folded and functional protein in cell lysate. Rapid depletion of ATP levels during cell lysis minimizes folding equilibria shifts by creating sufficient cellular chaperone holdase activity. The folding probes strategy is exemplified with retroaldolase (RA), a de novo designed enzyme, and transthyretin, a tetrameric plasma protein. This strategy revealed that partitioning of RA and TTR mutants between the aforementioned conformational states was strongly influenced by cytosolic proteostasis network perturbations, including expression of a polyglutamine protein.
Targeted mutagenesis of the oligopeptide repeat domain of the yeast prion Sup35 (754.3)
Authors: Knox James , Davis Emily , MacLea Kyle
Published: June 2021

The formation of prions in the baker’s yeast, Saccharomyces cerevisiae, is determined by amino acid composition rather than the primary sequence of amino acids. The infectious amyloid proteins known as prions undergo nucleation and propagation, two distinct activities critical for prion formation. The ability for prions to be transferred from cell to cell, or propagate, is of interest not only in yeast prions but also in prion diseases such as the mammalian spongiform encephalopathies. Prion formation has been widely studied in yeast prions, however, the fundamental mechanisms behind the specific process of propagation of prions from cell to cell are not yet understood. In the most well‐studied yeast prion, the prion form [PSI+] of Sup35, a domain of 5 ½ degenerate oligopeptide repeats called the oligopeptide repeat domain (ORD), has been shown to be important for prion propagation and to have a distinct amino acid composition as compared to the nucleation domain region. A library mutagenesis experiment has identified amino acids that favor or disfavor prion propagation in yeast cells. To confirm the results of the random library mutagenesis experiment, we generated several clones in which a portion of the ORD (the fourth oligopeptide repeat) was replaced with defined sequences expected to propagate or fail to propagate and these clones were assessed for their ability to propagate in vivo.
Serine hydrolase inhibitors block necrotic cell death by preventing calcium overload of the mitochondria and permeability transition pore formation (756.2)
Authors: Yun Bogeon , Lee HeeJung , Ghosh Momita , Cravatt Benjamin , Hsu Ku‐Lung , Bonventre Joseph , Ewing Heather , Gelb Michael , Leslie Christina C
Published: June 2021

Perturbation of calcium signaling occurs during cell injury and disease and promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, necrotic cell death and LDH release that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2 (cPLA2) or calcium independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2‐deficient fibroblasts was inhibited by the cPLA2 inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium loading. Inhibitors of calmodulin‐dependent protein kinase II, a mitochondrial Ca2+ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and by H2O2. Pyrrophenone blocked MCU‐mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium loading but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and OAG but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not fatty acid release that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell permeable serine hydrolase inhibitors that block MCU‐mediated mitochondrial calcium overload, MPTP formation and necrotic cell death.

Grant Funding Source: NIH HL34303, HL61378, HL50040, DA009789, DA033760, MH084512
Metalloprotease OMA1 is involved in fine‐tuning of mitochondrial bioenergetic function in embryonic development (756.4)
Authors: Khalimonchuk Oleh , Zahayko Nataliya , Rahn Jennifer , Fernandez Mario , Lewis Robert , Chan Sherine
Published: June 2021

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism ‐ intramitochondrial quality control (IMQC) is represented by conserved proteases distributed across mitochondrial compartments. Many aspects of IMQC are unclear. Together with the m‐AAA protease, OMA1 (ATP‐independent protease in the inner membrane (IM)) was implicated in depolarization‐induced cleavage of IM GTPase OPA1 and thus regulation of mitochondrial dynamics in response to stress. Genetic knockout of OMA1 in the mouse altered energy expenditure and thermogenesis. These defects were attributed to the impeded stress processing of OPA1.

We investigated the role of OMA1 in embryonic development using the zebrafish model. Analysis of the OMA1 morphant embryos revealed abnormal blood circulation and developmental defects of the heart and eyes. Surprisingly, the levels and processing of OPA1 were not significantly altered.

