bluuebunny/crossref_metadata_2025_split huggingface dataset:
Invariant BECN1 C‐x‐x‐C Motifs Are Essential For Starvation‐Induced Autophagy
Authors: Mukhopadhyay Shreya , Li Yue , Sinha Sangita 
Published: June 2021

Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1, a protein essential for autophagosome nucleation, contains two invariant C‐x‐x‐C motifs (18CxxC21 and 137CxxC140 in human BECN1) that bookend a large BECN1 intrinsically disordered region (IDR). We use circular dichroism spectroscopy to elucidate differences in secondary structural content of the wild‐type and CysTetrad mutant BECN1 IDR. Additionally, we are using small angle X‐ray scattering analyses to further understand the impact of the CysTetrad mutation on the structure of the BECN1 IDR. We also show that helicity of the BECN1 IDR increases upon addition of 2,2,2‐trifluoroethanol (TFE), suggesting that it may undergo binding‐associated helical transitions. Further, we use ICP‐MS to demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio under both, reducing and non‐reducing environments. Lastly, we show that both C‐x‐x‐C motifs are required, while the IDR is also important, for starvation‐triggered up‐regulation of autophagy.Support or Funding InformationNIH grants RO3 NS090939 and R15 GM122035 (S.C.S.)National Science Foundation grant MCB‐1413525 (S.C.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mortalin Caught in Action: Structures of the Nucleotide Binding Domain in the Apo, ADP‐bound and AMP+PPi‐bound States
Authors: Moseng Mitchell , Page Richard 
Published: June 2021

The heat shock protein 70, Hsp70, chaperone family plays key roles in cellular homeostasis and stress response. Hsp70s not only regulate the folding of nascent proteins, but also prevent aggregation, promote disaggregation, and refold misfolded proteins. The mitochondrial Hsp70, Mortalin, functions in a variety of processes such as mitochondrial protein import and quality control, Fe‐S cluster biogenesis, mitochondrial homeostasis, and regulation of p53, an important tumor suppressor. Mortalin has been implicated in the regulation of apoptosis, cell stress response, neurodegenerative diseases, and has an anti‐apoptotic role in cancer cells. We have elucidated crystal structures of the nucleotide binding domain of human Mortalin in the apo, ADP‐, AMP and AMP states at 2.8 Å, 2.78 Å, and 1.75 Å resolutions, respectively. The crystal structure of Mortalin‐NBD binding to the modified ADP homolog N6propargyl‐ADP has also been determined at 1.49 Å. The overall structures and active site organizations are similar, with a few key differences in side chain positions. Specific residues in the nucleotide binding pocket, which are not conserved in other Hsp70‐family members, lead to lower nucleotide affinity and slower turnover relative to cytosolic Hsp70. ITC and biolayer interferometry experiments performed on the R126W and Y128C disease variants of Mortalin to determine ADP binding and interactions with an inter‐domain linker peptide elucidate how the mutations change Mortalin's functions and how these interactions differ between Mortalin, Hsp70, and Hsc70. ITC experiments have also been performed to test how the MortalinNBD can accommodate modified ADP nucleotides. This structural and binding data may also contribute to the understanding of disease‐associated Mortalin mutations and to improved Mortalin‐targeting antitumor compounds.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An engineered E. coli ribosome with tunable translation rates enhances recombinant protein expression
Authors: Oza Javin P. , Soye Ben Des , Jewett Michael C. 
Published: June 2021

