bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosducin‐like protein and CCT interact with G protein β‐like, β propeller proteins (567.4)
Authors: Tsang Tsz Ming , Mak Wai Shun , Chan Tsz Yin , Lukov Georgi 
Published: June 2021

Previous studies have established that the cytosolic chaperonin complex, CCT and phosducin‐like protein (PhLP) are required for the proper folding of the G protein β subunit, and the assembly of the Gβγ subunit complex. The structure of the Gβ subunit is a seven bladed, β propeller. Previous studies have also shown that CCT has multiple binding partners, some of which have β propeller structure, similar to that of Gβ. Our objective is to investigate if proteins with seven bladed β propeller structure, similar to that of Gβ would associate with CCT and PhLP. We investigated the binding interaction of three Gβ‐like proteins, CDC20, TBL2 and MLST8, to CCT and PhLP. Using co‐immunoprecipitation approaches, we demonstrated that all three Gβ‐like proteins interact with CCT and PhLP. These findings suggest that CCT and PhLP may work together not only during the folding of the Gβ proteins, but they may be the folding machinery of all proteins with Gβ‐like, seven bladed β propeller structure.Grant Funding Source: BYUH Mentored Research and Student Research Associateship Funds
Mapping the binding interactions between a specialized Hsp70 chaperone and its Hsp40 co‐chaperone: a direct role for the Hsp40 HPD motif (567.5)
Authors: Alderson T. , Kim Jin , Markley John 
Published: June 2021

Proteins from the ISC operon of E. coli constitute the machinery used for the synthesis of iron‐sulfur clusters and their delivery to recipient apo‐proteins. [2Fe‐2S] cluster transfer from the holo‐scaffold protein IscU depends on ATP hydrolysis in the nucleotide‐binding domain (NBD) of HscA, a specialized Hsp70 chaperone. HscB, an Hsp40‐type co‐chaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer. However, the interactions between HscA and HscB have remained uncharacterized, and, moreover, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. We have created three variants of the isolated HscA NBD truncated at different residues within the linker, and have shown that the linker binds to the NBD and autoactivates ATP hydrolysis. Using solution‐state NMR spectroscopy and chemical shift perturbations, we mapped the apo‐HscA, HscA‐ADP and HscA‐ATP binding sites onto HscB. Our NMR data provide the first evidence of a structural role for the ubiquitously conserved J‐domain residues HPD of HscB, which we found selectively bind to the ATP‐bound form of HscA. Additionally, we have identified and characterized a previously unreported interaction between HscA and IscA, an iron‐binding protein putatively involved in [4Fe‐4S] cluster transfer, which competes with HscB for binding to HscA.Grant Funding Source: Supported by NIH grants U01 GM094622 and P41 GM103399
The structural basis for recognition and modification of the 30S ribosomal subunit by an antibiotic resistance methyltransferase (569.3)
Authors: Dunkle Jack , Vinal Kellie , Zelinskaya Natalia , Conn Graeme , Dunham Christine 
Published: June 2021

Ribosomal RNA contains modifications at specific nucleotides in all organisms, some of which control resistance to antibiotics. However, the mechanisms are not well understood controlling how modification enzymes recognize their specific target nucleotide and position it within the active site. We report the crystal structure of the pathogen‐derived aminoglycoside‐resistance rRNA methyltransferase NpmA bound to the 30S ribosomal subunit in a pre‐catalytic state. The structure reveals that NpmA recognizes conserved rRNA tertiary structure rather than RNA sequence. Additionally, the structure shows NpmA flips the target nucleotide from its position within the helical base stack to position it within the active site. The structure provides a general framework for investigating the mechanisms of other rRNA modification enzymes.Grant Funding Source: Supported by National Institutes of Health
Discovery of a novel Complex I biogenesis factor regulating mitochondrial gene expression (571.1)
Authors: Subrahmanian Nitya , Hamel Patrice 
Published: June 2021

