bluuebunny/crossref_metadata_2025_split huggingface dataset:
Contribution of MAP Kinases ERK1/2 in Beta Cell Function
Authors: Dioum Elhadji Mamadou , Schneider Jay W. , Cobb Melanie H 
Published: June 2021

Pancreatic islet development and beta‐cell function are dependent on the coordinated activity of multiple islet‐enriched transcription factors, including PDX‐1, NKX6.1, MafA and BETA2. Somatic mutations in these factors have been implicated in maturity‐onset diabetes of the young (MODY). Understanding of the molecular mechanisms of pancreatic beta‐cell function and integrity is necessary for the development of anti‐diabetic drugs. Activation of the mitogen activated protein kinases (MAPKs) Extracellular signal‐related kinases 1 and 2 (ERK1/2) in beta cells is required for insulin gene transcription and secretion. Using in vitro and cell culture approaches, we showed that ERK1/2 phosphorylate BETA2, PDX‐1 and MafA and modulate their DNA binding activity and ability to form ternary complexes required for insulin gene transcription and beta‐cell function.Isoxazole, a compound identified in a small molecule screen, shows improved beta‐cell function through chromatin remodeling and transcription factor activation in a pleiotropic pathway involving ERK1/2. We are exploring the molecular mechanisms involved in this signal transduction pathway.Source of funding:NIH R37 DK34128NIH RO1 DIC55310
Mapping and measuring molecular networks in cells
Authors: Aebersold Ruedi 
Published: June 2021

At a molecular level biological systems such as cell signaling or metabolism can be represented as dynamic networks of interacting molecules. In the living cell multiple such networks are concurrently present and active. These include the gene regulatory (transcriptional) networks, protein interaction networks, networks of enzymes and their substrates (e.g. protein kinase substrate networks), networks of microRNA and their substrates etc. Notably, most of these types of networks consist of or involve proteins. Therefore, the systematic analysis or the proteins that constitute a biological system, their interactions and the networks they constitute is a central theme of the emerging field of systems biology.Mass spectrometry is the most widely used method for the analysis of proteins and proteomes. In this presentation we will discuss new mass spectrometry based technologies for the analysis of different types of cellular networks and their dynamic change as a function of cellular state.
Reduced mitochondrial respiration and improved coupling in infarcted heart following stem cell therapy
Authors: Hughey Curtis , Ma Lianli , James Freyja , Young Pampee Paul , Wasserman David , Rottman Jeff , Shearer Jane 
Published: June 2021

AimTo determine the changes in mitochondria following stem cell therapy in the infarcted heart.MethodsC57BL/6J mice were randomly divided into three treatment groups, SHAM (n=8), MI (myocardial infarction)(n=9) and MI‐STEM (n=8) receiving direct myocardial injection of mesenchymal stem cells. Echocardiography was performed at baseline, 1 and 3 wk post‐infarct. Mitochondrial respiration (pmol/s*mgww/IUCS) in permeabilized fibers bordering the infarct was determined at 4wk following treatment. State 2 (absence of ADP) respiration, state 3 (ADP‐stimulated) respiration and state 4 respiration were examined. Citrate synthase activity (CS) was determined.ResultsMI‐STEM state 2 was lower than the MI and SHAM groups (20.98 vs. 29.46 and 32.45). State 3 was lower in the MI and MI‐STEM groups compared to the SHAM (77.01 and 68.46 vs. 97.18). Respiratory Control Ratio (RCR) was higher in the MI‐STEM compared to MI group (3.37 vs. 2.71) and state 4 was lower in the MI‐STEM compared to the SHAM group (67.93 vs. 96.23).ConclusionCell therapy does not prevent declines in state 3 respiration, however, the higher RCR and lower state 4 respiration suggests stem cell treatment reduces futile respiration and increases respiration efficiency. These findings show cell therapy to improve cardiac function by reducing the energetic mismatch between supply and demand following a MI. Research supported provided by CIHR.
In isolated rabbit renal proximal tubules, the antibiotic cephaloridine does not inhibit the mitochondrial uptake of succinate, a biomarker of its nephrotoxicity: A cellular metabolomic study with carbon 13 NMR
Authors: Baverel Gabriel , Cadi‐Soussi Sophia , Martin Guy 
Published: June 2021

