bluuebunny/crossref_metadata_2025 huggingface dataset:
Delivery of DNA encoding RNA Therapeutics to Alter the Expression of Oncogenic Transcripts in Glioblastoma
Authors: Gallagher Kevin R. , Falotico Sarah C. , Sivetz Nicole , Muralidharan Koushik , Khan Arbaz , Hicks Martin J.
Published: June 2021

Glioblastoma multiforme (GBM) is the most common central nervous system (CNS) malignancy. The median survival is only 14 months. It is often characterized by the activation of one or more tyrosine kinase receptors, particularly epidermal growth factor receptor (EGFR). This receptor is dysregulated in about 60% of GBM tumors. EGFR amplification, over‐expression and constitutive activation leads to uncontrolled growth and proliferation of GBM. Although a great deal is known about the aberrant biology exhibited by EGFR‐activated GBM, the application of therapies against the biologic processes is limited by the blood‐brain barrier, which restricts systemically administered therapies from reaching the brain. Although anti‐sense RNAs and small interfering RNAs can be used to target and silence gene expression, exogenously expressed RNAs are susceptible to extracellular and intracellular nucleases as well as activation of cellular immunity against foreign nucleic acids. To bypass these degradatory mechanisms, we take advantage of a natural noncoding RNA gene architecture and the miRNA expression pathway along with an anti‐sense targeted approach to alter EGFR expression. In addition, we make use of a polycistronic delivery system to express RNAs targeting splicing and alternative poly‐A signal/G‐rich elements of the EGFR transcript. DNA delivery vectors were transfected into human GBM cell lines. Results show that our vectors were expressed at high levels with subsequent reduction in full‐length EGFR mRNA expression and concomitant activation of alternative therapeutic isoforms. Current strategies include using the polycistronic delivery mechanism to target additional oncogenic transcripts and adapting to a mouse model of GBM.

Support or Funding Information

Bristol‐Myers Squibb, Johnson & Johnson, and the Independent College Fund of New Jersey

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A1543 is a Potential Bridge Between Cellular Redox State and RNA Processing
Authors: Thomas Patrick , Cameron Jeffrey C.
Published: June 2021

Synechococcus Sp. PCC 7002 is a rapidly growing, genetically tractable, photosynthetic microbe of industrial interest. Understanding how gene expression and carbon fixation are regulated in PCC 7002 is essential for its advancement as an industrial microbe. Towards that aim, we are investigating a cyanobacterial homolog to E. coli RNase II/R, A1543, that we hypothesize regulates gene expression and other cellular processes in response to changes in cellular redox state.

A1543 is an RNA binding protein with three RNA binding domains ‐ two OB folds and an S1 domain. Multiple alignment of PCC 7002 A1543 to RNase II/R homologues in other cyanobacteria show conservation across the genre of two cysteines. Threading of the A1543 sequence onto the E. coli RNase II crystal structure shows that these two cysteines sit in L12 and b2 of the first OB fold, within 4 angstroms of each other, and thus could potentially interact via a disulfide bond.

To characterize the role of A1543 in vivo we constructed a segregated A1543 knockout line, DA1543, which shows, as previously reported in the literature, defects in both pigmentation and growth. Additionally, we found that our DA1543 line shows resistance to the carbonic anhydrase acetazolamide compared to wild‐type. This phenotype had been characterized before in a related species, Synechocystis sp. 6803. We have found concentrations of acetazolamide that is lethal and inhibitory to both wildtype and DA1543 cells in several different growth conditions. Interestingly, at 3% CO2, acetazolamide has a greater effect on growth than in air for both cell lines.

A1543 is a potential link between the cellular redox state and RNA processing in cyanobacterial cells via changes of RNA binding behavior through oxidation or reduction of a disulfide bridge in a conserved OB‐fold. Understanding if this type of regulation is occurring and how it affects cyanobacterial gene expression and carbon fixation will enable further development of Synechococcus Sp. PCC 7002 as an industrial research organism.

Support or Funding Information

Patrick Thomas is supported by the NIH T32 Biophysics training grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting the Molecular Determinants that Mediate Protein/RNA Interactions at the Synapse
Authors: Mirkovic Jovan , Hossain Md. Faruk , Stolfi Alberto , Wang Ruizhi , Stitzel Henry , Christiaen Lionel , Ruggiu Matteo
Published: June 2021

The NOVA family of RNA‐binding proteins consists of neuron‐specific splicing regulators that are targeted in an autoimmune neurodegenerative motor disorder. They are sequence‐specific splicing factors, with a modular structure consisting of three KH domains that mediate protein‐RNA interactions.

