bluuebunny/crossref_metadata_2025 huggingface dataset:
Cryo‐EM analysis of full‐length receptor tyrosine kinase
Authors: Bai Xiaochen
Published: June 2021

Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors that have been shown not only to play a key role in regulating normal cellular processes, but also to be critical in the development and progression of many types of human cancer. To date, many crystal structures of individual domains of RTKs are available; however, the high resolution structural information for full‐length RTK proteins either in their apo‐state or in ligand‐bound states remains elusive. In addition, there are no structural detail about how the active full‐length receptor can bind the effector protein for the activation of downstream signaling pathway. Lacking a complete structure of full‐length receptor signaling complex hinder our understanding of how this important protein functions. We determine the structures of full‐length RTK proteins, in complex with their ligands and downstream proteins, by single particle cryo‐EM, as key to understand the critical role that this protein family plays in the physiology of the cell. Epidermal growth factor receptor (EGFR), RET, cMET and Insulin receptor (IR), the most studied members in the RTK family, are chosen as the targets for structure determination. These pathways comprise a complex and diverse signaling network, and play an essential role in normal cell proliferation, differentiation, development and motility. Aberrant activation can lead to both tumor growth and metastatic progression of cancer cells, which makes them promising candidate for cancer therapy. Determination of full‐length RTKs in apo‐ and ligand‐bound states shed light on its mechanisms of kinase activation, enhance understanding of the interaction between receptor and their ligand and inhibitors, and facilitate development of selective anti‐cancer drugs. Reconstitution of fully assembled signaling complex reveal the mechanism by which the active receptors initialize the downstream signaling transudation, and provide direct structural evidence for how the plasma membrane plays roles in this processing. Solving the structures of different members of the RTK family reveal similarities and differences in the structure‐function relationships within this receptor family, which can potentially lead to new strategies to selectively inhibit/modulate individual pathways for cancer therapy or concurrently target various pathways, an approach that has been suggested to improve therapeutic effectiveness.

Support or Funding Information

the Welch foundation, and CPRIT foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cholesterol sulfate alters astrocyte metabolism and offers some neuroprotective effects
Authors: Prah Jude k , Yang Shaohua
Published: June 2021

Introduction

Cholesterol sulfate (CS) is one of the most important known sterol sulfates in human plasma and present as a normal constituent in a variety of human tissues. In both the brain and periphery, CS serves as a substrate for the synthesis of sulfonated adrenal steroids such as pregnenolone sulfate and Dehydroepiandrosterone (DHEA) sulfate and as a constituent of many biological membranes including red blood cells where it functions as a stabilizing agent. It also acts as endogenous regulator of cholesterol synthesis. It is known that CS serves as a substrate for synthesizing other sterol sulfates in the brain. However, the role of CS in neurological insult and brain metabolism is unknown. Our goal in this study is to investigate the neuroprotective action of CS as well as its effect on brain energy metabolism.

Materials and Methods

Primary astrocytes were prepared from the cortex of postnatal day 0–2 C57BL/6 pups and seeded in Dulbecco's modified eagle medium (DMEM) with 10% FBS under normal glucose (5.5 mM). HT‐22 cells were maintained in high glucose (25 mM) DMEM supplemented with charcoal stripped FBS. The neuroprotective effect of CS and its role on cell metabolism were determined in primary astrocyte and HT‐22 cells using Calcien AM cell viability assay, flow cytometry, seahorse extracellular flux analysis, and metabolism assay kits.

Results

CS protects HT22 cells against glutamate toxicity and impact astrocyte metabolism by increasing ATP, and glycogen contents.

Conclusion

Our study demonstrated that CS have neuroprotective effect and modulate brain energy metabolism. Further studies are needed to determine the mechanisms underlying the neuroprotective action of CS and its action on brain energy metabolism.

Support or Funding Information

This work was partly supported by National Institutes of Health grants 1R21NS087209‐01A1 (SY) and R01NS088596 (SY).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histology and Organization of the Fascia
Authors: Gordon Marion K
Published: June 2021

The diameter of collagen fibrils is a contributing factor to the functional properties of fascia. This is demonstrated by pathologies such as Dupuytren's disease, where shortening of the palmar fascia causes contracture of fingers. Cell proliferation plus overproduction of collagen are hallmarks of the disease. Excess collagen production suggests a fibrotic disorder, however Dupuytren's is different from fibrotic pathologies such as lung fibrosis, because deposited fibrils are of smaller than normal diameters, while in lung fibrosis, diameters are larger.