In vivo evaluation of respiratory function in OMA1‐/‐ MEFs indicated loss of OMA1 leads to bioenergetic deficits without impairing basal mitochondrial efficiency. These results point to the importance of OMA1 in vertebrate development and indicate a role of this protease in the fine‐tuning of respiratory function that may extend beyond stress processing of OPA1.

Grant Funding Source: NIH‐NIGMS P30 GM103335
Interplay between O‐GlcNAc and acetylation regulates mitochondrial function (756.7)
Authors: Tan Ee Phie , Lanza Christopher , Swerdlow Russell , Slawson Chad
Published: June 2021

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a post‐translational modification involving an attachment of a single N‐acetylglucosamine residue to serine or threonine residues in nuclear and cytoplasmic proteins. The O‐GlcNAc modification is processed by O‐GlcNAc transferase (OGT), which adds the modification to the protein and O‐GlcNAcase (OGA), which removes it. Recent evident links the regulation of mitochondrial function to O‐GlcNAcylation. For example, we used a quantitative proteomics screen to determine that an OGT or OGA gain of function altered mitochondrial protein expression including proteins involved in the respiratory chain and TCA cycle. Furthermore, mitochondrial morphology and both cellular respiration and glycolysis were altered in these cells. Potentially, altered O‐GlcNAc is changing the levels of mitochondria acetylation. Acetylation is a common post‐translational modification that regulates proteins function. The objective of our study is to investigate if O‐GlcNAc is regulating the acetylation of mitochondrial proteins. Herein, we demonstrate that alterations to the cellular levels of O‐GlcNAc changes mitochondrial acetylation as well as the expression of SIRT3. These data demonstrate that alterations to the cellular homeostasis of O‐GlcNAc levels may have a profound effect on the mitochondrial acetylation.

Grant Funding Source: P30AG035982
Single organelle localisation microscopy in vascular smooth muscle reveals mitochondrial networks are multiple electrically‐discontinuous elements, with larger organelle dimensions and more extensive mitochondrial clustering occurring in hypertension (757.2)
Authors: Chalmers Susan , Saunter Christopher , Girkin John , McCarron John
Published: June 2021

Mitochondria vary from being small puncta to large interconnected networks. While more easily visualized in cultured cells, the precise structure of the organelle in native cells is poorly understood due to the cells’ complex organization and to imaging resolution limitations. In native cells, mitochondrion dimensions vary from ~0.5μm to ~7μm diameter. Here, the electrical changes arising from transient de‐ and re‐polarisations of membrane potential (‘flickers’) of single mitochondria within native vascular smooth muscle cells were visualised by fluorescence imaging and used to determine the organelles’ dimensions, position and relationship with Ca2+ signals. Cross‐correlation of changes in fluorescence intensity over a 40x40 pixel area around each image pixel was used to produce high‐resolution images of mitochondrial boundaries. This functionally resolved single organelle localisation microscopy (SOLM) reveals multiple electrically‐discontinuous mitochondrial elements within what appeared to be a single large organelle, i.e. a dense mitochondrial cluster. Significantly, both the size of individual mitochondria and extent of organelle clustering increased in resistance artery myocytes from spontaneously hypertensive (SHR) rats when compared to normotensive (WKY) controls. The Ca2+ signal evoked by plasma membrane depolarization was largest near mitochondrial clusters. In vascular smooth muscle, the extent of mitochondrial networking and organelle mobility correlates with cellular proliferation and disease. Here a previously unrecognised change in mitochondrial structure may contribute to changes in Ca2+ signalling and smooth muscle function characteristic of hypertension.