Cephaloridine is a model nephrotoxicant. In isolated rabbit renal mitochondria, cephaloridine (i) inhibits succinate uptake and (ii) reduces respiration. We have re‐examined this problem using isolated proximal tubules. Tubules were incubated in Krebs‐Henseleit buffer containing either 1,4‐13C‐succinate, or 2,3‐13C‐succinate or succinate + 13C‐bicarbonate without or with cephaloridine. Substrate removal and product formation were measured by enzymatic and carbon 13 NMR methods. Enzymatic fluxes were calculated by combining these results with an original mathematical model of renal succinate metabolism. Succinate was converted mainly into fumarate, malate, glucose and CO2. Cephaloridine induced an increase in the accumulation of fumarate, malate and a decrease in the production of glucose, CO2 and the level of ATP. Surprisingly, it did not change the removal of succinate. Fluxes through malate dehydrogenase, glucose 6‐phosphatase and citrate synthase were also diminished by cephaloridine.In conclusion, the absence of inhibition of mitochondrial uptake of succinate in intact renal proximal tubules strongly suggests that results obtained with isolated mitochondria do not necessarily apply to mitochondria in intact cells and, therefore, should be interpreted with great caution. These results also suggest that mitochondrial functions should be studied in intact cells.
ZBTB5 is a potential tumor suppressor in the absence of functional p53
Authors: Koh Dong‐In , Hur Man‐Wook 
Published: June 2021

Previously, we isolated and characterized a novel POZ domain Krüppel like zinc finger transcription repressor, ZBTB5 (Zinc finger and BTB domain containing 5). SAGE analysis showed that ZBTB5 expression is higher in retinoblastoma and muscle cancer tissues. Our data suggested that ZBTB5 is a potent transcription repressor of cell cycle arrest gene p21 and a potential proto‐oncogene stimulating cell proliferation.Interestingly, in the HCT116 p53 null cells lacking functional tumor suppressor p53, expression of ZBTB5 was greatly induced prior to induction of cell cycle arrest gene p21 when cells were expose to DNA damaging agent, etoposide. ZBTB5 potently activated transcription of p21 gene in the absence of p53. ZBTB5 and Sp1 synergistically activates transcription by acting on the p21 promoter lacking distal p53 binding elements both in the p53 null and p53 positive cells. Furthermore, ZBTB5 increased transcription of p21 in the presence of dominant negative mutant form of p53. Our data suggest that ZBTB5 has functional duality in the regulation of cell proliferation and can acts either as oncogene or tumor suppressor depending on presence or absence of functional p53.
UNRAVELING A POSSIBLE CELLULAR MECHANISM BEHIND LEPTOMENINGEAL AMYLOIDOSIS USING AS MODEL A HIGHLY UNSTABLE TRANSTHYRETIN TETRAMER
Authors: Foguel Debora , Azevedo Estefania , Palhano Fernando , Lima Flávia 
Published: June 2021

Transthyretin (TTR) is a 127‐residue homotetrameric β‐sheet‐rich protein that transports thyroxine in the blood and cerebrospinal fluid (CSF). In the present work we aimed to study A25T dissociation, aggregation and its role in leptomeningeal amyloidosis (LA). Using HPLC and native PAGE we monitored acrylodan‐labeled TTR aggregation in the plasma. The aggregates formed, when analyzed by AFM, Thioflavin T and Congo Red binding displayed amyloid structure. We solved the crystal structures of the wt and A25T and the comparative analysis shed light into the mechanism behind A25T highly amyloidogenicity: an expanded tetramer which is stabilized by a lower number of H‐bonds and hydrophobic interactions. Interestingly, in the presence of thyroxine and flufenamic acid (an anti‐inflammatory compound), two ligands of TTR, the structure of A25T was similar to that displayed by the wt protein. Since LA symptoms suggest a local inflammation, we questioned ourselves whether A25T aggregates can induce an inflammatory response in the CNS. Microglia primary cultures incubated for 48h with aggregates composed of A25T, underwent activation, phagocytosis (which could be blocked by cytochalasin D) and presented a significant nitric oxide and IL‐1 beta secretion. These results suggest that amyloid fibrils may have a role in LA pathogenesis, exacerbating tissue damage through inflammation.Support: CNPq and FAPERJ.
The effect of the sn‐2 acyl chain of diacylglycerol on its interaction with C1 domains and its signaling role
Authors: Kurilova Svetlana , Yoon Youngdae , Lee Park Joo , Bhardwaj Nitin , Lu Hui , Cho Wonhwa 
Published: June 2021