The minor fibrillar collagens, types III and V, FACIT collagen XIV, and certain proteoglycans regulate fibril diameters during fibrillogenesis, which occurs in steps: First, short heterotypic collagen fibrils with thin diameters are made; Second, short fibril segments gain length; Third, long fibril segments attain larger diameters by fusion with adjacent fibrils. The underlying problem in Dupuytren's disease may be the initiation of a wound healing state, with synthesis of a provisional matrix, but without the subsequent remodeling that returns the tissue to normal. By comparing the extracellular matrix components of diseased fascia with normal and fibrotic tissues, we gain clues about what molecules play important roles in the function, architecture and structural integrity of healthy fascia.
Fascia of the limbs ‐ histological and biomechanical considerations
Authors: Benjamin Michael
Published: June 2021

The lecture will commence with a general overview of the structure and functional significance fascia in both the upper and lower limbs and the importance of fascial planes in the limbs in relation to the spread of infection. Considerable emphasis will be placed on the work of Professor Frederic Wood Jones and in particular to his concept of a fascial ‘ectoskeleton’. The widespread role of fascia in dissipating stress concentration away from entheses (tendon/ligament attachment sites) will be a key aspect of the first half of the talk. Subsequent attention will be focussed on selected fascias of particular interest to the author ‐ including retinacula, the fascia lata and the plantar fascia. The intimate association of retinacula with wrap‐around tendons and their bony pulleys will be reviewed in relation to the concept of a ‘functional enthesis’ in order to explain the potential for fibrocartilage differentiation within fascia. In dealing with the fascia lata, the anatomical basis underpinning the hypothesis that iliotibial band syndrome is an overuse injury that can be better attributed to compression rather than to friction will be presented. Finally, in reviewing the plantar fascia, emphasis will be placed on its relationship to the Achilles tendon and to the heel fat pad and to its association with bony spurs and heel pain.
Human mitochondrial alanyl‐tRNA synthetase: A variant mode of RNA recognition
Authors: Borchert Karen , Anderson David , Natala Nakita , Zhu Yirong , Riley Lucas , Ener Maraia , Brown Julia , Chihade Joseph
Published: June 2021

Metazoan mitochondria contain tRNAs which are highly diverged in both sequence and structure from those found elsewhere in evolution. These tRNAs are recognized and specifically charged by nuclearly encoded mitochondrial aminoacyl‐tRNA synthetases. Our work is focused on characterizing the interaction between human mitochondrial alanyl‐tRNA synthetase (Hs mt AlaRS) and its tRNA substrate.

Mutational analysis of both the enzyme and tRNA substrate estabilished that contacts between the acceptor stem and the N‐terminal domains of the enzyme are relatively unimportant for establishing tRNA identity, in contrast to all previously characterized alanyl‐tRNA synthetases. However, the C‐terminal half of Hs mt AlaRS and the D‐acceptor domain of the tRNA are required for aminoacylation, suggesting that in the human mitochondrial system a second set of tRNA‐AlaRS contacts plays a role in determining tRNAAla identity. Because mitochondrial tRNAs are structurally fragile, we have used in‐line probing to validate RNA secondary and tertiary structure.

An important identity element in non‐mitochondrial tRNAAla is a G3:U70 wobble pair. Comparing N‐terminal AlaRS sequences from a variety of mitochondrial and non‐mitochondrial sources, some of which aminoacylate substrates that lack this element, has allowed us to identify residues associated with acceptor stem recognition.
The dynamics of the GroEL‐gp31 chaperonin complex studied with fluorescence spectroscopy
Authors: Calmat Stephane , Hendriks Johnny , van Heerikhuizen Harm , Schmidt Christoph F , van der Vies Saskia M , Peterman Erwin J.G.
Published: June 2021