Grant Funding Source: Supported by the Wellcome Trust (092292/Z/10/Z) and British Heart Foundation (PG/11/70/29086)
Defining the molecular requirements for phosphatidylserine decarboxylase activation (757.3)
Authors: Onguka Ouma , Ogunbona Oluwaseun , Claypool Steven
Published: June 2021

Phosphatidylethanolamine (PE) is a major membrane phospholipid that is critical for a multitude of biological processes and essential for life. One pathway of PE production occurs in the mitochondrion. Psd1p, the responsible enzyme, decarboxylates phosphatidylserine (PS) to PE. Psd1p is encoded in the nucleus and imported into the mitochondrion post‐translationally. The final active enzyme is produced by autocatalytic processing. Autocatalysis occurs at a conserved LGST motif. The result is a membrane anchored β‐subunit that remains associated with a small α‐subunit, which contains a catalytically essential pyruvoyl group on its N terminus. The goal of the present study was to further characterize Psd1p autocatalysis at the molecular level and define additional requirements for this process. Here, we demonstrate that neither mitochondrial lipids nor proteins are critical for Psd1p autocatalysis. Further, an autocatalytic mutant Psd1p assembles normally, indicating that Psd1p folding precedes autocatalysis. Finally, a temperature sensitive (ts) allele has perturbed autocatalysis at non‐permissive temperature, thus establishing the thermosensitive loss‐of‐function mechanism. Understanding the molecular requirements for Psd1p autocatalysis may uncover potential regulatory steps in Psd1p activity, which may be exploited to modulate cellular PE content.

Grant Funding Source: R01 HL108882‐01A1
Fatty acid chain elongation in palmitate‐perfused working rat heart: mitochondrial acetyl‐CoA is the source of two‐carbon units for chain elongation (758.2)
Authors: Kerner Janos , Minkler Paul , Lesnefsky Edward , Hoppel Charles
Published: June 2021

Rat hearts were perfused with [1,2,3,4‐13C4]palmitic acid (M+4) and the isotopic patterns of myocardial acylcarnitines and acyl‐CoAs analyzed using UHPLC‐MS/MS. The 13C‐enrichment in palmitoyl‐CoA and palmitoylcarnitine shows that little endogenous palmitate contributed to their formation. The stable isotope enrichment of acyl‐CoAs and acylcarnitines clearly shows that the perfused palmitate was β‐oxidized and chain elongated at the same time. The observed 13C enrichment in acetyl‐CoA and acetylcarnitine and M+6 stearoyl‐CoA and stearoylcarnitine suggests that the majority of two‐carbon units for chain elongation are derived from β‐oxidation of [1,2,3,4‐13C4]palmitic acid. These data are explained by conversion of the M+2 acetyl‐CoA to M+2 malonyl‐CoA, which serves as the acceptor for M+4 palmitoyl‐CoA in chain elongation and is supported by the identical 13C‐enrichment in mitochondrial acetyl‐CoA and malonyl‐CoA. Furthermore, isolated, intact rat heart mitochondria 1) synthesize malonyl‐CoA with simultaneous inhibition of carnitine palmitoyltransferase 1b, and 2) catalyze the palmitoyl‐CoA‐dependent incorporation of 14C from [2‐14C]malonyl‐CoA into lipid‐soluble products. In conclusion, rat heart chain elongate fatty acids using mitochondria‐derived two‐carbon units in a process localized to the outer surface of the mitochondrial outer membrane.

Support: NIH, PO1 AG15885.

Grant Funding Source: Supported by NIH
Activation of mitochondrial lysophospholipase by mitochondrial depolarization: role in organelle turnover (758.4)
Authors: Broekemeier Kimberly , Kocian Sara
Published: June 2021