One of the targets of NOVA is a neuron‐specific isoform of the ubiquitously expressed gene AGRIN. This splice variant, termed Z+ AGRIN, is secreted by neurons and is found at the synaptic cleft at the neuromuscular junction (NMJ), a synapse between a nerve cell and a muscle cell, and the best‐studied model synapse. By inducing the clustering of acetylcholine receptors (AChR) on the postsynaptic membrane, Z+ AGRIN promotes the formation, maintenance, and development of the NMJ. The Z exons are also of particular interest as they are potential mutation sites in congenital neuromuscular diseases.

The study of NOVA function in vertebrates is complicated by the presence of two NOVA genes termed NOVA1 and NOVA2. The tunicate Ciona robusta is a marine invertebrate that is the closest living relative to vertebrates. Interestingly, Ciona has only one Nova gene. We used the NOVA‐AGRIN paradigm to study the evolution of nerve cell specific protein‐RNA interactions. We cloned and characterized Nova proteins from Ciona and generated Agrin minigenes that recapitulate the action of Nova in a tissue culture cell system. Here, we identify the specific Agrin pre‐mRNA nucleotide sequences that mediate Nova‐dependent splicing of Z+ agrin in tunicates.

Support or Funding Information

National Institutes of Health

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 effect of conserved motifs on the structure and activity of the RNA‐binding protein LARP6c from Arabidopsis thaliana
Authors: Foster Jessica Sara , Foster Corina L. , Otte‐Petrill Courtney , Betancourt Francisco C. , Lewis Karen A. , Antonelli Cecile , Billey Elodie
Published: June 2021

La Related Protein 6 (LARP6) is an RNA binding protein that belongs to the La‐Related Protein superfamily (LARPS). LARP6 contains structural motifs that are conserved across vertebrates and invertebrates. In vertebrates, LARP6 regulates the translation of collagen mRNA and thereby the synthesis of type I collagen. However, the biological function of LARP6 in plants is not well understood. Plants express three LARP6 paralogs: LARP6a, LARP6b, and LARP6c. Our study focuses on Arabidopsis thaliana LARP6c (AtLARP6c), which contains three structural motifs: a poly‐A binding protein (PABP)‐interacting motif 2 (PAM2), an RNA‐binding domain consisting of the La module comprised of the La motif and RNA recognition motif (RRM), and a C‐terminal La‐and SUZ‐C associated (LSA) motif. We hypothesize that the PAM2 motif may affect the RNA binding activity of AtLARP6c. To test this hypothesis, we recombinantly expressed and purified three forms of AtLARP6c: full length AtLARP6c, AtLARP6c‐ΔPAM2, and the isolated La module. Limited proteolysis was used to probe the structural stability of the purified proteins, demonstrating that the three constructs were stably folded. Our current work is now focused on establishing electrophoretic mobility shift assays with biotinylated RNA ligands to test the role of the deleted protein domains on the RNA binding activity of our AtLARP6c variants. Previous published studies found that full length AtLARP6c binds to polyA, polyU, and polyC oligonucleotides. These same ligands are being used to directly test how the PAM2 and auxiliary domains of AtLARP6c affect the RNA binding activity of the La module. This investigation provides insight into potential regulatory mechanisms of RNA binding in the plant LARP6 proteins.

Support or Funding Information

Support provided by Texas State University and the National Institutes of Health ‐ National Institute of General Medical Studies (GM119096) to K.A.L.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Thermodynamic Analysis of ATP Dissociation in a DEAD‐Box Protein”
Authors: Yoder Lisa , Garcia Ivelitza , Patterson Jacob , Bardine Conner
Published: June 2021