Mitochondrial Complex I (CI), a proton pumping NADH:ubiquinone oxidoreductase, is the most complicated respiratory enzyme. The assembly of this highly conserved enzyme, consisting of more than 40 subunits of dual genetic origin, is an intricate process that is poorly understood. The objective of this study was to identify novel factors involved in CI biogenesis using Chlamydomonas reinhardtii, a unicellular photosynthetic alga, as an ideal model system. From a mutant screen, the amc11 mutant with a CI deficiency was isolated. In‐gel activity shows that amc11 accumulates a sub‐complex diagnostic of an assembly defect in the membrane arm of CI. The mutation in amc11 was mapped to a novel gene encoding a unique low complexity protein with no conserved domains. The role of this AMC11 locus in CI assembly was confirmed by complementation studies. In order to understand the basis for the CI deficiency due to loss of AMC11, the transcript levels of the five mitochondrial encoded CI subunits was determined. Interestingly, the accumulation of only the nd4 transcript, encoding a subunit in CI membrane arm was partially reduced, the levels of which are restored upon complementation with the WT nuclear gene. Also, substitution of mitochondrial nd4 gene with a reporter in the amc11 mutant results in loss of reporter protein levels. This suggests that loss of AMC11 function yields a specific defect in the translation of the nd4 transcript. This is in accord with previous studies showing that loss of function mutations in the nd4 gene yields a CI‐deficiency with accumulation of a subcomplex, similar to the observed phenotype in the amc11. Thus, our forward genetic screen has led to the discovery of AMC11, a novel nuclear‐encoded protein required for the translation of the mitochondrial nd4 transcript.Grant Funding Source: The Ohio State University
Insulin‐dependent mitochondrial Ca2+ uptake in skeletal muscle is quickly disrupted in high‐fat diet fed mice (572.3)
Authors: Contreras‐Ferrat Ariel , Llanos Paola , Jaimovich Enrique 
Published: June 2021

The mitochondrial function quickly responds to energy‐dense food supply in order to deal with the demand but the role of mitochondrial Ca2+ in these processes has not been understood.Male C57BL/6J mice were fed a normal chow diet (NCD) or a high fat diet (HFD) for 1 week. The insulin resistance was evaluated by IPGTT, fasting glucose and insulin, and HOMA‐IR. GLUT4myc‐eGFP was electroporated in the Flexor digitorum brevis muscle (FDB).Insulin induces an increase in cytoplasmic and mitochondrial Ca2+ signal in adult fibers. The uncoupler FCCP, releases Ca2+ from mitochondria to cytoplasm. Insulin‐dependent mitochondrial Ca2+ uptake is decreased in fibers from short‐term HFD fed mice while insulin‐dependent cytoplasmic Ca2+ increase appear to be faster and stronger. In fibers from NCD fed mice, insulin‐dependent mitochondrial Ca2+ uptake was inhibited by xestospongin B, a specific IP3R inhibitor. Using TMRE+ in the non‐quenching mode (5 nM), we found that the ΔΨm was larger in fibers from short‐term HFD fed mice v/s NCD derived fibers. Using TMRE+ in quenching mode (100 nM), we found that the TMRE+ released after uncoupler stimuli was higher in fibers from short‐term HFD fed mice v/s NCD fed mice. The glucose analogue (2‐NBDG) uptake and the redistribution of GLUT4myceGFP induced by insulin were decreased in presence of xestospongin or ruthenium red (MCU inhibitor). This effect suggest a retrograde regulation of insulin signaling by mitochondrial Ca2+ uptake.Grant Funding Source: Supported by FONDECYT 11130267 and ACT1111
Structural insights into the thermal stability of endo‐mannanase belonging to family 26 from Bacillus sp. CFR1601 (580.2)
Authors: Srivastava Praveen , Appu Rao Appu Rao , Kapoor Mukesh 
Published: June 2021

Structural insights into the thermal stability of endo‐mannanase belonging to family 26 from Bacillus sp. CFR1601 Praveen K. Srivastava, Appu Rao G. Appu Rao, Mukesh Kapoor: Department of Protein Chemistry and Technology, CSIR‐Central Food Technological Research Institute, Cheluvamba Mansion, Mysore‐570020.Endo‐β‐mannanase (endo‐1,4‐β‐mannosidase; EC 3.2.1.78) is the key enzyme that catalyzes random hydrolysis of β‐mannosidic linkages in mannans and heteromannans. Mannanases play an important role in modification of gums, oil drilling industry and in the preparation of pre‐biotics for food industry. An endo‐mannanase obtained from soil bacterium Bacillus sp. CFR1601 was purified to apparent homogeneity and characterized. Partial biochemical and molecular characterization of purified enzyme reveals that it belongs to the glycosyl hydrolase family 26 with highest specific activity (10,400 ± 100 IU/mg) in its class. The enzyme retains its structure and activity up to 700C with activation energy of 10.8 ± 1.0 kcalmol‐1. Based on kinetics of thermal inactivation, enthalpy, free energy and entropy were found to be 86.7± 0.0 kcalmol‐1, 24.192± 0.7 kcalmol‐1 and 186.3± 0.21cal mol‐1 K‐1, respectively. Thermal stability measurements in presence of additives suggested the role of surface charges in thermal inactivation. Modification of surface charges could lead to development of highly stable and active endo‐mannanase for food and oil drilling applications.Source of research support: Council of Scientific and Industrial Research (CSIR), New Delhi, India
Engineering a delta protein kinase C inhibitor that selectively inhibits interaction and phosphorylation of one substrate of the multi‐substrate kinase (582.3)
Authors: Qvit Nir , Disatnik Marie‐Helene , Mochly‐Rosen Daria 
Published: June 2021

