bluuebunny/crossref_metadata_2025_split huggingface dataset:
New copper binding components of blood plasma (992.1)
Authors: Dalphin Matthew , Salazar Dianne , Paik Samantha , Kidane Theodros , Vulpe Christopher , Page Kathryn , Linder Maria 
Published: June 2021

It has been assumed for some time that the main Cu‐containing proteins in blood plasma are ceruloplasmin (Cp), albumin and transcuprein/a2‐macroglobulin, Cp being the main ferroxidase (Fx) implicated in mediating efflux of iron from certain cells. Recent studies in our laboratory suggest the picture is much more complex, and that other known and unknown factors containing Cu are still to be identified. We had previously shown in mice that knocking out Cp only knocked down plasma Fx activity by half. We now have evidence that knocking out hephaestin (Hp) a membrane‐tethered Cp‐homologue reduces plasma Fx activity and a peak in the Cu profile obtained by size exclusion chromatography (SEC), indicating that solubilized Hp may be entering the circulation. Also, a patient with twice normal level of plasma Cu but Cp protein in the normal range had twice normal levels of Fx activity, suggesting that ratios of holo to apo‐Cp were elevated or that another Fx increased in response to the contraceptives. Use of the anticoagulant, EDTA, to collect plasma had a dramatic effect on SEC Cu profiles, moving a large proportion of Cu in larger components (but not Cp) to one of low molecular weight normally present in trace amounts, which is not Cu‐EDTA or metallothionein. We conclude that the proportions of plasma Cu associated with Cp and Fxs can vary widely and speculate that a copper peptide normally attached to other proteins is dislodged by EDTA.Grant Funding Source: HHMI 52007532
Structural and functional characterization of a heavy metal detoxifying ABC transporter (997.2)
Authors: Lee Jonas , Yang Janet , Zhitnitsky Daniel , Lewinson Oded , Rees Douglas 
Published: June 2021

The superfamily of ATP Binding Cassette (ABC) transporters includes importers and exporters that translocate a wide variety of substrates across the cell membrane. Although significant progress has been achieved in the structural analysis of ABC exporters, much less is known about how ABC exporters selectively recognize varying substrates, and couple its binding to ATP hydrolysis. We have addressed these questions by the crystallographic and functional characterization of a dimeric Atm1/ABCB7/HMT1/ABCB6 bacterial ortholog. Glutathione and it's derivatives are demonstrated to serve as substrates. In vivo, this transporter confers resistance to silver and mercury toxicity to E. coli, implicating a physiological role in cellular detoxification processes. The 2.4 Angstrom resolution structure allowed detailed mapping of the binding interactions highlighting the articulated design of ABC exporters. The key ligand interactions are positioned at the boundaries between structurally conserved elements, which links ligand binding to the conformational changes underlying ATPase‐coupled substrate translocation.Grant Funding Source: National Institutes of Health
Binding affinity of epithelial sodium channel subunits and peptides measured through surface plasmon resonance (997.5)
Authors: Gonzalez Andres , Peltier Grant , Booth Rachell , David Wendi 
Published: June 2021

Salt levels play a crucial role in regulating blood pressure, and cells are able to maintain salt levels via ion channels embedded within the plasma membrane. The Epithelial Sodium Channel (ENaC) is a heterotrimeric membrane protein that allows the passage of sodium, and this study’s aim is to further understand what structural aspects of ENaC are responsible for maintaining proper function of the channel. Potential residues critical for interaction between ENaC alpha, beta, and gamma subunits were identified and peptide segments containing the targeted residues were PCR amplified, ligated onto a pGEX‐4t‐2 vector, and transformed into Top10 E. coli. This vector contains a GST‐tag that was used to purify the peptides and immobilize them for further analysis by surface plasmon resonance (SPR). Binding between sets of peptides predicted to be important for stabilizing interactions in ENaC was investigated using SPR. One peptide from a potential interacting set of peptide regions was immobilized through the GST‐tag and binding with the corresponding partner (with GST‐tag removed) was quantified. Two interactions ‐one between the alpha‐palm and beta‐thumb, and the other between the beta‐palm and gamma‐thumb regions of ENaC ‐ were analyzed. The alpha‐palm and beta‐thumb interaction had a measured KD of 2.9 x 10‐8 M, whereas the interaction from the beta‐palm, gamma‐thumb region was in the micromolar range for KD. Additional sets of peptides have been expressed and will be analyzed for binding interactions. Results obtained from this initial study will be compared to those obtained from a mutation screen of ENaC function to confirm whether the predicted residues play a crucial structural role related to function.Grant Funding Source: Supported by NIH R15 GM86798 (R. Booth & W. David) and Welch Foundation Departmental Grant AI‐0045.
Novel Role for PTEN in Regulating Non Homologous End‐Joining Factor 53BP1 to Suppress Replication Fork Instability (LB120)
Authors: Schlacher Katharina , Guo Wen , Jasin Maria , Wu Hong 
Published: June 2021