RNA plays an essential role in many cellular functions. Similar to proteins, RNA function is strictly determined by the overall structure and dynamics of the RNA macromolecule. Thus, many cellular proteins or accessory factors are associated with RNA folding. One example of these RNA binding proteins are DEAD‐Box proteins. DEAD‐Box proteins utilize ATP hydrolysis to unwind and reanneal RNA duplexes to promote correct folding during RNA processing. DEAD‐Box proteins possess two conserved RecA‐like domains that bind ATP and RNA. In addition, these proteins also contain N‐ and/or C‐ terminal domains (NTD and CTD, respectively). Peripheral domains in DEAD‐Box proteins are proposed to regulate ATP hydrolysis as well as confer RNA specificity. Thus, the NTD and/or CTD can alter the ATP binding pocket and subsequently alter ATP association and/or dissociation. For example, Rok1p, a yeast DEAD‐Box protein, contains both a NTD and a CTD. The NTD in Rok1p is suggested to affect the structure and stability of the protein. Therefore, the dissociation rate of a fluorescence ATP analog was determined through transient kinetics and stopped‐flow spectroscopy under various thermal conditions. Resulting trends conform to double exponential fits suggesting a two‐step mechanism. The observed slow rate was consistent with a conformation change in the protein that allows rapid dissociation. The rate in which the conformation change occurs was observed to be temperature dependent. The current hypothesis centers on the ability, of the Rok1p peripheral domains, to affect the slow structural change needed for ATP dissociation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Cysteine Residue Thiol Status of t‐DARPP, A Protein Involved in Chemoresistance
Authors: Aldana‐Mendoza Jesus Antonio , Momand Jamil Antonio
Published: June 2021

The gene PPP1R1B expresses t‐Darpp, a protein that activates protein kinase A and the AKT pathway in breast cancer cells. t‐Darpp, when overexpressed in breast cancer cells, also confers resistance to trastuzumab, a therapeutic that binds to the Her2 receptor. Although t‐Darpp expression is relatively high in gastric, breast and colon cancers, its structure is unknown. Human t‐Darpp has two cysteine residues that can be exploited to explore t‐Darpp structure, Cys36 and Cys119. Methoxypolyethylene glycol‐maleimide (Mal‐PEG) was used to determine whether recombinant t‐Darpp cysteine thiols are on the surface of the protein and to find out if they are reversibly oxidized. Later, a quantitative analysis was conducted using Ellman's test. 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (Ellman's reagent) was used to determine whether recombinant t‐Darpp cysteine thiols are free or not. Both studies suggest that the two cysteine thiols are either buried or oxidized. Further work will be conducted to distinguish between these two possibilities.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein lysine acetylation is a regulatory mechanism for Bacillus subtilis multicellularity
Authors: Reverdy Alicyn , Chen Yun , Chai Yunrong
Published: June 2021

Lysine acetylation is a post‐translational modification that alters the charge, conformation, and stability of proteins. Published lists of acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of function, including central metabolism, DNA transcription and binding, chemotaxis, and cell size. Bacillus subtilis is a non‐pathogenic soil bacterium, and model organism for sporulation, motility, biofilm formation, and general multicellular development. The genetic regulation of these processes has been extensively studied, thus, we aimed to investigate the role of lysine acetylation in multicellular development (or biofilm formation) in B. subtilis. To further investigate acetylation, we generated an acetylome under biofilm‐inducing conditions and identified acetylated proteins involved in multicellularity; specifically, swarming and biofilm formation. Upon deletion of known genes for acetylation, cells had significantly slower swarming kinetics and a significantly smaller final swarming radius compared to wildtype. This suggests that acetylation impacts bacterial swarming. Biofilm is a mixture of cells and secreted matrix consisting of exopolysaccharides, proteins, and nucleotides that assists bacterial communities in colonization and survival. Genetic analysis of acetylation in biofilm formation demonstrated that lack of acetylation impacts biofilm development as shown by a decrease in matrix production. Targeting the acetylated biofilm proteins, we performed site‐directed mutagenesis of known acetylated lysine residues and assayed for biofilm formation. Two biofilm proteins, YmcA, a biofilm regulatory protein, and GtaB, an enzyme for biosynthesis of a precursor sugar for exopolysaccharides, demonstrated severe biofilm defects upon mutation of lysine residues. This suggests the importance of YmcA Lys64 and GtaB Lys89 and Lys191 for function in biofilm formation. In summary, we believe that protein lysine acetylation is a novel regulatory mechanism linking cell metabolism to bacterial multicellular development.