Using E. coli for the expression of recombinant proteins in their soluble active form continues to be a challenge in biotechnology. Issues including low yields of full‐length, soluble, and functional proteins fundamentally limit discovery, development, and manufacturing of commercially, industrially, and medically relevant proteins. In many cases, improvements in protein solubility have been achieved by codon optimization, over‐expression of chaperone proteins, solubility tags, and by varying expression conditions. Optimization of these parameters is often done iteratively and on a case‐to‐case basis, which can be slow and expensive. To overcome limitations in recombinant protein expression, we report a genomically engineered strain of E. coli with a tunable ribosome enabling modulation in translation rates under isothermic conditions. Additionally, we show that cell extracts from this strain of E. coli can support a cell‐free protein synthesis platform capable of on‐demand production of recombinant proteins. This platform technology enables two key advances. First, we provide quantitative evidence that tuning protein synthesis rates improves soluble fractions of traditionally intractable proteins such as the human MEK1 and IRKβ. In each case, soluble fractions increased from <20% to >80% of total protein when compared with expression in BL21 cell extracts. 2) This platform technology is plug‐and‐play, allowing the user to identify the optimal translation rate condition for a desired protein in a high‐throughput manner, which can then be scaled‐up for desired protein yields. The timescale from DNA template to the optimized protein expression condition is 24–48 hours, notably faster than traditional methods involving cell transformations, and optimization of expression times and temperatures. Additionally, our data suggest that this approach obviates the need for solubility tags, enabling the expression of proteins in their wild‐type forms. Our work provides a new tool for studying protein folding in the context of ribosomal kinetics, and a new platform for manufacturing traditionally difficult proteins.Support or Funding InformationThis work was supported by DARPA and the the David and Lucille Packard Foundation Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Mechanism of cis Amide Bond Stabilization in Phosphoserine‐Proline Sequences
Authors: Raniszewski Nicole R. , Ganguly Himal K. , Zondlo Neal 
Published: June 2021

Phosphorylation is a reversible covalent modification to biomolecules that is central to protein‐protein interactions, signal transduction, and gene expression. When improperly regulated, phosphorylation can trigger oncogenesis and neurodegenerative diseases such as Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE). Phosphorylation can also act as a structural switch for cis‐trans isomerization of peptide bonds: through intraresidue phosphate‐amide hydrogen bonds, phosphorylated residues promote compact trans conformations. In other contexts, phosphorylated Serine (pSer) residues can promote cis peptide bonds through unknown mechanisms. To investigate the basis for these structural effects, a series of X‐pSer‐Pro‐Z peptides was synthesized with modifications on the proline, N‐terminal to pSer (X), and C‐terminal to Pro (Z).Dipeptides remove influences from secondary and tertiary protein structures, allowing elucidation of key molecular interactions that affect the cis conformer. Two 4S‐substituted hydroxyproline derivatives, Fmoc‐ 4S‐iodophenyl‐hydroxyproline and Fmoc‐4S‐fluoroproline, were synthesized and incorporated into the peptide. These substitutions promote the cis‐Pro conformation via intraresidue stereoelectronic effects. The initial peptides were examined with C‐terminal amides, while modifications of the N‐terminus allowed for control of sterics. Formyl, acetyl, pivaloyl, 2‐iodobenzoyl, and 4‐iodobenzoyl moieties were included in peptides to provide varying degrees of steric demand to promote alternate conformations at the N‐terminus.The peptides were analyzed by 1H NMR to quantify the effects of proline, N‐terminal modifications, and phosphorylation on structure. TOCSY and ROESY 1H NMR spectra were employed to differentiate the cis and trans peaks and characterize the conformational changes. Structural changes as a function of phosphate protonation state were identified by pH‐dependent NMR experiments. The downfield shift in the amide region with increasing pH implied a Hydrogen bonding interaction between the phosphate oxygen and its own amide hydrogen. The equilibrium constant for cis‐trans isomerization, Ktrans/cis, increased with increasing pH for the acetyl and 4‐iodobenzoyl derivatives. Pivaloyl and 2‐iodobenzoyl derivatives showed an opposite trend, increasing the population of cis with increasing pH. N‐terminal 2‐iodobenzoyl and acetyl groups show greater preference towards the cis at various pH with respect to other examined peptides. Stabilization of cis amide bonds upon phosphorylation can be used to design tunable scaffolds for peptide conformations.Support or Funding InformationDavid M. Heitzer Award, DODThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of novel monomeric L‐threonine 3‐dehydrogenase and elucidation of product release mechanism
Authors: Motoyana Tomoharu , Nakano Shogo , Yamamoto Yuta , Tokiwa Hiroaki , Asano Yasuhisa , Ito Sohei 
Published: June 2021