PTEN is one of the most frequently inactivated tumor suppressors with unresolved connections to DNA repair processes that prevent genomic instability. Using single molecule DNA spreading, we here show that PTEN suppresses replication fork instability by promoting efficient replication restart of stalled replication forks, a phenotype mirroring ATM and BLMs helicase functions. Moreover, we find that similar to BLMs, PTEN suppresses replication stress dependent DNA bridges. Previous studies show non‐homologous end‐joining (NHEJ) protein and ATM phosphorylation target 53BP1 in complex with BLMs at stalled fork, an interaction requiring phosphorylation of 53BP1. Yet, the role of 53BP1 at stalled forks remains ill‐defined. Strikingly, 53BP1 is highly phosphorylated in PTEN proficient cells upon replication stalling, but not in cells lacking PTEN. We further find 53BP1 to be readily recruited to nuclear foci upon replication stalling. Yet foci formation is compromised in PTEN defective cells, implying PTEN controls a function for 53BP1 when replication forks are stalling. Lastly, we reveal a direct requirement for 53BP1 in restarting stalled replication forks. These data reveal a surprising function for NHEJ factors controlled by PTEN to suppress replication fork stability and genomic instability.Grant Funding Source: Supported by 1K22CA175262‐01 (K.S.) and UO1CA164188 (H.W.)
Substitutions in the Ω‐Loop of TEM β‐Lactamase Change the Substrate Profile of the Enzyme (LB140)
Authors: Stojanoski Vlatko , Hu Liya , Chow Dar‐Chone , Prasad BV , Palzkill Timothy 
Published: June 2021

Serine β‐lactamases are bacterial enzymes that hydrolyze β‐lactam antibiotics. Mechanistically, serine β‐lactamases are similar to serine proteases such as chymotrypsin. They both utilize acylation and deacylation of an active site serine in their mechanism of catalysis. TEM‐1, a common plasmid‐encoded serine β‐lactamase, catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a previously identified triple mutant of TEM‐1, 165‐TyrTyrGly‐167 (wild type165‐TrpGluPro‐167) with substrate specificity altered from preferential hydrolysis of ampicillin to that of ceftazidime. When compared to chymotrypsin, the Glu166Tyr substitution in the TEM‐1 triple mutant is analogous to a substitution of the general base His57 in chymotrypsin that results in an enzyme that maintains function. Our findings agree with previous observations of altered substrate specificity of the triple mutant, which displays increased hydrolysis of ceftazidime. Additionally, enzyme kinetic analyses show that the hydrolysis of ceftazidime follows a branched pathway characteristic of substrate‐induced reversible inactivation. Structural analysis of the triple mutant reveals enlargement of the active site making it more accessible for larger substrates. Furthermore, in the crystal structure of the triple mutant, Tyr166 is within hydrogen bond distance to Ser70 suggesting involvement in the catalytic mechanism of the enzyme. These findings indicate that Glu166 general base residue can be substituted in the mechanism of serine β‐lactamases. More generally, this provides insights into alternate mechanisms for reactions catalyzed by serine hydrolases.Grant Funding Source: Supported by NIH
Novel Transporters for Enzyme Replacement Therapy (LB148)
Authors: Tong Wenyong , Hamil Kristina , Tor Yitzhak , Esko Jeffrey 
Published: June 2021