The GroEL‐gp31 chaperonin complex, composed of the E. coli GroEL and the bacteriophage T4 encoded gp31, is essential for the folding of the T4 major capsid protein (gp23). Interestingly the E.coli GroEL‐GroES complex cannot satisfy the folding requirements of gp23. Although the amino acid sequence of gp31 and GroES is only 14% identical, their structure is quite similar. Cryo electron microscopy and image reconstruction of the GroEL‐gp31‐ADP complex revealed that the folding cavity is larger than that of the GroEL‐GroES‐ADP complex (Clare, JMB 2006), which is consistent with the size of the capsid protein (56kDa) being close to the maximum size thought to fit inside the folding cage. The folding of gp23 requires a gp31‐dependant cis‐folding mechanism (Bakkes PNAS 2005) but little is known about the dynamics of the GroEL‐gp31 chaperonin complex. Using stopped‐flow fluorescence spectroscopy, we show that the chaperonin‐assisted refolding of eGFP occurs with the same rate and yield with both GroES and gp31. We also show that the dissociation kinetics of gp31 from GroEL are faster than GroES, and that the binding of the gp23 capsid protein accelerates the ATPase cycle of the GroEL‐gp31 chaperonin complex.
Trafficking of neuroligin mutant proteins associated with autism
Authors: De Jaco Antonella , Miller Meghan , Comoletti Davide , Dubi Noga , Taylor Palmer
Published: June 2021

Mutations in the synaptic adhesion proteins, neuroligins (NLs) and neurexins have been found to play an important role in the etiology of autistic spectral disorders. We characterized the NL3‐Arg451Cys mutation found in two affected brothers. The exposed Cys causes retention of the protein in the endoplasmic reticulum (ER) reducing processing and delivery to the cell membrane. A new alteration found linked to autism is a deletion of exon 4 in the NL4 gene. We show by confocal fluorescence microscopy that this deletion causes defective intracellular trafficking of the mutated protein that is retained in the ER. To define molecular mechanisms involved in trafficking of NL proteins, we investigated protein‐protein interaction mechanisms between NLs and ER‐resident chaperones by immunoprecipitation. We identify calnexin and calreticulin as mayor players in the retention of NL3‐Arg451Cys protein. The use of glycan processing inhibitors shows that the binding to calnexin is important in the degradation pathway of the NLs. Other candidate partner proteins involved in the trafficking of wild type and mutant proteins are identified by 2D‐electophoresis and mass spectrometry. Metabolic labeling of NL3 has been used to study the half life of wt and mutant proteins and the sequence of association with single chaperones. Understanding the mechanisms by which these proteins are retained within the cell should provide important insights into cellular abnormalities that contribute to autism.

This work was supported by NIH R37‐GM18360 and U0‐1 ES10337 to PT and pilot research award to DC.
ERp29: A luminal chaperone that facilitates CFTR trafficking in response to 4‐Phenylbutyrate
Authors: Suaud Laurence , Miller Katelyn , Alvey Lora , Yan Wusheng , Robay Amal , Hubbard Michael , Rubenstein Ronald C
Published: June 2021

Sodium 4‐Phenylbutyrate (4PBA) improves the aberrant intracellular trafficking of deltaF508‐CFTR in Cystic Fibrosis (CF) epithelial cells. We hypothesized that 4PBA causes a complex cellular adaptation leading to altered expression of molecular chaperones and improved deltaF508 intracellular trafficking. To identify elements of this cellular response, we performed differential display RT‐PCR on RNA extracted from IB3‐1 bronchiolar epithelial cells (genotype deltaF508/W1282X) treated for 0–24 hours with 1 mM 4PBA. We isolated a cDNA clone for ERp29 (29 kDa ER Lumenal Protein) that had a time‐dependant increase in mRNA expression. ERp29 is a ubiquitous, conserved thioredoxin homolog that is prominently expressed in lung. IB3‐1 cells treated with 4PBA had increased ERp29 mRNA expression by ribonuclease protection assays and protein expression by immunoblot. In coimmunoprecipitation experiments, ERp29 was associated with deltaF508‐CFTR in IB3‐1 cell lysates, but not detectably with wt‐CFTR in lysates of T84 intestinal epithelial cells. In oocytes, modest ERp29 overexpression increased the functional expression of wt and deltaF508‐CFTR. This effect was lost at higher levels of ERp29 overexpression. The increased wt‐CFTR functional expression corresponded to increased expression of CFTR at the oocyte surface. These data are consistent with increased ERp29 expression facilitating ΔF508 trafficking and functional expression in CF epithelial cells in response to 4PBA.