IntroductionL‐Threonine (L‐Thr) metabolism is not well studied, however, plays some part in the lipid synthesis of Trypanosoma brucei and the growth of mouse embryonic stem (ES) cells. L‐Thr is initially catalyzed by L‐threonine dehydrogenase (TDH) which is a NAD+‐dependent enzyme catalyzing dehydrogenation of hydroxyl group of L‐Thr to 2‐amino 3‐ketobutyrate (AKB). TDH belonging to short chain dehydrogenase/reductase family has high specificity toward L‐Thr; the specificity is regulated by switching between open state and closed state by binding of substrate (1). On the other hand, the product release mechanism remains to be elucidated. In this study, we discuss the reaction mechanism associated with structural change at the active site by combinational analysis, such as high resolution X‐ray crystal structure, quantum mechanical (QM) calculation method and kinetic analysis.MethodsIn silico screening was performed using INTMSAlign (2) to obtain TDH (mtTDH) derived from metagenome library. At first, we evaluated enzymatic properties of mtTDH. Four crystal structures of mtTDH were determined at 1.25 – 1.9 Å resolution: apo, binary (NAD+‐binding) and two ternary (l‐Ser‐NAD+‐binding and AKB‐NADH‐binding). For the AKB‐NADH‐binding structure, the Inter‐Fragment Interaction Energy (IFIE) were calculated by the Fragment Molecular Orbital (FMO) method. We also performed site‐directed mutagenesis (S74A, S111A, Y136F, T177A, D179A, and D179N) and kinetic studies to elucidate the roles of active site residues.Results & DiscussionThe crystal structure of mtTDH was obtained as a monomeric form; this is consistent with the result of gel‐filtration chromatography. Based on these results, we concluded that mtTDH is monomeric TDH. Structural comparison of the four structures revealed that ligand (AKB or l‐Thr) and eight molecules (six residues and two molecules of water) appeared to form hydrogen bonding interactions each other. In order to evaluate these interactions quantitatively, IFIEs were calculated by FMO method which is a kind of QM calculation method. As a result, surprisingly, D179 had a repulsive interaction with the AKB. Kinetic analysis of mtTDH variants, D179A and D179N, exhibited the remarkably decrease in activity in spite that D179 works against formation of interaction with the ligand. Taken together, D179 may be a sensor detecting the completion of the reaction and important for switching between open state and closed state (3).Support or Funding InformationThis work was supported by JSPS KAKENHI Grants 16K18688, 17KT0010, and 17K06931 and JST by ERATO Grant JPMJER1102.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioactive Fungal Screening to find Efficient Biomass‐Degrading Enzymes
Authors: Vazquez Uriel Adan , Conrad Dale , Ma Hongyan , Woodard Toni , Wu Si 
Published: June 2021

Traditional fuels such as coal and gasoline are limited and can produce harmful effects to the environment. Bioethanol from biomass (i.e., plant‐based materials) degradation is a sustainable and renewable form of fuel that aims to reduce these effects. The fungal secretome, known as a pool of biomass degrading enzymes, can be utilized to discover industrial enzyme candidates useful for biofuel production. The overall purpose of our research is to pre‐screen a pool of fungi collected from the Great Lakes, select candidates that can break down biomass (i.e., cellulose) efficiently, and characterize the biomass degrading enzymes. In this study, we first selected seven different fungal species, and grew them onto carboxymethyl cellulose (CMC) plates. A specific dye called Congo red was used to monitor the cellulose activity after either a five‐day or twelve‐day growth period. A clear zone present around the central fungal growth indicated the degradation of the cellulose in the culture medium. Fungal activity was measured using a ratio of enzyme activity (outer ring) and the fungal growth (central circle). The enzyme activities of these strains were compared to Aspergillus niger, a known industrial fungal strain. Overall, there were four different species that demonstrated higher cellulase activity in comparison to A.niger in the CMC assay. Additionally, the secretome and cell lysate of A. niger was prepared to determine the source of the biomass‐degrading enzymes through an enzymatic activity assay. Preparation of the secretome and lysate were obtained through ultra filtration and bead beating, respectively. Both methods resulted in acquiring a highly concentrated solution of each protein samples. It was determined that the biomass‐degrading enzymes were secreted from the fungal cells rather than contained within the cell. Future directions include secretome preparation of the four identified fungal strains to verify their biomass hydrolysis capabilities.Support or Funding InformationThis work was supported NSF award 1359457 to Dr. Si Wu.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sulfur respiration in A. fulgidus? Kinetics and Structure of an A. fulgidus NAD‐dependent Poly/Persulfide Reductase.
Authors: Shabdar Sherwin , Castineiras Ana , Desir Neyissa , Crane EJ , Sazinsky Matthew 
Published: June 2021

