bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular chaperones disperse Pab1 hydrogel more quickly than misfolded aggregates
Authors: Yoo Haneul , Pilipenko Evgeny , Drummond D. Allan 
Published: June 2021

In budding yeast, S. cerevisiae, many environmental stresses such as heat trigger aggregation of intracellular proteins and mRNA into massive particles, many of which are then recruited to stress granules (SGs). During the recovery period, a set of molecular chaperones called heat shock proteins re‐solubilizes the constituent proteins back to the cytosol.Although this aggregation has long been interpreted as resulting from stress‐induced protein misfolding, recent results offer an alternative view. We recently demonstrated that poly(A)‐binding protein (Pab1 in budding yeast), a classical marker for SGs, demixes into hydrogel droplets in vitro upon exposure to physiological stress conditions. Demixing involves phase separation and gelation, and is an adaptive response to stress. Here we show that the Hsp104 disaggregase system disperses Pab1 hydrogels with unparalleled efficiency relative to the model aggregate firefly luciferase. We isolate the precise proteins necessary for efficient dispersal. In an important methodological advance, we introduce a new fluorescence anisotropy assay for kinetic monitoring of the conversion of inert Pab1 hydrogels into soluble RNA‐binding Pab1 monomers. Our assay reveals that hydrogel dispersal into functional monomers is markedly more rapid than disaggregation of a model misfolded protein. Our results raise important questions as to the identity of the native substrates of protein disaggregase systems, the mechanisms by which these systems recognize their substrates, and the molecular features which control the efficiency of molecular dispersal of phase‐separated endogenous structures.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insertion of human mtHsp70 (mortalin) into liposomes resembling mitochondrial membrane.
Authors: das Dores Silva Paulo Roberto , Cauvi David M. , Borges Julio Cesar , da De Maio Antonio 
Published: June 2021

Heat shock proteins (hsp) are important components of the cellular chaperone pool, playing an important role in protein folding, intracellular traffic, and degradation among others. The Hsp70 family of hsp present a 40–60% sequence identity among its prokaryotic and eukaryotic members. The human mitochondrial Hsp70 (mtHsp70), also named mortalin, is mainly involved in the transport of proteins from the cytosol into the mitochondrion matrix. It is also present in the cytoplasm where it is involved in the p53 sequestration. Prior studies have shown that the human Hsp70 and Hsc70 have an unusual capacity for interacting with lipid membranes opening ion conductance pathways. Since mtHsp70/mortalin is a key player in the translocation of polypeptides across the mitochondrial membrane, we investigate whether this protein could also get inserted into lipid membranes. To test this hypothesis, we performed interactions studies with recombinant mtHsp70/mortalin and liposomes, resembling the composition of the mitochondrial membrane. Recombinant human mtHsp70/mortalin was incubated with liposomes (100nm) made of lipids contained in the mitochondrial membrane: palmitoyl‐oleoyl phosphocholine (POPC), palmitoyl‐oleoyl phosphoserine (POPS), and palmitoyl‐oleoyl phosphoethanolamine (POPE), and cardiolipin (CL). In addition, the effect of the co‐chaperone hHep1 on liposome insertion was analyzed. MtHsp70/mortalin displayed a great predilection for POPS and CL and a low affinity for POPC and POPE, suggesting that the proteins that it has high specificity for are negatively charged lipids. The interaction of mtHsp70/mortalin with CL may be very relevant for the interaction with the mitochondrial membrane that is enriched in CL (up to 15% of total lipids). In contrast, the co‐chaperone hHep1 interacted with liposomes made of all the lipids with high prominence for POPC. In all, our data have shown that mtHsp70/mortalin, as well as other human Hsp70s, are prone to interact with membranes and that perhaps is also the case of the inner mitochondrial membrane and the CL lipid plays a central role in this interaction.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) Grants R01 GM098455‐04 and R01 GM114473‐01 and FAPESP process: 2016/22477‐1This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tuning Hsp70 Function: Investigating the Ability of Hsp40/Hsp70 Extragenic Suppressors to Promote Prion Propagation in Yeast
Authors: Kamiya Erina , Schilke Brenda A. , Craig Elizabeth A. , Hines Justin K. 
Published: June 2021