Mitochondria contain a Ca2+‐independent phospholipase A2 (iPLA2) that is activated by loss of membrane potential (ΔΨ). Accumulation of unsaturated fatty acids, determined using heptadecanoic acid as internal standard, was used to quantitate iPLA2 activity. In these incubations, accumulation of saturated fatty acids was also observed. Of interest was whether mitochondria contained lysophospholipase activity and additionally, if activity was regulated similarly to iPLA2 or if appearance of saturated fatty acids was determined simply by lysophospholipid availability. Using 1‐heptadecanoylphosphatidylcholine, 10 mg/mL as substrate and pentadecanoic acid as internal standard it was determined that Percoll purified mitochondria contain lysophospholipase activity that is regulated similarly to iPLA2. When mitochondria are energized, activity is near zero and is up‐regulated when mitochondria are depolarized by a variety of de‐energizing conditions. Fatty acids were released from exogenous and endogenous substrate in nearly equal amounts. At 10 nmol lysophospholipid/mg, there was minimal effect on mitochondrial bioenergetic parameters. Activity is relatively resistant to inhibition by BEL, an inhibitor of iPLA2. Coordinated activation of lipid degrading enzymes by loss of ΔΨ may be involved in the removal of poorly functioning mitochondria by the Parkin/Pink‐1 dependent pathway.
NFκB‐dependent Bcl3 regulates survival in cutaneous T‐cell lymphoma (762.1)
Authors: Chang Tzu‐Pei , Singha Bipradeb , Sanacora Shannon , Reddy Himavanth , Vancurova Ivana
Published: June 2021

Cutaneous T cell lymphoma (CTCL) is characterized by high constitutive activity of NFκB, which promotes cell survival and resistance to apoptosis. Increased expression of the proto‐oncogene Bcl3 belonging to IκB family is associated with the pathogenesis of different types of human cancer, yet, the regulation and function of Bcl3 in CTCL have not been previously studied. Here, we show that human CTCL Hut‐78 and HH cells express high protein levels of Bcl3, which is localized both in the cytoplasm and in the nucleus. NFκB subunits p65 and p50 are highly recruited to the Bcl3 promoter, indicating that Bcl3 expression is regulated by NFκB p65/p50 heterodimers in CTCL cells. Proteasome inhibition by bortezomib (BZ), which induces apoptosis in CTCL cells, inhibits Bcl3 expression. The BZ‐decreased Bcl3 expression is associated with decreased recruitment of NFκB p65 and p50 subunits to the endogenous Bcl3 promoter, but with increased recruitment of NFκB p52. Importantly, suppression of Bcl3 levels by siRNA induces apoptosis and down‐regulates expression of the anti‐apoptotic genes cIAP1 and cIAP2 in CTCL cells. Bcl3 is specifically recruited to the endogenous cIAP1 and cIAP2 promoters, and BZ inhibits this recruitment. These data provide first insights into the regulation and function of Bcl3 in CTCL, and indicate that Bcl3 has an important pro‐survival role in these cells.

Grant Funding Source: Support by NIH grants AI085497 and CA173452 to I.V.
Prohibitin overexpression in adipocyte induces mitochondrial biogenesis, leads to obesity development and affects whole‐body metabolism in a gender specific manner (765.3)
Authors: Ande Sudharsana , Nguyen K. Hoa , Padilla‐Meier G. Pauline , Wahida Wahida , Nyomba B.L. Grégoire , Mishra Suresh
Published: June 2021

Objective: Adipocyte is the primary cell in the body where excess energy is stored as triglycerides. To perform this specialized function adipocytes rely on their mitochondria. However, the role of adipocyte mitochondria in the regulation of adipose tissue homeostasis and its impact on metabolic regulation is not understood.

Results: We developed a transgenic mouse model, namely “Mito‐Ob”, overexpressing prohibitin in adipocytes. Mito‐Ob mice developed obesity due to upregulation of mitochondrial biogenesis in adipocytes. Interestingly, Mito‐Ob female mice developed more visceral fat than Mito‐Ob male mice. However, female mic preserved insulin sensitivity and had high adiponectin levels, whereas male mice became insulin resistant, had compromised brown adipose tissue structure and function and low adiponectin levels. Mechanistically, we found that prohibitin overexpression enhances the crosstalk between mitochondria and nucleus and facilitates mitochondrial biogenesis.