Support or Funding Information

National Science Foundation and Northeastern University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards Visualizing Citrullinated Proteins in SDS‐PAGE Gels Using Phenylglyoxal‐Based Chemistries
Authors: Thompson Kourtland A. , Grant Jennifer E.
Published: June 2021

Citrullination is the deimination of arginine, a post‐translational modification that is the root cause of rheumatoid arthritis and multiple sclerosis. The result of this modification in these diseases is an aberrant immune response to the citrullinated proteins which results in the disease phenotype. Citrullination is notoriously difficult to visualize due to the minimal mass upon modification, and the limited options for chemical derivatization of citrulline over arginine. Furthermore, amino acid specificity patterns defining protein citrullination remain obscure, although protein secondary structure maintain an influence (Olson et al., 2017, Comp. Biol. Chem., 70:107–115). The goal of this project is to design an assay system that allows visualization of citrulline‐modified proteins on an SDS‐PAGE gel, to aid in understanding of citrullination patterns within proteins and the discovery of citrullinated proteins.

A model system has been designed to detect citrullinated peptides using SDS‐PAGE and related technologies. Peptides likely to be highly citrullinated by PAD4, like histone H3, were synthesized; citrullinated versions of peptides were also prepared to serve as positive controls. We also examined the feasibility of actively citrullinating full‐length proteins as part of the methods development process. The basis of the assay is to use PAD4 to citrullinate peptides and proteins in an in vitro system, and then employ phenylglyoxal‐based chemistry to detect citrullination events. The design of peptides and proteins, optimization of conditions for citrullination proteins in vitro by PAD4, and use of SDS‐PAGE gels in visualizing citrullinated proteins, are presented.

In sum, developing more direct methods for visualization of citrulline, like the SDS‐PAGE method, will facilitate identification of proteins susceptible to citrullination events.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying the Dimerization of the FNR Transcription Factor by Mutant Analysis at the 130 and 140 Residue Positions
Authors: Kazmouz Sobhi , Moore Laura J.
Published: June 2021

The Fumarate Nitrate Reductase (FNR) transcription factor is the main anaerobic switch in Escherichia coli bacteria. In the absence of oxygen, the FNR protein exists as a dimer with a [4Fe‐4S] cluster and binds to DNA to regulate transcription of genes needed for anaerobic respiration. When oxygen is present, however, the [4Fe‐4S] cluster degrades causing FNR to return to its inactive monomeric form. Examination of the crystal structure of Alivibrio fischeri FNR, a close variant of E. coli FNR, suggested that the dimerization and, thus, activation of FNR is dependent upon a salt bridge interaction between the Asp 130 residue and the Arg 140 residue.[1] Site‐directed mutagenesis was used to analyze how various mutations at those two particular residues can affect FNR activity. Analysis of double mutants where the 130 and 140 residues were swapped showed that the salt bridge interaction may not be playing a role in FNR dimerization since the double mutants had no FNR activity. Therefore, additional mutations were constructed to study the importance of the 130 and 140 residues in promoting dimerization. Data from β – galactosidase assays carried out on an E. coli reporter strain to measure FNR activity suggest that only Arg 140 is essential for regulating dimerization. Single mutants in which Asp 130 was replaced by hydrophobic and polar amino acids display FNR activity almost the same as that of the wild type FNR while the same mutations that replaced Arg 140 resulted in almost no FNR activity. Furthermore, alanine and leucine double mutants at positions 130 and 140 displayed FNR activity even higher than the wild type FNR activity, further suggesting a salt bridge is not required for dimerization.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural analysis of the active site and DNA binding of human cytidine deaminase APOBEC3B
Authors: Hou Shurong , Silvas Tania V. , Leidner Florian , Nalivaika Ellen , Yilmaz Nese Kurt , Schiffer Celia A.
Published: June 2021

APOBEC3s proteins (A3s), a family of human cytidine deaminases, protect the host cell from endogenous retro‐elements and exogenous viral infections by introducing hypermutations. However, their ability to mutate genomic DNA makes them a potential cancer source. Of the 7 human A3s, A3A, A3B and A3H have been implicated as an endogenous cause for multiple cancers. Among all A3s, A3B and A3A share highest sequence identity, but have distinct deamination activities with A3B about 15‐fold lower activity compared to A3A. Over the past few years, our lab along with other groups has determined several structures of apo as well as DNA‐bound A3A and A3B complexes. These structures revealed the molecular determinants of nucleotide specificity and importance of the loops around the active site, including the critical loop 1, in DNA binding. However, the current A3B‐DNA co‐crystal structure is that of a chimera with loop 1 from A3A. Hence, the molecular mechanism for how A3B binds to DNA within the context of native loop 1 and how A3B regulates its deamination activity resulting in much lower activity compared to A3A still remain unknown. Here we identified an auto‐inhibited mode of A3B that restricts its activity, revealed the molecular mechanism for DNA binding to A3B, as well as the sequence specificity upstream of substrate cytidine. Using structural analysis and molecular dynamics, we revealed a unique hydrogen bonding network in A3B loop 1. This network auto‐inhibits A3B's activity by locking the active site in a DNA‐binding incompatible mode. In addition, the critical residues for DNA binding to A3B have been identified through molecular modeling and simulations. The results were verified experimentally by binding assays with different engineered A3B mutants. Overall, structural analysis of the active site and DNA binding of A3B demonstrate how A3B structurally regulates its deamination activity and will potentially lead to discovering A3B‐specific inhibitors that may be developed as anti‐cancer drugs to benefit cancer therapeutics.