Hsp70s are a versatile class of molecular chaperones, essential for a variety of cellular functions ranging from protein folding and transport to iron‐sulfur cluster biogenesis. Hsp70s function with obligate Hsp40 co‐chaperones, also called J‐proteins, which stimulate Hsp70 ATPase activity via their characteristic J‐domains. In Saccharomyces cerevisiae, the J‐protein Sis1 is essential for cell viability and the propagation of prions. However, the means by which Sis1 drives specific functions of Hsp70 are largely unknown. We recently reported the discovery of gain‐of‐function substitutions in both the J‐protein Ydj1 and the Hsp70 Ssa1 which allow cell survival without Sis1. Here we present additional data that support the ability of the suppressor mutants to substitute for Sis1‐dependent roles, specifically the maintenance of yeast prions. Specifically, we tested the ability of the two most robust suppressor mutations in both Ydj1 and Ssa1 to maintain two prions, [PSI‐+] and [RNQ+], and included both phenotypically strong and weak variants of [PSI‐+] in our analysis. Interestingly, we found similar results for all mutants: both Ydj1 mutants could substitute for the propagation of strong [PSI‐+], but not weak [PSI‐+] nor [RNQ+]. Similarly, we found that the Ssa1 mutants could not support the maintenance of weak [PSI‐+] nor [RNQ+]. Unfortunately, due to the severe [PSI‐+] toxicity in the compromised strain bearing strong [PSI‐+], we could not confidently determine the ability of the Ssa1 mutants to support strong [PSI‐+] propagation. Further investigation of additional Ydj1 and Ssa1 mutants may provide greater resolution regarding the specific functions required of J‐proteins by distinct prions for propagation.Support or Funding InformationThis work was supported by National Institutes of Health (https://www.nih.gov/) grants GM31107 and GM27870 (EAC) and R15GM110606 (JKH). This work was also supported by the Lafayette College Chemistry Department, the EXCEL research scholarship program, and the John F and Dorothy M Dorflinger Summer Research Endowment Fund (JKH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heparan sulfate chain length modulates the cellular targeting of prions in vivo
Authors: Aguilar‐Calvo Patricia , Bapat Jaidev , Sevillano Alejandro Manuel , Sandoval Daniel Rogelio , Dwyer Chrissa , Esko Jeffrey , Sigurdson Christina J. 
Published: June 2021

Prion diseases are fatal neurodegenerative disorders caused by the misfolding of the physiologically expressed prion protein into an insoluble form that accumulates as amorphous aggregates (subfibrillar prions) or amyloid fibers (fibrillar prions). Identical prion protein sequences can misfold into different conformational variants or strains that affect specific neural cell populations, leading to a wide variety of clinical signs and brain regions targeted. The mechanisms underlying the cell targeting of prion strains are unknown. Heparan sulfate (HS) molecules are glycosaminoglycans abundantly expressed in the brain and that profoundly vary in length and sulfation depending on the cell type. HS co‐localize with prion plaques of Creutzfeldt–Jakob disease (CJD), Gerstmann‐Sträussler‐Scheinker (GSS) syndrome, and sheep scrapie, and promote prion replication and fibril formation in vitro. Paradoxically, HS have been used to slow or even prevent prion formation in CJD patients and scrapie‐infected rodents. We hypothesize that the interaction of HS with prions is a major determinant underlying the selective cell vulnerability in prion diseases. Here we characterized the HS length that most effectively modulates the in vitro prion conversion into the aggregated state. We also determined how manipulating the HS chain length shifts the cell tropism of prion strains in vivo. While longer HS chain lengths led to more efficient prion conversion for subfibrillar strains (RML and ME7), intermediate lengths were more effective for the fibrillar strain mCWD. Ext1+/‐ mice expressing shorter HS molecules only altered prion disease phenotype when inoculated with the fibrillar prion mCWD, affecting the disease progression and neuropathology. Together these results suggest that the specific length of the HS together with the prion aggregate conformation determine the binding affinity of prions to HS, and underlie the specific cellular targeting in the CNS. These results have implications for other misfolding proteins such as amyloid‐β and α‐synuclein, which also interact with HS.Support or Funding InformationNIH, NINDS R01 grant 069566 Creutzfeldt–Jakob disease Foundation Fundación Ramón ArecesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decoding the Prion‐Chaperone Interaction Network in Yeast: Prion‐specific Chaperone Functions Promote Amyloid Diversity
Authors: Hines Justin K. 
Published: June 2021