Mucopolysaccharidoses (MPS) are inherited metabolic diseases caused by a deficiency in degradative enzymes that reside in lysosomes. A few of these disorders can be treated by enzyme replacement therapy. However, significant challenges remain because key organs and tissues remain inaccessible to intravenously injected enzymes. We are exploring the utility of guanidinoglycosides as carriers in vivo in mice. The approach is based on our previous observation that guanidinylated neomycin (GNeo) can deliver high molecular weight proteins across cell membranes and into lysosomes via cell surface heparan sulfate proteoglycans. All cells highly express heparan sulfate proteoglycans, suggesting the potential utility of GNeo‐conjugates for delivery to multiple tissues. Our study demonstrates that conjugation of guanidinylated glycosides to lysosomal enzymes can facilitate entry into the brain after intranasal administration, thus bypassing the blood brain barrier. This novel delivery system might be applicable to a variety of lysosomal storage disorders as well as for the delivery of antibodies and growth factors to the brain.Grant Funding Source: Supported by an NIH LDN post‐doctoral fellowship (W.T.) and grants from the MPS Society and from Improved Therapies for MPS I Program through the University of Pennsylvania (to J.D.E.).
X‐ray structure of the human mitochondrial kinase ADCK3 elucidates the kinase fold of the ancient UbiB family (LB173)
Authors: Reidenbach Andrew , Stefely Jonathan , Saunders Jaclyn , Johnson Isabel , Oruganty Krishnadev , Barber Grant , Li Sheng , Kannan Natarajan , Bingman Craig , Pagliarini David 
Published: June 2021

The UbiB family of kinases represents an estimated 25% of the protein kinase‐like (PKL) family members in the microbial kingdom, and members of this family are required for the biosynthesis of coenzyme Q from E. coli to humans. We present the first structure of a UbiB family member, the human mitochondrial kinase ADCK3, which is mutated in a human cerebellar ataxia (ARCA2) associated with coenzyme Q deficiency. The structure of ADCK3 reveals that the signature, invariant KxGQ motif of UbiB kinases interacts directly with the kinase active site, in close proximity to residues important for nucleotide binding. Differential scanning fluorimetry (DSF) and deuterium exchange mass spectrometry were used to confirm that this motif is important for proper nucleotide binding, and to establish that ADP binding has a global stabilizing effect on the kinase. Last, mapping ADCK3 patient mutations onto our structure suggests how these mutations can destabilize the kinase. We purified and analyzed the thermal stability of eight such ARCA2 patient mutations using DSF and found that all but three of these proteins are less stable than wild type ADCK3. Our findings form the foundation for studying the enzymatic activity of the UbiB family and for investigating how disruptions in this kinase can give rise to a human neurodegenerative disorder.Grant Funding Source: A.G.R. was supported by NIH Chemistry‐Biology Interface Training Grant NIGMS T32 GM008505, D.J.P. was supported by NIH Grant U01 GM94622 (Protein Structure Initiative: Biology, Mitochondrial Protein Partnership) and a Searle Scholars Award
Hypoxia impairs maturation of the Na,K‐ATPase (LB180)
Authors: Capri Joseph , Tokhtaeva Elmira , Sun Haying , Angulo Martin , Dada Laura , Sznajder Jacob , Kaplan Jack , Whitelegge Julian , Vagin Olga 
Published: June 2021

Renal tissue hypoxia is a major cause of acute renal failure. The goal of this study was to elucidate the mechanism(s) underlying role of the Na,K‐ATPase in the adaptive response of renal cells to a hypoxic stress. Kidney microsome membranes were isolated from mice exposed to 7% O2 (hypoxia) or to room air for three days, proteins were extracted by a non‐ionic detergent, immunoprecipitated using anti‐Na,K‐ATPase antibodies, and analyzed by nLC‐MS/MS and Western blot analyses. Hypoxia did not change the total amount of the Na,K‐ATPase, as was assessed by comparing the Mascot scores and confirmed by a Western blot analysis. In contrast, hypoxia significantly decreased the amount of the Na,K‐ATPase‐co‐immunoprecipitated ER chaperones and enzymes presumably involved in the maturation of the Na,K‐ATPase, including BiP, Hsp40, calnexin, PDI, glucosidase, mannosidase and DnaJ. Consistent with these results, a significant retention of the Na,K‐ATPase was observed in cultured epithelial cells exposed to hypoxia. The results suggest that the hypoxia‐induced ER retention of the Na,K‐ATPase slows down the maturation and hence the delivery of the newly synthesized pumps to the plasma membrane, resulting in a decrease in ATP consumption as an adaptive response to hypoxia.Grant Funding Source: Supported by 1R01HL113350‐01A1
Structural investigation of the folding of an immunoglobulin domain on the ribosome using NMR Spectroscopy (LB197)
Authors: Cassaignau Anais , Cabrita Lisa , Waudby Christopher , Wang Xiaolin , Launay Hélène , Camilloni Carlo , Karyadi Maria‐Evangelia , Chan Sammy , Vendruscolo Michele , Dobson Christopher , Christodoulou John 
Published: June 2021