Support or Funding Information

NIH R01GM118474

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FRET‐based Measurements of Protein Multimerization in Pyruvate Carboxylase
Authors: Koza Anya Lei , Hakala Joshua , St. Maurice Martin
Published: June 2021

Changes in environmental conditions can affect protein multimerization which, in turn, may alter an enzyme's activity. The multi‐domain homotetrameric enzyme pyruvate carboxylase (PC) serves as a model to study protein multimerization. Past studies on PC have shown that it equilibrates between different oligomeric forms, but that it is active exclusively as a tetramer. When diluted to low concentrations, the PC equilibrium shifts to favor inactive monomeric and dimeric oligomerization states[1],[2]. Since the in vitro activity of PC depends on its oligomerization state, it stands to reason that regulation of oligomerization states offers a way to control the enzyme activity in vivo.

In a heterogeneous population, smaller PC oligomers can reassociate into a population containing mixed heterotetramers. Controlled manipulation of PC multimerization will aid in developing hybrid tetramers that would serve as a tool to reveal key mechanistic insights into inter‐subunit coordination during catalysis. Past remixing studies have failed to precisely control the assembly of heterogeneous tetramers. This research will contribute to a more complete description of the factors influencing PC multimerization by using a FRET‐based approach to measure the assembly of PC tetramers under a wide array of remixing conditions. A non‐conserved residue (Leu 339) on the surface of the biotin carboxylase (BC) domain of Staphylococcus aureus PC (SaPC) was mutated to a Cys for site‐specific maleimide‐based labeling with either a Cy3 or Cy5 fluorophore. These site‐specifically incorporated fluorophores will form a FRET pair when the BC domain of one PC population dimerizes with a labeled BC domain of another PC population en route to re‐associating into a heterogeneous tetramer. Preliminary characterization revealed no significant difference in the maleimide‐based labeling of WT SaPC and L339C in SaPC. However, the addition of ADP along with the BC domain transition state analogues, phosphonoacetate (PPA), dramatically reduced the labeling of WT SaPC without affecting labeling of SaPC L339C. The site‐specifically labeled FRET pair of Cy3 and Cy5 offers a method to measure PC multimerization, which enables the investigation of conditions that alter the formation of the heterogeneous PC tetramer. These studies represent the first efforts to carefully control remixing of PC through manipulation of PC oligomerization states and may provide insight into ways to control the oligomerization states in other oligomeric proteins.

Support or Funding Information

This work was supported by the National Institutes of Health grant GM117540.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reconstitution of a Tail‐anchored Mitochondrial Membrane Protein
Authors: Bakkum Amber L. , Hill Blake
Published: June 2021

Tail‐anchored (TA) proteins have a single transmembrane domain (TMD) located on the C‐terminus with the bulk of the protein facing the cytosol; in contrast with the conventional membrane protein that is translated directly into the membrane of interest. TA proteins are involved in organelle division, vesicle trafficking, and apoptosis. TA proteins lack an N‐terminal signal sequence and exhibit wide sequence variation in the TMD. Thus, TA proteins are likely inserted into the membrane post translationally. We asked if we can reconstitute Fis1, a mitochondrial outer membrane TA protein, in a detergent micelle to determine its structure. We were able to purify soluble recombinant Fis1 in the absence of detergent. Preliminary results suggest that in the absence of detergent, Fis1 exists in a soluble aggregate that can be disrupted by addition of detergent. This soluble aggregate is likely facilitated by the TMD of Fis1. We further show that in the detergent micelle Fis1 is oriented such that the cytoplasmic domain faces the solvent, reminiscent of the orientation on the mitochondrial surface. Using properly reconstituted Fis1 in detergent micelles, we collected CD, DLS, NMR, and EPR data with the goal of determining a high‐resolution structure of Fis1 in the membrane, which is an outstanding question in the mitochondrial fission field. More generally, this work has many implications for TA membrane protein reconstitution.