Rationale: Many kinases phosphorylate multiple substrates. To assess the role of an individual phosphorylation event without using laborious mutagenesis studies, selective pharmacological tools that inhibit phosphorylation of specific one substrate are needed. We focused on delta protein kinase C (δPKC) because δPKC activation after heart attack leads to cardiac damage. We reasoned that since pyruvate dehydrogenase kinase (PDK) phosphorylation by δPKC leads to inhibition of ATP production, it might be critical in the injurious effect δPKC after heart attack. To determine if of the many substrates of δPKC, PDK is the substrate that mediates this injurious effect in the heart, we designed a selective inhibitor of δPKC binding and phosphorylation of PDK.Methods: We designed a short peptide inhibitor that mimics a unique interaction between PDK and δPKC to selectively inhibit δPKC phosphorylation of PDK. This protein‐protein interaction inhibitory peptide (PPIIP) was used in vitro, in cells and in intact heart, in models of heart attack.Results: PDK/δPKC interaction was inhibited by PPIIP in vitro and in vivo. PDK/δPKC PPIIP selectively inhibited phosphorylation of PDK without affecting phosphorylation of several other δPKC substrates. Treatment with PDK/δPKC PPIIP inhibited myocardial infarction‐induced PDK phosphorylation without inhibiting the phosphorylation of other δPKC substrates. Further, PDK/δPKC PPIIP treatment led to a 50% reduction in infarct size, in release of cardiac enzyme (a measure of cell lysis) and in JNK phosphorylation, all markers of cardiac injury.Conclusion: PDK/δPKC PPIIP is a selective inhibitor of PDK phosphorylation by δPKC. Of the several δPKC‐mediated phosphorylation events following heart attack, δPKC‐induced PDK phosphorylation is sufficient to cause cardiac injury as a selective inhibitor of this phosphorylation event alone was sufficient to provide great reduction in δPKC‐mediated cardiac injury after heart attack.
Mass spectrometry characterization of trypanothione and novel peptides of medical importance isolated from Acanthamoeba polyphaga (585.1)
Authors: Ondarza Raul 
Published: June 2021

This paper presents unequivocal results about the presence of trypanothione and its precursor glutathione‐spermidine from the opportunistic human pathogen Acanthamoeba polyphaga.They were isolated by RP‐HPLC as thiol‐bimane derivatives and characterized using matrix‐assisted laser desorption ionization‐time‐of‐flight (MALDI‐TOF/TOF).Besides trypanothione, we want to report four new peptides in trophozoites, a tetrapeptide, a hexapeptide, a heptapeptide and a nonapeptide. Trypanothione and two of the thiol peptides, the hexapeptide and heptapeptide, are oxidized since the reduced forms increase in amount when the normal extract is treated by DTT or by electrolytic reduction that convert the oxidized forms to reduced ones. On the other hand, they disappear when the amoeba extract is treated with NEM or when the amoeba culture is treated with various inhibitors of NADPH‐dependent disulfide reducing enzymes.Comparison of the thiol peptides, including trypanothione from A. polyphaga with extracts from human lymphocytes showed that they are not present in the latter. Therefore, some of the peptides here reported could be used as antigens for rapid detection of these parasites.In regard to the presence of the enzymes that synthesize and reduce trypanothione in A. polyphaga we suggest that they can be used as drug targets.Grant Funding Source: no grant funding
Computational fluid dynamic modeling to predict wall shear stress in the cephalic vein of human subjects (588.1)
Authors: Hammes Mary , Boghosian Michael , Kevin Cassel , Hines Jane 
Published: June 2021