Correct protein folding is key to a functional proteome, as globular proteins must acquire their correct three‐dimensional native fold in order to fulfil their biological role. A significant amount of folding occurs co‐translationally on the ribosome and this process is likely to differ from folding of isolated proteins not least through the attachment to the ribosome and the vectorial emergence of the nascent polypeptide from the ribosomal exit tunnel. We are using solution‐state Nuclear Magnetic Resonance (NMR) spectroscopy to study structural aspects of the co‐translational protein folding of stalled ribosome‐bound nascent chain complexes (RNCs) which mimic the progressive emergence of the nascent polypeptide and its acquisition of structure.The current state of our study on the co‐translational folding of a model immunoglobulin domain will be presented including some of the structural models built from chemical shift data of RNCs. These findings contrast with analogous studies of C‐terminal truncations of this domain, indicating some exciting differences between folding on the ribosome and that in bulk solution.[1] Waudby CA, Launay H, Cabrita LD &amp; Christodoulou J. Protein folding on the ribosome studied using NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 74, 57‐75 (2013).
Identification of a Sperm acrosome associated protein (SPACA7) splice‐variant in human testis (LB212)
Authors: Rashid Mohammad , Parimi Ramya 
Published: June 2021

Transcriptome in a eukaryotic cell can vary based on the alternative splicing (AS) of genes giving a cell unique characteristic. AS can be a mechanism of gene regulation at the transcriptional level or it may be a way of generating diverse proteins from the same gene allowing them to function differently. Alternative splicing may well be a reason to develop male infertility, for example, studies on X‐linked gene glucocorticoid‐induced leucine zipper (GILZ) that is essential for survival and differentiation of spermatogonia shows six genetic variants of GILZ and two of them are involved in male infertility. Recently Sperm Acrosome Associated 7(SPACA7) protein has been identified in mouse. The analysis of SPACA7 using MyHits motif scan indicated the presence of a putative anaphylatoxins‐like domain motif in its N‐terminal domain that may be responsible for such activities like penetration of the zona pellucida. The human counterpart of SPACA7 gene is located on chromosome 13 (13q34) and its product is a195 amino acid protein.Sperm acrosome structure is formed on the sperm head known to be specifically involved in the process of egg‐sperm fusion, called acrosome reaction, where it is believed to release the enzymes that allow the sperm to fuse with the egg.The role of SPACA isoforms is still under considerable debate. SPACA7 is also known to be unique compared to other isoforms of SPACA that it is exclusively expressed in testis. SPACA7 in human has never been cloned till date and its specific role is still unknown. We have sub‐cloned a SPACA7 splice variant in our laboratory while screening a human testis cDNA library to study protein‐protein interaction in the regulation of human inducible nitric oxide synthase exploiting yeast and mammalian two hybrid assay systems. We hypothesize that this specific variant of SPACA7 may be the preferred version of the protein that interacts with iNOS to modulate nitric oxide synthesis during acrosome reaction.Grant Funding Source: NIH/ARRA
Functional effects of a pathogenic mutation in Cereblon on the regulation of protein synthesis via the AMPK‐mTOR cascade (LB223)
Authors: Park Chul‐Seung , Lee Kwang Min , Yang Seung‐Joo , Choi Ja‐Hyun 
Published: June 2021