Supported by grants from NIDDK.
Cloning and expression of a putative metacaspase gene from Schizophyllum commune
Authors: Carrese Alexander James , Fox Melissa M. , Fox Kristin M.
Published: June 2021

Caspases are essential players in mammalian apoptosis, but their role in the fungal life cycle is not well understood. If the role of apoptosis can be examined in simpler organisms, hopefully it will aid in elucidation of the role of the details of this process in overall development. Using degenerate PCR based on the sequence of the yeast metacaspase, YCA1, a putative metacaspase has been identified in S. commune, and its genomic sequence has been determined. Because the entire open reading frame is not present in the cDNA library, it is being constructed through splicing by overlap extension PCR with the genomic DNA and the partial cDNA clone as templates. At this point, the cDNA has been successfully amplified. Further work will involve amplifying three missing exons and constructing the full gene.
Nuclear proteins from Leishmania tarentolae can bind to a putative tRNA promoter element
Authors: Jennings Kimberly , Alfonzo Juan , Pitula Joseph
Published: June 2021

Trypanosomatids are the cause of many diseases across the world. Leishmania is a specific trypanosomatid that is the causative agent of Leishmaniasis. Trypanosomes are unique in their tRNA transcription in that, two transcription factors, TFIIIC and TFIIIB, which were believed to be necessary for RNA Pol III recognition, are not found in genomic databases. To find out if there are any tRNA transcription factors that bind to the promoter region of the tRNA gene, we conducted band‐shift assays using nuclear extracts from Leishmania cultures and an oligonucleotide sequence of the promoter region. The assays revealed three distinct protein‐ DNA complexes. By using increasing amounts of NaCl, we found that higher levels of salt (>0.15M) lead to an inhibition of protein binding. We also used competition assays to test the specificity of the complexes using unlabeled duplex and a PCR fragment. We conclude that complex C is the most stable complex and may not be specific to the oligonucleotide sequence. Further assays will need to be conducted to characterize the two remaining complexes.
Acrolein inhibits cytokine gene expression by alkylating cysteine and arginine residues in the NF‐κB1 DNA‐binding domain
Authors: Lambert Cherie , Li Jimei , Yang Teng‐Chieh , Harvey Jean , Freed Brian
Published: June 2021

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The α,β‐unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of IL‐2, IL‐10, GM‐CSF, IFN‐γ and TNF‐α by human T cells, but did not inhibit production of IL‐8. Acrolein inhibited induction of NF‐κB DNA‐binding activity following mitogenic stimulation of T cells, but had no effect on induction of NFAT or AP‐1. Acrolein inhibited NF‐κB1 (p50) binding to the IL‐2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2,000‐fold more potent than crotonaldehyde in blocking DNA binding to an NF‐κB consensus sequence. Matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys61 and Arg307) in the DNA‐binding domain. Crotonaldehyde reacted with Cys61, but not Arg307, while the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor. Supported by NIEHS grant ES05673 and by a grant from Philip Morris Incorporated.
Evolution of Cooperativity in the Phosphagen Kinase Family
Authors: McGinley Marisa Patryce , Fraga Dean , Snider Mark
Published: June 2021

The phosphagen kinases (PK) family of enzymes are widely distributed in various animal, protozoan, and bacterial taxa. They catalyze the reversible transfer of a phosphoryl group onto a guanidine acceptor and have an important role in cellular energy homeostasis. Two large groups dominate the PK family; arginine and creatine kinases. Arginine kinases (AK) are found in bacterial and invertebrate organisms and typically function as monomeric enzymes. Conversely, creatine kinases (CK) are found in some invertebrates and all vertebrates and are homodimeric enzymes. Recent studies have indicated that dimeric CKs may display cooperativity between subunits. This study investigated the nature of cooperativity in a broad range of species to better understand the possible evolution of this trait. Measurements of cooperativity using an isothermal titration calorimeter (ITC) have been completed on the dimeric AK found in the sea cucumber, Sticopus japonicus and indicate that this species displays cooperativity. Studies are currently underway comparing these results with any cooperativity seen in two protoflagellar CKs found in the sponge, (Tethya aurantia). These are thought to be at the base of the lineage leading to both the cytoplasmic and flagellar CKs and many allow us to hypothesize about the origin of this property. This project was funded by NSF‐RUI #0344432.
Characterization of the arginine kinase family of Caenorhabditis elegans
Authors: Andrus Valerie Marie , Thomas David , Meulia Tea , Snider Mark , Fraga Dean
Published: June 2021