Conclusions: Our data suggests a critical role of prohibitin and adipocyte mitochondria in adipose tissue homeostasis and reveals gender differences on the effect of adipocyte mitochondrial remodeling on whole‐body metabolism. Targeting adipocyte mitochondria may provide new therapeutic opportunities for obesity treatment.
Establishment of biomarkers for radiation damage after computer tomography in children (766.4)
Authors: Halm Brunhild , Lai Jennifer , Pagano Ian , Tiirikainen Maarit , Cooney Robert , Turner Helen , Brenner David , Zohrabian Vatche , Franke Adrian
Published: June 2021

Objective: Computer tomography (CT) involves ionizing radiation (IR) and increases cancer risk, particularly in children. We tested whether blood markers are altered in 17 children (0.25‐6 y) undergoing CT.

Methods: Blood drawn before and 1 hour after CT were analyzed for: plasma antioxidants (ascorbic acid (AA); tocopherols; carotenoids; coenzyme Q10), plasma redox status (oxidized/reduced Q10; dehydroascorbic (DHAA)/total AA ratios) by HPLC/DAD, total antioxidant capacity by ORAC, expression of genes hypothesized to be usable as biodosimeters by microarray chip arrays, cytokine levels (10plex) by ELISA, and γ‐H2AX foci formation in lymphocytes (DNA double strand break biomarker) by immunofluorescence.

Results: All major plasma lipid soluble antioxidant levels decreased after CT (p<0.05) while oxidized and reduced Q10 levels increased but not significantly. Mean AA levels increased (134%), DHAA levels decreased (29%), and total antioxidant capacities in lipophilic and hydrophilic plasma extracts remained unchanged after CT similarly to cytokine levels. The ERP29 gene was significantly altered and dose‐dependent increases in γ‐H2AX foci were observed after CT (p<0.05, n=3).

Conclusion: IR influences the antioxidant systems of young children exposed to diagnostic CT scans and γ‐H2AX foci indicate IR damage most sensitively.

Grant Funding Source: This research was supported by a UHCC seed grant.
A redox‐centered view of radiation resistance in head and neck squamous cancer (768.1)
Authors: Mims Jade , Zhao Weiling , Reisz Julie , Devarie Baez Nelmi , Kuremsky Jeffrey , Punska Elizabeth , Williams Kristin , Arcaro Kathleen , Tsang Allen , Furdui Cristina
Published: June 2021

The central issue of resistance to radiation remains a significant challenge in the treatment of cancer despite improvements in treatment modality and emergence of new therapies. To facilitate the identification of molecular factors that elicit protection against radiation, we developed a matched model of radiation resistance for head and neck cancer (HNC) and characterized its properties using selective chemical probes for protein oxidation, omics technologies and complementary assays. Functional network analysis of proteomics data identified DNA replication and base excision repair, ECM‐receptor interaction, and regulation of actin cytoskeleton as significantly up‐ or down‐regulated networks in resistant (rSCC‐61) HNC cells. Up‐regulated proteins in rSCC‐61 included enzymes involved in lipid metabolism and antioxidant system. Redox proteomics analysis using biotin‐tagged chemical probes and mass spectrometry identified decreased oxidative damage of the antioxidant system in rSCC‐61 consistent with the cells increased ability to withstand a radiation insult. Further metabolomics and epigenetics studies of this system show that multiple pathways converge to produce the rSCC‐61 phenotype and point to the function of the antioxidant system as major regulator of resistance to ionizing radiation in rSCC‐61, a phenomenon confirmed by analysis of HNC tumor samples.
Regulation of histone deacetylase 8 substrate specificity (768.14)
Authors: Pitcairn Carol , Gattis Samuel , Fierke Carol
Published: June 2021