Yeast prions are heritable protein‐based elements, most of which are amyloid aggregates of functional proteins. Their propagation in cell populations is dependent upon the fragmentation of these aggregates by a core set of chaperone proteins including the J‐protein/Hsp40 Sis1, Hsp70s, and Hsp104. Prions form distinct amyloid structural polymorphisms, called ‘strains' in mammalian systems and ‘variants' in yeast. Recently we and others have uncovered significant complexity in the chaperone requirements of various yeast prions and prion variants, demonstrating that, in contrast to Hsp104 and Hsp70, which have general roles, J‐proteins represent a prion‐specific component of the prion‐propagation machinery. Most notable has been a direct demonstration that the persistence of alternative conformations of prions is dependent on the action of different molecular chaperones. Because J‐proteins often act as targeting factors for Hsp70s, they may constitute the first chaperone response to the presence of amyloid, but a critical barrier to advancing the understanding of chaperone function in prion biology is that the fundamental chaperone requirements for most yeast prions remain unidentified, precluding a comprehensive understanding of how protein sequences give rise to amyloids with distinct patterns of chaperone interaction. Here we will present our latest undergraduate‐generated data regarding: 1) the unusual role of the J‐protein Swa2 in the propagation of the [URE3] prion and our proposal of a novel chaperone complex involving Hsp90, and 2) the conservation of the prion‐maintaining J‐protein functions of both Sis1 and Swa2 in plants. These findings suggest that amyloid structures of alternative prion conformations have unique features that are differentiable by chaperone proteins, revealing a previously unappreciated level of additional complexity that may be exploitable for therapeutic intervention.Support or Funding InformationThis work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R15GM110606. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disulfide bridge formation contributes to histone ligand recognition by the ATAD2 bromodomain
Authors: Evans Chiara , Gay Jamie , Eckenroth Brian E. , Carlson Samuel , Lloyd Jonathan T. , Glass Karen C. 
Published: June 2021

The ATPase Family, AAA domain‐containing protein 2 (ATAD2) bromodomain has canonical bromodomain structure, consisting of four α‐helices. ATAD2 is known to be up‐regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate cancer. ATAD2 is a co‐activator of the androgen and estrogen receptors, as well as MYC and E2F transcription factors. Recently, ATAD2 was shown to read newly synthesized diacetylated marks during DNA replication, and ATAD2 overexpression further increases ATAD2 levels, cell proliferation, and survival genes. Furthermore, up‐regulation of ATAD2 is strongly correlated with poor patient prognosis. This makes the ATAD2 bromodomain an innovative target for cancer therapeutics, and several inhibitors are currently in development. Interestingly, the ATAD2 bromodomain contains two cysteine residues near the base of the bromodomain‐binding pocket between helices αB and αC. We hypothesized that disulfide bridge formation between residues C1057 and C1079 might regulate the interaction of the ATAD2 bromodomain with its histone ligands. X‐ray crystallography confirmed formation of a disulfide bridge in the ATAD2 bromodomain. The role of the disulfide bridge in ATAD2 bromodomain stability was explored through mutagenesis, codon optimization, and expression experiments. To investigate the impact of disulfide bridge formation on histone ligand recognition by the ATAD2 bromodomain, isothermal titration calorimetry was carried out to characterize binding affinities of histone ligands using the ATAD2 bromodomain protein with and without an intact disulfide bridge. This study demonstrates the functional effect of disulfide bridge formation upon histone ligand recognition by the ATAD2 bromodomain, and presents additional structural information, which may prove essential for effective inhibitor development for the treatment of many cancers.Support or Funding InformationNIGMS R15 215GM104865, GlassStudent Summer Research Award, ACPHS Graduate Research Assistantship, EvansThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The amyloid‐β peptide in Alzheimer's disease: molecular interactions and structure conversions
Authors: Gräslund Astrid 
Published: June 2021