Phosphagen kinases aid in cellular regulation of energy homeostasis through the transfer of high‐energy phosphates to acceptor compounds such as creatine or arginine for later use when energy demands are high. Two significant subfamilies of PKs include arginine kinases and creatine kinases. The physiological roles of the CK family have been extensively studied, but less is known about the physiological roles of AKs. C. elegans is a well‐developed model system that can be used to develop our understanding of the evolution of the physiological roles of AKs. In this study, the activities of four separate AK isomers, isolated from C. elegans, were characterized and found to be similar to other AKs, validating the use of this model system. We are now attempting to determine if the localization pattern of these AKs is similar to what has been observed in CKs. One C. elegans AK isoform was identified as being potentially mitochondrially localized on the basis of putative signal sequences observed at the N‐terminus. Preliminary immunohistochemistry results indicate that this isoform is restricted in tissue expression to muscle and possibly nervous tissue, indicating that tissue restricted expression of isoforms in this family evolved early. Our current efforts are directed at determining if it is resident in the mitochondria.
Use of VX and V‐analog compounds to probe the spatial requirements of the active site of wild type and mutant human paraoxonase 1
Authors: Otto Tamara Caviston , Yeung David T , Cerasoli Douglas M , Lenz David E
Published: June 2021

Human paraoxonase 1 (HuPON1) exhibits catalytic activity toward the organophosphorus nerve agent VX, but the catalytic mechanism mediating this activity is unresolved. To examine the effect of substrate volume on activity, the hydrolysis of five analogs of VX was examined. The analogs were designed such that the O‐ ethyl alkyl group of VX was replaced with O‐propyl (V1), O‐isopropyl (V2) or O‐isobutyl (V3) on the retained group, or the 2‐diisopropyl alkyl groups were replaced with 2‐diethyl (V4) or 2‐dimethyl (V5) on the leaving group. Both Km and kcat values for V1, V2, and V3 decreased as the retained group increased in size. In contrast, reducing the size of the leaving group (V4 and V5) had little effect on Km and progressively decreased kcat. The H1 15W mutant of HuPON1 hydrolyzed V1, V4 and V5, whereas no turnover of V2 or V3 was detected. Further analyses determined that V3 was recognized as a competitive inhibitor of VX hydrolysis by the H115W mutant. The results indicate that the volume of both the retained and leaving groups can affect binding of these substrates to HuPON1 and also suggest that the R‐chain of the retained group may directly interact with residue 115. Together, the results allow an estimate of the size of the HuPON1 binding pocket to be made.

This work was supported by NIH Centers of Excellence Grant U‐54 NS 058183‐01 Center for Catalytic Bioscavenger Medical Defense Research. TCO was a NRC research fellow.
Oligomeric state and utilization of various substrates by a Bcp‐class peroxiredoxin from Escherichia coli
Authors: Reeves Stacy A , Parsonage Derek , Nelson Kimberly J , Poole Leslie B
Published: June 2021

Peroxiredoxins (Prx) possess a conserved active site structure centered around the catalytic residue (the “peroxidatic” cysteine) which forms a cysteine sulfenic acid upon reaction with peroxides; this cysteine subsequently forms a disulfide bond with the putative resolving cysteine (either inter‐ or intramolecular) and/or the reductant during reductive recycling. In Escherichia coli, bacterioferritin‐comigratory protein (Bcp) is a peroxiredoxin responsible for catalyzing the reduction of hydrogen peroxide and alkyl hydroperoxides. A second cysteine is located near the active site of E. coli Bcp (conserved in 48% of Bcp homologs) which may or may not serve as a resolving cysteine during catalysis. Analytical ultracentrifugation studies of reduced and oxidized Bcp showed that E. coli Bcp exists as a monomer in solution, eliminating the possibility of intersubunit disulfide bond formation. During thioredoxin‐dependent peroxidase activity studies conducted by stopped flow spectroscopy, a linear rate increase was observed with increasing reducing substrate along with at least six‐fold higher turnover rates than those previously reported. An exploration of other potential reductants of Bcp also indicated that comparable rates were observed with either glutaredoxin (Grx1) or thioredoxin, indicating that Grx1 likely also acts as a reductant of Bcp in E. coli. Supported by NIH RO1 GM50389 to LBP.
Metal‐triggered hydrogelation of self‐assembling β‐hairpin peptides for bioremediation
Authors: Knerr Patrick James , Nagarkar Radhika P. , Schneider Joel P.
Published: June 2021