Lysine acetylation, catalyzed by lysine acetyltransferases (KATs or HATs), is an important post‐translational modification of proteins such as histones, transcription factors, nuclear regulators, and cytoplasmic proteins. The modification is reversible; hydrolysis is catalyzed by a group of enzymes called histone deacetylases (HDACs). Lysine deacetylation is important for regulating various cellular processes, and aberrant deacetylation is implicated in cancer. Histone deacetylase 8 (HDAC8) is one of the metal‐dependent HDACs, and initial studies identified Zn(II) as the catalytic metal ion in the active site. Recent in vitro studies have shown that HDAC8 is activated not only by Zn(II), but also by Co(II) and Fe(II). We propose that in vivo regulation of HDAC8 activity may be modulated by metal switching. Using pull‐downs of HDAC8 over‐expressed in tissue culture cells and mass spectrometry, we are developing methods to determine the in vivo metal ion cofactor(s) of HDAC8, and preliminary data indicates there is a mixture of iron and zinc present. We are also investigating the mechanism by which phosphorylation of Ser39 on HDAC8 regulates HDAC8 activity by exploring potential changes in substrate specificity compared to wild type HDAC8 using peptide libraries.

Grant Funding Source: Supported by grants from the National Institutes of Health
Unique biocatalysis: synthesis of a carbon‐nitrogen bond by carbon‐sulfur bond synthetic enzymes (768.15)
Authors: Kobayashi Michihiko , Abe Tomoko , Hashimoto Yoshiteru , Zhuang Ye , Kumano Takuto
Published: June 2021

We have studied several enzymes involved in cleavage and synthesis of a C‐N single or triple bond (e.g., nitrilase, nitrile hydratase, amidase, isonitrile hydratase, N‐substituted formamide deformylase, and aldoxime dehydratase).

During the studies on the degradation of toxic nitrile with a cyano functional group (carbon‐nitrogen triple bond) in the industrial microorganism Pseudomonas, we found that acyl‐CoA synthetase (AcsA) is one of enzymes involved in the metabolic pathway. AcsA ligates acid with CoA to form acyl‐CoA with a carbon‐sulfur bond. However, when L‐cysteine was used as a substrate instead of CoA, N‐acyl‐L‐cysteine was surprisingly detected as a reaction product. This finding demonstrates that AcsA synthesizes an amide bond comprising the amino group of cysteine and the carboxyl group of the acid. AcsA formed a variety of N‐acyl‐compounds, when various acids and cysteine‐analogues were used as substrates. Enzymes of the adenylate‐forming enzyme superfamily, which AcsA belongs to, were also found to catalyze the similar reaction. Furthermore, we discovered that adenylation domains of nonribosomal peptide synthetases, which belong to the same superfamily showed the peptide‐bond synthetic activity. Various dipeptides and oligopeptides can be synthesized by the adenylation domains. Here, we would like to propose the reaction mechanism underlying these new carbon‐nitrogen bond synthetic reactions.
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
Probing the catalytic promiscuity in tautomerase 1 and 2 in Methylibium petroleiphilum PM1 (769.15)
Authors: Heussner Derek , Terrell Cassidy
Published: June 2021

Certain hydrocarbons created as industrial byproducts, specifically in energy production and oil refinement, are toxic to the vast majority of organisms. Developing remediation strategies to solve this problem is of particular importance to the maintenance of multiple ecosystems. A unique adaptation made by the genome of the bacterium Methylibium petroleiphilum PM1 codes for parallel metabolic pathways which enable the breakdown of numerous toxic hydrocarbon sources. Two isozymes in these parallel pathways, Tautomerase 1 and Tautomerase 2, share remarkably similar 3‐dimensional structure, but only 35% primary sequence identity. Structural and kinetic differences between these isozymes raise exciting questions about possible substrate and metabolic pathway differentiation. In this study, we probe the catalytic promiscuity of Tautomerase 1 and 2, testing the hypothesis that these enzymes discriminate between hydrocarbon sources, thus offering a metabolic advantage to the organism. The non‐canonical substrate trans‐β‐chloroacrylic acid (TCA) was chosen for study based on previous research of active site structural homologs. The reaction was monitored by 1HNMR spectroscopy and UV‐vis spectroscopy. Results to date show that Tautomerase 1 catalyzes the dehalogenation of TCA with greater efficiency than Tautomerase 2, however the overall rates for both enzymes are particularly slower than with the canonical substrate. These initial results also trend towards faster rates for both Tautomerase 1 and 2 compared to the rates of other members of the 4‐OT family and their catalysis of TCA. Current work includes the incorporation of several quantitative methods for extracting kinetic parameters, while also testing other atypical substrates with differing stereochemistry and halogen groups for catalytic differentiation between the 4‐OT homologs.
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 & 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.
Functionalization of gold nanorod‐based immunosensors for label‐free bio‐detection with high sensitivity and specificity (780.7)
Authors: Gomez‐Rivera Francisco , Wang Yanyan , Tang Liang
Published: June 2021