The amyloid β (Aβ) peptide consists of 39–43 residues and is the major component of the neuritic plaques in the brains of Alzheimer's disease (AD) patients. It is possible to study the peptide self‐aggregation process (“amyloid formation”, structures and kinetics) using biophysical methods such as NMR, fluorescence or CD spectroscopy. The interactions of Aβ(1–40) with small molecules, or metal ions such as Zn(II) or Cu(II), that modulate the aggregation process can be studied in semi‐stationary states by these methods. Kinetic effects on the amyloid formation process can be followed by fluorescence, after labeling the material rich in β‐structure by the amyloid‐specific dye Thioflavin T. The β‐hairpin structure of the peptide with a mainly unstructured N‐terminus appears to be a basic unit for formation of the larger amyloid structures associated with insoluble fibrils. Smaller oligomeric peptide aggregates, possibly the major cell toxic agents, can be studied by Soft Ionization Mass Spectrometry.Fluorescence Correlation Spectroscopy (FCS) shows the heterogeneity of the aggregation process of ThT‐labeled Aβ, in terms of time dependent amyloid aggregate sizes, on a single particle level. Modulation of the amyloid formation by Zn(II) or Cu(II) ions binding to specific residues in the N‐terminus of the Aβ peptide can be followed by the biophysical methods, showing changes in structures as well as kinetics for the aggregation process.Understanding the basic properties and reactions of the major participants in the chemical processes leading to AD gives a firm basis for the future development of agents that may be used in therapies against AD.Support or Funding InformationThe Swedish Research Council, The Alzheimer Foundation, The Magn. Bergvall FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Screening for novel long‐chain bacterial esterase activity
Authors: Jozwiakowski Jakob Leland , Johnson R. Jeremy 
Published: June 2021

Hydrolases are ubiquitous cellular enzymes with more than 1200 different hydrolases, >1/3 of all enzyme structures in the PDB, and 13 different enzyme commission subclasses. Hydrolases are also fairly promiscuous and can also catalyze hydrolysis reactions on varied substrates including esters, amides, thioesters, phosphoric acid esters, and acid anhydrides. All of these reactions originate from the same α/β hydrolase protein fold and utilize the catalytic triad, suggesting great plasticity and generality in this reaction. Using a fluorogenic mimic of palmitic acid linked molecules, we performed a high‐throughput screen in Escherichia coli searching for previously unidentified depalmitoylase activity. Simultaneous analysis of forty‐five different hydrolase knockout strains with this fluorogenic probe identified a single uncharacterized esterase with high specificity for the probe. To confirm this reactivity, this hydrolase was heterologously expressed, purified to homogeneity, and characterized for its detailed substrate specificity using a library of fluorogenic esters. Our screen highlighted the potential novel hydrolase reactivity present within even model organisms like E. coli that could have applications in protein engineering or biological imaging.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Molecular Determinants of Heterocycle Formation in Hybrid Nonribosomal Peptides/Polyketides
Authors: Dowling Daniel P. , Kung Yan , Croft Anna , Taghizadeh Koli , Kelly Wendy , Walsh Christopher T. , Drennan Catherine L. 
Published: June 2021