Peptides have been designed which undergo intermolecular self‐assembly to form rigid, fibrillar hydrogel networks. These peptides are unstructured and soluble in aqueous solution, but can be triggered to fold into an amphiphilic β‐hairpin conformation under specific environmental conditions. Once folded, these hairpins self‐assemble to form the hydrogel. Introduction of metal‐binding functionality into such a system allows for the development of materials in which hydrogelation can be controlled with particular metal ions. This work reports a β‐hairpin peptide incorporating two strategically‐placed cysteine residues, known to bind a host of metal ions including arsenic and zinc. Equimolar addition of methylarsonous acid to an aqueous peptide solution triggers folding and self‐assembly at low peptide concentration (1.0 wt %). Metal‐dependent behavior has been demonstrated using circular dichroism, FTIR, TEM and oscillatory shear rheology. It is envisioned that this system could be responsive to other cysteine‐binding toxic metals, such as mercury and lead. Since the peptide sequence includes only natural L‐amino acids, this peptide can potentially be synthesized biologically and used for large‐scale bioremediation of polluted environments. Funding has been supplied by the Beckman Foundation, the HHMI Undergraduate Biological Sciences Education Program and the NIH.
Model of how gating electron delocalization along the main chain impacts the formation of secondary structure in proteins
Authors: Dwyer Donard S.
Published: June 2021

Factors that determine formation of secondary structure in proteins are incompletely understood. We have proposed that the electronic properties of amino acid side chains are important in this process because they regulate electron delocalization along the main chain through substituent effects. A model is presented for how these electronic effects direct the formation of secondary structure. The electronic properties of the amino acid side chains (e.g., inductive and field effects) determine the electron density at the Cα atom, a key node in the transmission pathway. When the electron density at Cα is high, electron delocalization (transmission) is facilitated, which decreases bond length favoring extended conformations. Similar effects are seen in molecular electronic devices. Conversely, when the electron density at Cα is low, electron delocalization is interrupted along the main chain, thus increasing bond length and rotational flexibility. Electron density at the Cα atom is inversely correlated with preference for α‐helical structure. Additional factors (e.g., hydrophilicity) also affect electronic parameters including bond orders. Thus, the electron density along the main chain determines the fold.
Crystal Structure of the Naturally Fused Coral Lipoxygenase and Allene Oxide Synthase
Authors: Gilbert Nathaniel Carson , Niebuhr Marc , Tsuruta Hiro , Bordelon Tee , Dassey Adam , Bartlett Sue , Newcomer Marcia
Published: June 2021

A naturally occurring bi‐functional protein from Plexaura homomalla links sequential catalytic activities in an oxylipin biosynthetic pathway. The C‐terminal 8R‐lipoxygenase (8‐LO) domain catalyzes the transformation of arachidonic acid (AA) to the corresponding 8R‐hydroperoxyeicosatertaenoic acid, and the N‐terminal allene oxide synthase (AOS) domain promotes the conversion of the hydroperoxide intermediate to the product allene oxide (AO). The structures of the independently expressed 8‐LO and AOS domains have been reported, and previous work has indicated that there are three calcium binding sites that upon coordination promote lipid binding. We report here the crystal structure of the full length bi‐functional enzyme in a calcium bound conformation. Small angle X‐ray scattering data indicate that the two catalytic domains that transform AA to AO associate to form a complex that recapitulates the structure of the bi‐functional protein in the absence of their covalent linkage. However, membrane binding experiments indicate that the covalent tether is required for Ca2+‐dependent membrane targeting of the sequential activities. Furthermore, membrane targeting is accompanied by a conformational change as monitored by specific proteolysis of the linker that joins the AOS and 8‐LO domains. Our data support a model in which Ca2+‐dependent membrane binding releases the non‐covalent interactions between the AOS and 8‐LO domains and suggest that the membrane and AOS binding sites on the 8‐LO domain overlap.
Amyloid‐beta peptide toxicity in neuronal‐like, differentiated PC12 cells: calpain‐calpastatin, caspase and membrane damage
Authors: Kosower Nechama S , Vaisid Tali , Elkind Esther , Barnoy Sivia
Published: June 2021