Support or Funding Information

NIH R01‐GM067180

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NMR structural studies of membrane proteins in bilayer environments
Authors: Radoicic Jasmina , Park Sang Ho , Opella Stanley J.
Published: June 2021

Membrane protein research focused on studying the structure, function, and interactions of proteins in membrane environments by NMR spectroscopy will be presented. Sample preparation is challenging because membrane proteins are generally low expressing in heterologous systems, hydrophobic, and difficult to re‐fold into their active conformations – any variation from the native structure results in inactive and/or mis‐folded protein. Typically, the purification or extraction of these proteins requires the use of detergents, which can be detrimental to their structure and function. Therefore, an important goal is the development and implementation of detergent‐free environments for NMR studies of membrane proteins.

The main membrane environments used for NMR studies include micelles, bicelles, liposomes, and nano‐ and macro‐discs. Studying the same protein in different environments can reveal significant differences in the structure, conformation, and orientation of the protein. For example, the HIV‐1 protein Vpu, studied using static oriented sample solid state NMR, in different lipid bicelle environments resulted in different tilt angles of the protein in the model membrane as well as the absence (or presence) of a kink in the transmembrane helix, depending on the bilayer‐forming phospholipids. Another example is the structure obtained for the hepatitis C protein p7. In DPC micelles, the protein is a hexamer whereas in DHPC micelles the protein is a monomer, having the typical two‐transmembrane spanning protein topology.

A macrodisc is a detergent‐free model membrane system composed of phospholipid bilayers surrounded by a peptide, protein, or styrene‐maleic acid (SMA) polymer belt, with a diameter of > 30nm. They serve to immobilize and magnetically align embedded membrane proteins. Examples of proteins having between one and seven trans‐membrane helices will be described, including G‐protein coupled receptors (GPCRs).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TPS domain folding causes activation of a newly defined hemolytic functional domain
Authors: Wade Gina Mary , Weaver Todd Michael , Grilley Daniel Patrick
Published: June 2021

Proteus mirabilis is a pathogenic opportunistic Gram‐negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a two‐partner secretion (TPS) pathway to simultaneously transport and activate HpmA across its outer membrane and release it into the blood of a host where it destroys red blood cells (hemolysis). Like all other TPS proteins, HpmA contains an N‐terminal TPS domain (≈300 residues) essential for secretion and a separate C‐terminal functional domain (>1000 residues) responsible for virulent functions. To understand the functional relationship between these domains and define the boundaries of the virulent hemolytic domain in HpmA, we made N‐terminal, internal, and C‐terminal deletions. In the absence of secretion, the unfolded full‐length HpmA requires a folded TPS domain to act as a template protein to achieve its hemolytically active form. Using a Template Assisted Hemolytic Activity (TAHA) assay, we can quantitate the effect of the deletions on HpmA function. Unfolded proteins lacking a TPS domain at the N‐terminus show no activity in TAHA assays. This suggests that TPS domain folding is required for activation of hemolytic activity. To determine if there is a direct linkage between TPS domain folding and hemolytic activation, we created internal deletions immediately C‐terminal to the TPS domain. These demonstrate how TPS domain folding is coupled to activation of hemolytic function. We also show that a significant segment (>300 residues) can be removed from the C‐terminus and not affect the hemolytic function. These results have allowed us to define the C‐terminal boundary of the hemolytic functional domain. Results of this research have allowed us to locate regions of HpmA necessary for its hemolytic function and characterize how TPS domain folding is required to induce hemolytic activation. These studies have expanded our working model for HpmA secretion, activation, and function.

Support or Funding Information

This research was generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity Grant (GMW).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

This work was supported by National Institutes of Health (NIH) Grants R01 GM098455‐04 and R01 GM114473‐01 and FAPESP process: 2016/22477‐1

This 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 Information

This 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 Information

NIH, NINDS R01 grant 069566 Creutzfeldt–Jakob disease Foundation Fundación Ramón Areces

This 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 Information

This 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 Information

NIGMS R15 215GM104865, Glass

Student Summer Research Award, ACPHS Graduate Research Assistantship, Evans

This 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 Information

The Swedish Research Council, The Alzheimer Foundation, The Magn. Bergvall Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.