Gold nanoparticles have shown great potential applications in biomedical diagnosis and therapeutic treatments due to their shape and size‐dependent optical properties. However, densely packed CTAB double layer (surfactant used to prevent nanoparticle aggregation) on the nanorod surfaces present challenges for effective biofunctionalization to setup a nanoparticle‐based platform with high detection sensitivity. In this study, we systematically studied the functionalization of gold nanorods (GNRs) with various bioconjugate linkers in pursuit of developing nanorod‐based immunosensors with maximal sensitivity and specificity. First, GNR chips were fabricated by chemically binding nanorods onto silanized glass substrate; then the GNRs were functionalized with carboxylic acid or amine group via thiol linkers for antibody conjugation. The effects of thiol linkers with different lengths such as 11‐mercaptoundecanic acid (MUDA), 4‐aminothiophenol (4‐ATP), 2‐mercaptopropionic acid (MPA), cysteamine hydrochloride (CAE) and 6‐mercaptohexanoic acid (MHA) were investigated. GNR nano‐biochip exhibited high detection sensitivity (0.3017/ IgGnM), low detection limit (1.3 nM) and high specificity, with the features of label free and simple detection mechanism. The optimized functionalization of GNR chip was applied to develop a nano‐biosensor to detect human serum IgG as a model system. Since the localized surface plasmon resonance is highly sensitive to the biological binding on the nanorod surface, this mechanism provides a label‐free biodetection, eliminating signaling labels such as fluorophore and radioactive agents. The miniaturized nano‐biochip is attractive for rapid medical diagnostics in clinics, due to its user‐friendly and cost‐effective features.
Dispersion of purified single wall carbon nanotube in polyethylene glycol and polypyrrole for an improved efficiency of a glucose biosensor (780.8)
Authors: Goornavar Virupaxi , Ezeagwu Christian , Ezeagwu Dexter , Pradhan Aswini , Ramesh Govindarajan
Published: June 2021

Abstract Single wall carbon nanotubes (SWCNTs) are a class of materials that have been utilized in the field of sensors, drug delivery, bioimaging etc. SWCNTs have been used in the field of sensors because of their aspect ratio and the network they form for a faster electron transfer. The presence of impurities such as transition metals, amorphous carbon and structural defects hinders the process of electron transfer. The purification of SWCNTs thus becomes necessary for such applications. Here we present the purification process of SWCNTs that does not damage the tubes or shorten the tubes. These purified SWCNTs are later dispersed in polyethylene glycol (PEG) and pyrrole (Py) at a concentration of 1mg/ml and later coated onto a glassy carbon electrode (GCE) for the detection of glucose. The GCE/SWCNTs was coated with glucose oxidase (3mg/ml) and dried at room temperature for 90 min and glucose is amperometrically detected in 0.05M PBS at 0.7V versus Ag/AgCl. The GCE/SWCNT/PEG gave a detection limit of 0.434 µM and a current sensitivity of (0.08164± 0.001129 µA mM‐1, r2=0.9975). The GCE/SWCNT/Py gave a detection limit of 0.9617µM with a sensitivity of (0.04189± 0.00087 µA mM‐1, r2=0.9944). These results prove that purification of SWCNTs with a mild oxidative step does not hamper the electron transfer rate and the dispersion of SWCNTs obtained helps in the entrapment of glucose oxidase without needing an additional protective layer.
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