PNDORs are a large class of homodimeric NADH and FAD‐dependent enzymes that generally function to reduce small molecule substrates at a catalytic cysteine positioned at the si face of the isoalloxazine ring. The substrate preference and reactivity vary greatly depending on surrounding catalytic residues and additional structural elements. Two closely related PNDORs have been found to have in vitro S0 reducing activities and have been correlated with in vivo S0 reduction – CoADR and Npsr. Both enzymes catalyze the general reaction of S80, Sn2− or CoA‐SnH + NAD(P)H‐> S2− + NAD(P)+ + CoA‐SH. The most obvious structural difference between the two enzymes is the addition of a rhodanase‐like domain on the Npsr which contains a surface‐located cysteine that we have shown to be the initial sulfur‐accepting site. CoADR is widely distributed throughout the thermophilic and hyperthermophilic archaea, while the Npsr is not found in the archaea, with the exception of an Npsr homologue found in the sulfate‐reducing thermophile Archaeoglobus fulgidus. Unlike the other CoADR and Npsr‐containing organisms, A. fulgidus cannot respire S0, and is actually inhibited by S0. Sequence homology suggests that this A. fulgidus Npsr should be a per/polysulfide reductase, however, the lack of a sulfur‐reducing phenotype suggests 1) the catalytic function of this enzyme is changed, and it fulfills a new function, or 2) the polysulfide reductase activity of the enzyme in vivo is not sufficient to confer the ability to respire S0 in the context of the A. fulgidus cell, and the enzyme's persulfide/polysulfide reductase activity fulfills a different role, perhaps acting in an antioxidant or detoxification reaction. To differentiate between these two possibilities and better understand archael sulfur respiration processes here we report the kinetics, substrate preferences and structure A. fulgidus Npsr.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant No. 1518306This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis of nonproteinogenic amino acids oxyvinylglycines
Authors: Li Bo , Patteson Jon B. , Dunn Zachary D. 
Published: June 2021

Nonproteinogenic amino acids (NAAs) are well known for their potent biological activities and unique chemical functionalities that are not encoded in proteins. NAAs are produced across all kingdoms of life, and many NAAs have broad applications in agriculture, nutrition, and medicine. The biosynthetic chemistries for many NAAs, however, remain largely unknown. I will discuss our recent results on reconstituting the biosynthesis of a class of NAAs called oxyvinylglycines, which exist as free amino acids and act as antimetabolites. Our work has uncovered unique enzymatic transformations involved in the biosynthesis of oxyvinylglycines. Understanding the biosynthetic chemistry of NAAs could facilitate the discovery of new enzyme catalysts and engineered biosynthesis of NAA‐containing natural products.Support or Funding InformationThis work is supported by National Institutes of Health (R00 GM099904), National Science Foundation (CHE1654678), the Rita Allen Foundation, and the University of North Carolina at Chapel Hill.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Design and Simulation of a Cyclized Dimeric Multipass Transmembrane Protein
Authors: Aldana‐Mendoza Jonathan Agust , DeGrado William Agust 
Published: June 2021