Many secondary metabolites from natural sources contain thiazole or oxazole derivatives that stem from the precursor amino acids cysteine, serine or threonine, and how Nature generates these azolines depends on the biosynthetic strategy used by that organism. Here, we explore azoline formation within hybrid nonribosomal peptide (NRP) and polyketide (PK) compounds, a class of molecules that are extremely beneficial for drug discovery. Epothilones are example hybrid NRP/PK products that demonstrate antitumor activity by stabilizing microtubules. They are biosynthesized through the action of EpoA‐F from Sorangium cellulosum, in which the EpoA and EpoB proteins act as the starter unit to generate the 2‐methylthiazole functionality. EpoB contains a cyclization (Cy) domain capable of catalyzing both chain extension and cyclization reactions to yield a heterocycle. We report crystal structures for a didomain of the EpoB Cy domain tethered to its N‐terminal docking domain, which have implications in locating carrier protein binding sites, identifying phosphopantetheine binding tunnels, and determining residues involved in catalysis. Site‐directed mutagenesis of active site residues have identified key amino acids required for catalysis. X‐ray crystal structures of the EpoB Cy domain and related systems provide an opportunity to analyze critical similarities and differences that may guide future bioengineering efforts for green chemistry applications.Support or Funding InformationU.S. National Institutes of Health [1R15GM123425‐01 to D.P.D.]. C.L.D. is a Howard Hughes Medical Institute Investigator. Support for this research was provided by National Institute of Environmental Health Sciences, NIH Core Center Grant P30‐ES002109.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthetic biology, biosynthesis, enzymology, (meta)genomics, et al. Learning from Nature.
Authors: Agarwal Vinayak 
Published: June 2021

Nature is an organic chemist. The language in which we interact with Nature is chemistry, with small molecule natural products being the alphabets of this language. Natural products range from antibiotics and pharmaceuticals that we use in the clinic, to toxins and pollutants that pervade our environment. Research in the Agarwal lab seeks to understand how natural products are produced, what are the biological forces driving their syntheses in Nature, and how we can leverage this knowledge to drive scientific progress and human well‐being. We employ a combination of chemical and biochemical approaches, including but not limited to microbial genetics and metagenomics, structural and biochemical enzymology, and synthetic and analytical chemistry.Natural product biosynthesis is a treasure trove of novel enzymology. We will describe a total enzymatic reconstitution of the biosynthesis of a polybrominated marine natural product. Crystal structures of biosynthetic enzymes provide deep insights into mechanisms of key enzymatic transformations and allow for structure guided engineering of catalysts. These findings set the stage for the development of methods for mining complex metagenomic datasets to tackle the immense biological complexity of natural product sources.Support or Funding InformationNIH R00ES026620This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved Synthesis of N‐methyl Cadaverine
Authors: Anderson Kayla Nicole , Moaven Shiva , Cozzolino Anthony , D'Auria John C. 
Published: June 2021

Polyamines are abundant in nature, and the methylation of polyamines is a common enzymatic step in plants, especially plants containing alkaloids. This includes the family of compounds called granatane alkaloids. Pomegranate (Punica granatum) is one of the most commonly known granatane alkaloid‐producing plants. Pomegranate extracts are widely used for their antioxidant, anti‐inflammatory, and anticarcinogenic effects. [1] One of the proposed enzymes in the hypothetical pathway for the formation of granatane alkaloids is cadaverine N‐methyl transferase.[2] It is important to find a method of synthesis for N‐methyl cadaverine (N‐methylpentane‐1,5‐diamine) and other N‐methyl polyamines, because they can be utilized as a standard for quantification purposes as well as providing a positive control when evaluating enzymatic activities of putative alkaloid producing N‐methyl transferases. The synthesis of N‐methyl cadaverine was employed by utilizing a substitution reaction between N‐methylbenzylamine and 5‐bromo‐valeronitrile, removing the protecting group (or benzyl ring) attached to the methylated amine, and reduction of the nitrile. [3]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.