Background Cephalic arch stenosis (CAS) leads to access failure in hemodialysis patients with brachiocephalic fistula. The objective of this study was to use computational fluid dynamic modeling (CFD) to predict wall shear stress (WSS) in fistula access to determine the effects of increased arterial flow in the cephalic arch. Methods A venogram, Doppler and whole blood viscosity were performed pre‐fistula and at 3 months. Geometric measurements of arch angle and vein diameter were made. CFD of the cephalic arch was created to predict wall shear stress throughout the vessel. Results Eleven subjects receiving hemodialysis were studied. The arch angle decreased with a mean average of 133° at baseline to 116° at three months (p = 0.002). The venous diameter increased from 0.26 cm to 0.44 cm (p = 0.002). The blood flow increased from 3.95 cm/sec to 47.14 cm/sec (p < 0.001). A comparison of the CFD streamline plots between baseline and three months showed angle reduction, coupled with high flows leading to flow separation in most cases at three months. Flow separation was associated with a change in flow direction and low WSS. Conclusion Fistula creation results in increased arterial pressure and flow in the native vein. The vein dilates over three months with an observed decrease in [LOCAL] WSS made worse by the acute angulation of the arch, both contributing to intimal hyperplasia and CAS. Grant Funding Source: Supported By: NIH‐NDK 1 RO1 DK090769
Imaging enzymes at work: metabolic mapping by enzyme histochemistry to study functional mechanisms of the isocitrate dehydrogenase IDH1 mutation in glioblastoma (59.3)
Authors: Van Noorden Cornelis 
Published: June 2021

Activity localization of NADP+‐ dependent isocitrate dehydrogenases (IDH) was studied quantitatively using metabolic mapping and image analysis to study the metabolic consequences of the IDH1 mutation in glioblastoma, the most aggressive form of brain tumors. The mutation is found in 70‐80% of secondary glioblastoma patients. Metabolic rewiring of cells due to the mutation causes gliomagenesis by the production of 2‐hydroxyglutarate. On the other hand, glioblastoma patients with the IDH1 mutation have a significant survival benefit of approx. one year. We found that the NADPH production capacity in glioblastoma is reduced by 38% when IDH1 is mutated (Bleeker et al. 2010). We hypothesized that this reduced production capacity of NADPH, a major metabolic involved in detoxification processes, renders glioblastoma cells less resistant against irradiation and chemotherapy and thus prolong patient survival (Baldewpersad Tewarie et al. 2013). We also demonstrated with metabolic mapping that cellular metabolism is rewired in IDH1‐mutated glioblastoma cells by elevated mitochondrial activity (Navis et al. 2013). Further metabolic mapping of human and rodent tissues showed that IDH activity is responsible for 65% of the NADPH production capacity in human brain and only for 15% in rodent brain whereas the pentose phosphate pathway was responsible for 85% of the NADPH production capacity in rodent tissues (Atai et al. 2011). It explains why the pentose phosphate pathway was always linked with cancer and not IDH because most studies on metabolism and cancer have been performed in rodents. This study clearly shows the importance of metabolic mapping as a histochemical tool to understand the consequences of a mutation in the gene of an enzyme such as IDH1.Grant Funding Source: none
A novel NUT translocation partner binds to BRD4 and is necessary for the blockade of differentiation in NUT midline carcinoma (59.8)
Authors: French Christopher , Walsh Erica , Kuhnle Simone , Rahman Shaila , Grayson Adlai , Lemieux Madeleine , Howley Peter 
Published: June 2021

NUT midline carcinoma (NMC) is an aggressive type of squamous cell carcinoma that typically harbors BRD4/3‐NUT fusion oncogenes which encode chimeric proteins that block differentiation and maintain tumor growth. However, in 30% of cases NUT is fused to yet to be identified non‐BRD gene(s). Using RNA sequencing, we identified a novel gene fused to NUT in a NMC cell line (1221), fusing a novel 5’ coding sequence to the NUT gene. Like Brd4/3, this gene encodes a protein that is also involved in epigenetic regulation, and we find that siRNA knockdown of this protein leads to differentiation of the 1221 cells as well as of three BRD4‐NUT‐positive NMC cell lines. We have established that the novel NUT fusion protein encoded by this translocated gene binds to BRD4. We have mapped the regions of Brd4 and of the novel fusion protein involved in complex formation and are exploring whether association of the novel protein with Brd4 is required for the blockade of differentiation in NMC. Differentiation of TC‐797 cells induced by knockdown of BRD4‐NUT is abrogated by enforced expression of the novel NUT‐fusion gene. Together, these findings identify a novel BRD4‐NUT‐interacting protein whose expression is required for the maintenance of the undifferentiated state in NMC, and when fused to NUT, is oncogenic and can recapitulate the function of BRD4‐NUT to block differentiation.Grant Funding Source: NCI
Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma (591.2)
Authors: Li Menglin , Zhao Mindi , Gao Youhe 
Published: June 2021