Initially identified as a protein implicated in human mental deficit, cereblon (CRBN) was recently recognized as a negative regulator of adenosine monophosphate‐activated protein kinase (AMPK) in vivo and in vitro. Here, we present results showing that CRBN can effectively regulate new protein synthesis through the mammalian target of rapamycin (mTOR) signaling pathway, a downstream target of AMPK. Whereas deficiency of Crbn repressed protein translation via activation of the AMPK‐mTOR cascade in Crbn‐knockout mice, ectopic expression of the wild‐type CRBN increased protein synthesis by repressing endogenous AMPK. Unlike the wild‐type CRBN, a mutant CRBN found in human patients, which lacks the last 24 amino acids, failed to rescue mTOR‐dependent repression of protein synthesis in Crbn‐deficient mouse fibroblasts. These results provide the first evidence that Crbn can activate the protein synthesis machinery through the mTOR signaling pathway by inhibiting AMPK. In light of the fact that protein synthesis regulated by mTOR is essential for various forms of synaptic plasticity that underlie the cognitive functions of the brain, the results of this study suggest a plausible mechanism for CRBN involvement in higher brain function in humans, and they may help explain how a specific mutation in CRBN can affect the cognitive ability of patients.Grant Funding Source: Supported by Ministry for Health and Welfare [HI13C1412] and Ministry of Science, ICT & Future Planning of Korea [2007‐0056157] [2011‐0028665] of Korea.
Hyperglycemia down‐regulates the cgmp‐dependent protein kinase i expression in cytotrophoblasts (LB241)
Authors: Ahsan Gias 
Published: June 2021

Objective: The signaling pathway of nitric oxide (NO), cGMP, and cGMP‐dependent protein kinase (PKG) down‐regulates under diabetic conditions. High glucose exposure reduces PKG activity in smooth muscle cells; however, there is no data in cytotrophoblasts (CTBs). The present study evaluates the effect of high glucose on PKG‐I expression in CTBs. Study Design: Human CTBs (Sw. 71) were treated with 100, 150, 200, 300 or 400 mg/dL glucose for 72h. Some cells were pretreated with a p38 inhibitor (10 μM SB203580). After the treatment, the expression of protein kinase G1α (PKG1α), protein kinase G1β (PKG1β), soluble guanylate cyclase 1α (sGC1α), and soluble guanylate cyclase 1 β (sGC1β) were measured in the cell lysates by western blot analysis. The p38 MAPK phosphorylation was evaluated by western blot. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: The expression of PKG1α, PKG1β, sGC1α, and sGC1β were significantly down‐regulated (p<0.05) in CTBs treated with 蠅150 mg/dL glucose compared to basal (100 mg/dL) that were attenuated by SB203580. The p38 MAPK phosphorylation was up‐regulated (p<0.05) in excess glucose‐treated CTBs. Conclusions: Exposure of CTBs to excess glucose down‐regulates the PKG. The attenuation of hyperglycemia‐induced down‐regulation of PKG proteins by SB203580 pretreatment suggests the involvement of stress signaling in this process.Grant Funding Source: Mohammad Uddin
Purification of Histidine‐tagged and GST tagged proteins using ÄKTA™ start (LB264)
Authors: Haskins Kristi , R Sangeetha , Rao Devavratha , Rao Veena 
Published: June 2021

Expression of recombinant proteins using affinity tags is a commonly used purification strategy. Histidine and glutathione‐S transferase (GST) are the most common and versatile of these tags. Histidine tags are small and therefore less disruptive to the protein structures and functions. GST tags enhance solubility of the target protein, hence improving the yield.Manual purification of tagged proteins often face issues with time to purify, low reproducibility and low yield etc. In addition a researcher who is purifying manually needs to spend time in setting‐up the run, sample loading, collection and measuring the final protein concentration.We describe here the purification Histidine‐tagged and GST‐tagged protein using ÄKTA start with Frac30 fraction collector and UNICORNTM start system control software.We purified an N‐terminal Histidine tagged protein expressed in E.coli on a pre‐packed HisTrapTM FF 1mL column. The purification method was completely automated for unattended operation by applying large volume of sample using pump and collecting peak fractions using Frac30 fraction collector. We have consistently obtained high levels of purity (> 95%) of the tagged protein, which is 1.6 fold higher when compared to a conventional manual purification method.In the second example, we describe the purification of GST tagged protein expressed in E.coli on a pre‐packed GSTrapTM FF 1ml column using ÄKTA start with Frac30 fraction collector. The target protein was purified using pre‐defined method workflow available from the instrument display. The result file that was stored on USB memory stick is imported for viewing and report generation using UNICORN start software.ÄKTATM start is the newly developed laboratory scale protein purification system that can address the challenges faced during manual purification. Automated purification of tagged proteins has showed higher yields and purity with high consistency.