bluuebunny/crossref_metadata_2025_split huggingface dataset:
Vacuolin‐1 inhibits autophagy by activating rab5 (737.7)
Authors: Yue Jianbo , Lu Yingying 
Published: June 2021

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with wide ranges of human diseases, e.g. cancer and neurodegenerative diseases. A large number of small chemicals that modulate autophagy have been widely used to dissect this process and some of them, e.g. chloroquine (CQ), might be ultimately applied to treat a variety of autophagy associated human diseases. Here we found that the small chemical vacuolin‐1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin‐1 was less toxic but was at least 10 folds more potent in inhibiting autophagy compared to CQ. Vacuolin‐1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal‐lysosomal degradation. Treatment of cells with vacuolin‐1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content, yet vacuolin‐1 had little effect on vacuolar ATPase activity. Instead, vacuolin‐1 treatment markedly activated Rab5 GTPase activity. Expression of a dominant negative mutant of Rab5A or Rab5A knockdown abolished vacuolin‐1 induced autophagosomal‐lysosomal fusion blockage, whereas expression of a constitutive active form of Rab5A inhibited the lysosomal‐autophagosomal fusion. These data indicate that vacuolin‐1 activates Rab5 to suppress autophagosomal‐lysosomal fusion. Vacuolin‐1 and its analogues present a novel class of drug that can potently and reversibly modulate autophagy.Grant Funding Source: This work was supported by Research Grant Council (RGC) grants (HKU 782709M, HKU 785911M, HKU 769912
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.
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
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.
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.