Transmembrane (TM) proteins play critical roles in cell surface markers, receptors, and can serve as channels for molecular transport. Therefore characterizing this structural class of proteins is important for understanding a wide variety of cellular functions. With the influx of solved structures of alpha helical TM proteins, we are better equipped to address the unique structural characteristics of these proteins. We hypothesize that a de novo design and synthesis of a tm protein cold be carried out with accurate atomic level prediction of its tertiary structure. We based our design on the characterized GX6G motif of alpha‐helical proteins; to set parameters for searching naturally occurring alpha‐helices in the PDB. Then using these naturally occurring proteins to analyze any sequence preference given the alpha‐helical structure. We plan on validating our design and simulation through the synthesis of this peptide using solid phase Fmoc synthesis. Structure analysis will be performed on the product through circular dichroism, infrared/vibrational, and NMR spectroscopy. The synthesis and accurate prediction of this alpha‐helical tm protein, will demonstrate our capacity to predict structures of tm proteins that contain the sequence/structural motifs through in cooperation in a rational design. Enabling us to rationally design tm proteins, or small molecules that target tm proteins, and therefore have powerful therapeutic potential.Support or Funding InformationNIH Grant # GM 49001, CSU‐LSAMP is supported by the National Science Foundation under Grant # HRD‐1302873 and the CSU Office of the ChancellorThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Design Of A Mimic Against Neurodegenerative Diseases Based On VEGF‐D
Authors: Mason Christopher , Bibi Najma , Zahran Mai 
Published: June 2021

The basis of this research is the use of a computational approach to design drugs based on the tertiary structure of the protein VEGF‐D. Vascular Endothelial Growth Factor‐D has been discovered to have a profound role in neuronal growth in which it demonstrates its ability to repair memory and improve cognitive functions. This discovery of VEGF‐D's new function has fascinated researchers as it shows promising results of being a possible cure against neurodegenerative diseases such as Alzheimer's however, its inability to bypass the blood brain barrier has intrigued our collaborators to develop a set of peptides that may induce the same reaction as VEGF‐D. The purpose of this research is to analyze the structural and functional details of VEGF‐D complexed to its receptor VEGFR‐3 at atomic level, design smaller drugs that have the potential to act as VEGF‐D, and identify the best modeled peptide that interacts most favorably with the receptor to present to our collaborators for experimental testing. This work involves the designing of 14 small peptides for which molecular dynamic simulations were performed to investigate the behavior of the peptides along the trajectory. Information obtained from dynamics simulation denote peptide 56 as the most suitable candidate to induce the same reaction as VEGF‐D.Support or Funding InformationNew York College Of Technology, NSFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of Iron in Biological Samples using Sector‐Field vs. Quadrupole Inductively Coupled Plasma Mass Spectrometry
Authors: AL‐Ogaidi Hussein A. , Ketterer Michael E. , Ragan Emily J. 
Published: June 2021

Inductively coupled plasma mass spectrometry (ICPMS) has become the preferred technique for trace and ultra‐trace determination of elemental concentrations in a wide variety of sample matrices. In our studies of iron (Fe) uptake and metabolism in insect cells, we evaluate different ICPMS procedures to measure pg – ng quantities of Fe in whole insects and Drosophila melanogaster S2 cells (Sg4 isolate); samples are digested and mineralized by heating with nitric acid (HNO3) at 90°C in fluoropolymer containers. The determination of Fe by ICPMS is well‐known to be affected by interferences from polyatomic ions; e.g., the species 40Ar16O+ forms in the plasma and directly impacts the most abundant 56Fe isotope. We evaluate two ICPMS approaches for performing reliable low‐level Fe determinations: A) quadrupole ICPMS with a collision cell system for attenuation of polyatomic ions; and B) a sector field ICPMS system capable of producing high resolving powers (m/Δm ~ 10,000). The latter system is easily capable of physically separating the 56Fe+ peak (55.94 amu) from interfering 40Ar16O+ (55.96 amu), and can generate solution detection limits of ~ 0.01 mg L−1, which is far superior to the ~ 1 mg L−1 detection limits attainable by quadrupole ICPMS with a collision cell in the kinetic energy discrimination mode. We describe the relative merits of using internal standardization with 59Co vs. isotope dilution with a 57Fe‐enriched spike solution. Standard operating procedures for the determination of Fe using SF‐ICPMS will be discussed.Support or Funding InformationNSF grant IOS‐1656407This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.