Diacylglycerol (DAG) is a signaling lipid that interacts with the C1 domain in various target proteins, including protein kinases C, and Ras guanyl nucleotide‐releasing proteins, thereby recruiting them to the plasma membrane. Although these proteins can all bind DAG, some are known to target other intracellular membranes. To understand the basis of this differential membrane targeting, we explored the possibility that a difference in the acyl chain composition of DAG in the plasma membrane and other membranes may affect the C1‐DAG interaction. To test this notion, we measured the binding of the various C1 domains to DAGs with different sn‐2 acyl groups by fluorescence correlation spectroscopy (FCS). The C1 domains were expressed as an enhanced green fluorescence protein‐fusion protein to enhance protein stability and allow FCS measurements. FCS measurements as well as the molecular dynamics simulation show that the sn‐2 acyl group of DAG has significant and differential effects on the membrane binding properties of C1 domains, which is consistent with the differential subcellular localization of the C1 domains.Supported by NIH GM76581
Phosphatidylserine binding is important for plasma membrane targeting of 3′‐phosphoinositide‐dependent kinase‐1 pleckstrin homology domain
Authors: Lucas Nathan Thomas , Kim Hee‐Yong , Cho Wonhwa 
Published: June 2021

3′‐Phosphoinositide dependent kinase (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide‐3‐kinase activation. An increase in phosphatidylinositol‐3,4,5‐trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol‐3,4‐bisphosphate (PtdIns(3,4)P2) is thought to recruit PDK1 and its substrates to the 3′‐phosphoinositide‐rich regions of the plasma membrane for subsequent phosphorylation. PDK1 contains a C‐terminal catalytic domain and an N‐terminal pleckstrin homology (PH) domain that was reported to have high affinity for PtdIns(3,4,5)P3 and PtdIns(3,4)P2. To better understand how lipids regulate the subcellular localization and activation of PDK1, we rigorously investigated the membrane binding properties of PDK1 PH domain by surface plasmon resonance analysis. The results show that the PDK1 PH domain has high affinity for not only PtdIns(3,4,5)P3, PtdIns(3,4)P2 and phosphatidylinositol‐4,5‐bisphosphate (PtdIns(4,5)P2) but also for phosphatidylserine (PS). We identified the separate binding sites for phosphoinositides and PS through molecular modeling and mutagenesis and analyzed the differential effects of phosphoinositides and PS in membrane recruitment of PDK1.Supported by a NIH grant GM68849.
Mass Spectrometry and its contribution to hybrid structure determination
Authors: Robinson Carol 
Published: June 2021

Standard proteomics techniques are unable to describe the stoichiometry, subunit interactions and organization of assemblies since many are heterogeneous, present at low cellular abundance and frequently difficult to isolate. We have combined two existing methodologies to tackle these challenges: affinity purification and nanoflow ESI‐MS. We use methods designed to maintain non‐covalent complexes within the mass spectrometer to provide definitive evidence of interacting subunits based on the masses of complexes and subcomplexes generated by perturbation both in solution and gas phases. Structural models will be presented for oligomeric protein complexes with different degrees of structural information including the human U1snRNP and eIF3 complexes. These models will then be examined within the context of their function.Recent developments in mass spectrometry have added a further dimension to our studies of protein complexes: that of their collision cross‐section. Using ion mobility mass spectrometry we have been able to add spatial restraints to our models validating our models with measurements of collision cross–sections.Very recently we have had a considerable breakthrough which has enabled us to preserve intact membrane complexes in the gas phase. This enables us to establish lipid and nucleotide binding and to define the stoichiometry and post translational modifications within the intact transmembrane regions of a number of complexes. I will demonstrate some of the advantages of this approach by presenting recent insights into the structures of intact V‐type ATP synthases.
Interaction analysis of TcrX/Y two component system from Mycobacterium tuberculosis
Authors: Bhattacharya Monolekha , Biswas Ashis , Das Amit Kumar 
Published: June 2021

TcrX/Y is one of the twelve two component system (TCS) present in Mycobacterium tuberculosis. The objective was to investigate the TcrX/Y interaction by in silico studies, pull down assay, radioactive phosphotransfer, surface plasmon resonance as well as crosstalk analysis of TcrY with TcrA ‐ a non‐cognate response regulator. Sequence alignment of TcrY with other histidine kinases revealed His256 as the residue responsible for autophosphorylation. Modeled structure of TcrX predicts that Asp59 may be involved in phosphotransfer with His256 of TcrY. Docking analysis pointed to the interaction of TcrY/His256 with TcrX/Asp54. TcrY/Y dimerisation was also observed by in silico and crosslinking studies. Autophosphorylation of TcrY has been observed followed by the phosphate transfer to TcrX. The phosphorylation process required divalent metal ions like Mg2+ or Ca2+ ions as evident from the radioactive phosphorylation studies. Interaction was not observed in TcrY/A suggesting the signal transduction process is specific in TcrX/Y system. TcrY hydrolyzes ATP and the Km value has been found to be 10 mM. TcrX/Y interaction has been determined by surface plasmon resonance and dissociation constant (KD) was evaluated to be 3.6 μM. We conclude from our results that TcrX/Y are part of the same signal transduction pathway without their involvement in crosstalk with non‐cognate counterpart.The work was funded by Department of Biotechnology, Govt. of India.
TCF‐1 Regulation By A Wnt‐Kinase Network In Colon Cancer
Authors: Najdi Rani , Theisen Heidi , Atcha Fawzia , Edwards Robert , Marsh J. Lawrence , Waterman Marian L. 
Published: June 2021