Biomarker studies commonly monitor the composition of plasma, which is under strict homeostatic control, while urine is not. Urine is a partial filtrate of blood; and its composition reflects systemic physiology. Here we show that changes in the coagulation status of blood can be more sensitively detected in urine than in plasma. Plasma and urine protein composition was analysed by LC‐MS/MS and Western blot in six SD female rats before and after treatment with heparin or argatroban. LC‐MS/MS and Western blot analyses demonstrated drug‐induced increases in transferrin and hemopexin levels in urine but not in plasma. Our data indicates that urine may serve as a source for more sensitive detection of protein biomarkers than plasma.Grant Funding Source: Supported by the National Basic Research Program of China
Analysis of the oligomerization propensity of prohibitin1 (603.1)
Authors: Padilla‐Meier Gay Pauline , Meier Markus , Stetefeld Jorg , Mishra Suresh 
Published: June 2021

Prohibitins (PHB) are highly conserved and ubiquitously expressed proteins in eukaryotic cells. The prohibitin family exist in two isoforms PHB1 (also known as BAP32) and PHB2 ( also known as BAP37 and REA). The PHB1 and PHB2 together form large ring shaped complexes in the inner‐mitochondrial membrane, although PHB has also been reported to be localized in the nucleus and the cell membrane. PHB proteins have been implicated in mitochondrial biogenesis as well as scaffold for mitochondrial proteins. Details on the structure and function of PHB1 are still unclear. To better understand the functional mechanisms of PHB1 , we conducted biophysical analysis on recombinant human PHB1 and a truncated form of PHB1 (aa 52‐197). Our results show that PHB1 variants can form oligomers, and that varying salt concentration can modulate the polymerization of PHB1. Furthermore, addition of iron to PHB1 in solution affects the stability of the PHB1 oligomers. These data suggest that PHB1 could have diverse oligomeric forms depending on its local environmentGrant Funding Source: Supported by: Manitoba Health Research Council,
Role of RKTG, a novel Golgi localized receptor, in Gβγ regulated transport of secretory proteins from the Golgi to the plasma membrane (604.4)
Authors: Hewavitharana Thamara , Wedegaertner Philip 
Published: June 2021

Recent studies have revealed a role for G protein betagamma signaling at the Golgi via protein kinase D (PKD) regulating fission of plasma membrane (PM) destined secretory vesicles from the trans Golgi network (TGN). The mechanism by which the Gbetagamma subunits are recruited to and activated at the Golgi in this process remains elusive. Recent studies reveal that a newly characterized Golgi localized, membrane protein belonging to the Progestin and AdipoQ Receptor (PAQR) family, RKTG (Raf Kinase Trapping to the Golgi), interacts with the Gbeta subunit and sequesters Gbetagamma to the Golgi thereby regulating the functions of Gbetagamma. Here we show that over expression of RKTG causes vesiculation of the Golgi, while a Gbeta binding mutant of RKTG does not cause vesiculation. Also, the C‐terminal fragment of GRK2 (GRK2ct), which interacts with, and inhibits Gbetagamma signaling, and Gallein, a small molecule inhibitor of Gbetagamma, were both able to inhibit Golgi vesiculation in the presence of RKTG. Furthermore, a dominant negative form of PKD (PKD‐DN) and a pharmacological inhibitor of PKD, Go6976, also inhibited RKTG mediated vesiculation of the Golgi. Collectively, these results reveal a novel role for the newly characterized, Golgi localized RKTG receptor, in regulating Gbetagamma at the Golgi, thus controlling Golgi to PM protein transport via the Gbetagamma‐PKD signaling pathway.Grant Funding Source: NIH GM056444