TCF‐1 and TCF‐4 are the major transducers of Wnt signaling in the intestine and the oncogenic drivers of colon cancer. However, knock‐out of the TCF1 gene leads to intestinal tumors while knockout of TCF‐4 suppresses growth and maintenance of crypt stem cells. These phenotypes suggest that TCF‐4 is Wnt‐promoting and TCF‐1 is a tumor suppressor. We show that a truncated dominant negative isoform of TCF‐1 (dnTCF‐1) predominates in the normal colon and is equally distributed between nuclear and cytoplasmic compartments in normal crypts. However, in primary human colon tumors, TCF‐1 is often cytoplasmic while TCF‐4 remains nuclear. This shift to the cytoplasm is regulated by a CaMKII/TAK1/NLK kinase cascade that triggers secretion of a Wnt ligand to selectively export TCF‐1 from the nucleus. In colon cancer, TCF‐1 expression is altered so that dnTCF‐1 is downregulated while to full‐length, Wnt‐promoting isoforms are upregulated. Our study shows that control of nuclear export is an important mechanism for regulating the balance of TCFs and that a Wnt/CaMKII kinase signal plays a key role in modulating Wnt activity in the human colon.Supported by NIH
Regulation of the LEF1 gene's alternative promoter
Authors: Pate Kira T , Yokoyama Noriko N , Waterman Marian L 
Published: June 2021

LEF1 is a DNA‐binding transcription factor and a downstream component of the Wnt signaling pathway. The LEF1 gene has multiple promoters and produces two distinct proteins: a full‐length, growth‐promoting form produced from promoter 1 (P1), LEF1, and a truncated, growth‐suppressing form produced from promoter 2 (P2), dnLEF1. In tissues where LEF1 is normally expressed, both promoters are active, producing both forms of LEF1. However, in colon cancer, LEF1 is aberrantly expressed, and only the full‐length, growth‐promoting protein is produced. P1 is a Wnt target, explaining its transcriptional upregulation in Wnt‐driven colon cancers. P2 is silent because it is actively repressed. A genomic region upstream of P2 that is necessary for its repression has been delimited, however, the protein components and exact mechanism of repression are unknown. In this study we describe one component of the repressor machinery, identified through site‐directed mutagenesis of the LEF1 core promoter 2. A YY1 binding site at the +25 location was identified, which, when mutated, greatly reduces repressor function. The binding of YY1 to the +25 site in vitro is shown by gel shift, while ChIP suggests in vivo binding of YY1 within a colon cancer cell line. The functional role of YY1 is confirmed through siRNA knockdown of YY1, which results in loss of repressor function. These findings suggest a pivotal role for YY1 in repression of P2.Supported by NIH.
The Parkinson's disease associated LRRK2 exhibits weaker in vitro phosphorylation of 4E‐BP compared to autophosphorylation
Authors: Kumar Azad , Greggio Elisa , Beilina Alexandra , Kaganovich Alice , Chan Diane , Wolozin Ben , Cookson Mark 
Published: June 2021

Mutations in the gene encoding leucine‐rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). LRRK2 is a multi‐domain protein containing a central catalytic core, with GTPase and kinase domains, surrounded by a number of protein‐protein interaction domains. An important step forward in the understanding of the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological kinase substrates. In the present study we examined phosphorylation of 4E‐BP (eukaryotic initiation factor 4E (eIF4E)‐binding protein), a recently proposed substrate for LRRK2 and confirmed that LRRK2 is capable of phosphorylating 4E‐BP in vitro. The PD related LRRK2‐G2019S mutant was ~2 fold more active than wild type protein. Interestingly, LRRK2 autophosphorylation, which we have mapped to the GTPase domain, was stronger than 4E‐BP phosphorylation under conditions of molar excess of 4E‐BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E‐BP at the same site as LRRK2. Overall, our results suggest that 4E‐BP is a relatively poor direct substrate for LRRK2 compared to autophosphorylation. We suggest that increased 4E‐BP phosphorylation reported in some systems may be related to p38‐mediated cell stress rather than direct LRRK2 activity.This research was supported by the Intramural Research Program of the NIH, National Institute on Aging
Modification of a 3‐d deuterated‐retinol dilution (DRD) equation for estimation of vitamin A (VA) stores in humans
Authors: Duca Frank Anthony , Green Michael H 
Published: June 2021

Isotope dilution analysis has been successfully used to estimate vitamin A stores in humans via the Furr‐Olson DRD equation: estimated total body stores = F × dose × {S ×a × [(1/D:H) −1]} (Am J Clin Nutr 1989;49:713). The method requires a blood sample 21 d after isotope administration and involves several assumptions (factors F, S and a). Using VA tracer data from rat and human studies, we refined a modified DRD equation (J Nutr 2008; 138:971) that uses a 3‐d sample, a time that is more feasible in the field. We found that the VA specific activity ratio in plasma vs. stores (factor S in DRD equation) varies with VA status in both rats and humans. Also, the amount of tracer irreversibly lost between dose administration and blood sampling (factor a) increases as status decreases. In contrast, retention (factor F) increases as status increases. Applying these findings to the original DRD equation, we developed a new equation in which S, F, and a vary with a subject's 3‐d plasma VA deuterium:hydrogen (D:H) ratio. For data from humans of varying age, gender and ethnicity, VA stores predicted by the new equation agree better with estimates from the original 21‐d Furr‐Olson equation than do values obtained using other published 3‐d DRD equations. Thus the new equation provides an improved tool to determine VA stores in humans.Funded by the College of Health and Human Development, Penn StateGrant Funding Source: The College of Health and Human Development
Hepatic iron loading increases rat ZIP8 expression
Authors: Wang Chia‐Yu , Nam Hyeyoung , Jenkitkasemwong Supak , Duarte Stephanie 
Published: June 2021

Zip8 is a member of the ZIP (Zrt‐, Irt‐like Protein) family of metal‐ion transporters. Zip8 amino acid sequence shares approximately 60% homology to Zip14, which can transport both zinc and iron. Similar to Zip14, overexpression of rat Zip8 in HEK293T cells significantly enhances the uptake of iron. In the present study, Western blot analysis revealed ~3‐fold higher levels of Zip8 in iron‐loaded rat liver compared to iron‐normal controls (mean non‐heme iron concentrations of 1813 vs. 30 μg/g liver, respectively, n=6/group). Higher Zip8 protein levels were not associated with elevated Zip8 transcript abundance, suggesting post‐transcriptional regulation. In the H4IIE rat hepatocyte cell line, we further demonstrated that Zip8 is glycosylated and detectable at the cell surface. These data suggest that Zip8 may contribute to hepatic iron transport, particularly during states of iron overload. Supported by NIH.Grant Funding Source: NIH
Zinc Deficiency Induces Apoptosis but Zinc Induces Necrosis in Rat Hepatoma Cells
Authors: Sankavaram Kavitha , Chong Leelyn , Bruno Richard S , Freake Hedley C 
Published: June 2021

Altered concentrations of intracellular zinc influence cellular homeostasis and are believed to modulate apoptosis. We reported earlier that both zinc supplementation and DTPA (diethylenetriaminepentaacetic acid) induced zinc deficiency generate reactive oxygen species (ROS) and decrease cell viability in rat hepatoma (H4IIE) cells. In this study, we examined the effects of altered zinc availability on mode of cell death, signaling pathways and associated morphological changes in these cells. Fluorescence microscopy of cells with Hoechst 33342 and PI indicated that DTPA induced apoptosis but zinc induced both necrosis and apoptosis. Cells were also stained with both PI and FTTC‐labeled annexin V and then analyzed by flow cytometry. The results confirmed that DTPA induced apoptosis (20%) whereas zinc induced both necrosis (50%) and apoptosis (10%). Zinc induced necrosis prior to apoptosis and at a greater rate. Features of apoptosis, such as chromatin condensation, nuclear fragmentation, elevated levels of p53, p21, and Fas/Fas ligand mRNA accompanied DTPA and zinc induced cell death. Decreased expression of Bcl2 and elevated expression of Bax were observed with DTPA but not with zinc treatment. These results indicate that zinc deficiency leads to mitochondrial dependent apoptosis, modulating ROS and apoptosis proteins in H4IIE cells. However, elevated zinc leads predominantly to cell necrosis.Grant Funding Source: UConn Research Foundation