Amyloid‐beta peptide (Aβ) is central to the pathogenesis of Alzheimer's disease (AD). It is toxic to neurons, possibly via causing initial neuronal membrane and synaptic damage. Although implicated in AD, calpain and caspase interaction or the role of the specific calpain inhibitor, calpastatin, are little understood.

Objective:

to examine effects of Aβ 25–35 (sAβ) on the proteases, on calpastatin and on the membrane in neuronal‐like cells.

Results:

sAβ‐treated cells exhibited primarily cell membrane damage (varicosities along neurites, enhanced membrane permeability to propidium iodide (PI), no apparent nuclear changes of apoptosis, and little poly(ADP‐ribose) polymerase (PARP) degradation). The sAβ‐induced damage contrasted with the staurosporine‐induced damage (nuclear apoptotic changes, PARP degradation, no PI permeability). sAβ led to caspase‐8 and calpain activation (with cross‐talk between them), promotion of calpastatin degradation (by caspase‐8 and by calpain), and enhanced degradation of fodrin (mainly by calpain). Calpastatin overexpression attenuated the sAβ‐toxicity (lowering PI permeability and fodrin degradation).

Conclusions:

Aβ causes primarily neuronal membrane damage, responding to calpain/caspase‐8 activation and calpastatin loss. Increased expression of calpastatin and/or decrease in the proteases may serve to ameliorate early symptoms of AD.
PDMD: A complex II‐regulated form of mitochondrial dysfunction in metabolically exhausted cells
Authors: Hawkins Brian John , Petrenko Nataliya B , Patel Vickas V , Davis Christiana , Madesh Muniswamy
Published: June 2021

Functional impairment of mitochondria contributes to many human diseases including myocardial infarction, stroke, cancer, aging and neurodegeneration. However, the underlying mechanisms of mitochondrial dysfunction are not well understood. Our studies elucidate a mode of mitochondrial depolarization that is independent of cyclophilin D, Bcl‐2 family proteins, extra mitochondrial energy, or high amplitude swelling. We termed this mode of mitochondrial dysfunction proton‐dependent mitochondrial depolarization (PDMD), which prominently manifests in ischemic cells. The evolutionarily conserved PDMD is dependent on dissipation of the mitochondrial proton gradient and can be solely reversed by the complex II substrate, succinate. Human fibroblasts lacking mitochondrial‐encoded complex I and F0F1‐ATPase subunits still maintain mitochondrial membrane potential by a complex II‐driven mechanism. This route of reversible mitochondrial functional impairment provides a potential target for the treatment of mitochondrial diseases and rejuvenation of exhausted cells in acute injury.
Genotoxic Stress Modifies the RNA Polymerase II CTD Code to Regulate Alternative Splicing
Authors: Wang Jean Y. J. , Lee Shun J.
Published: June 2021

The majority of cancer therapeutic agents are genotoxins that damage DNA. Genotoxic drugs, e.g., doxorubicin, activate the DNA damage response pathways to alter a wide spectrum of cellular processes. The ubiquitously expressed ABL tyrosine kinase is activated by DNA damage to phosphorylate RNA Polymerase II at the C‐terminal repeated domain (CTD), which consists of the heptad sequence YSPTSPS iterated 52 times. Differential phosphorylation of the CTD dictates its interaction with various factors, underlying the concept of a CTD code for transcription regulation. We show doxorubicin stimulates ABL‐dependent CTD tyrosine phosphorylation to disrupt interaction with p72 helicase leading to inhibition of RNA splicing at CD44 variable exon 5. The human genome contains twenty thousand genes but its protein‐coding capacity is greatly expanded through alternative splicing. The finding that DNA damage alters the CTD code to regulate splicing adds a new level of complexity to how cells respond to chemotherapeutic drugs.
Kinases in Diabetes and Hypertension
Authors: Cobb Melanie H. , Cummings Staci , Wauson Eric , Heise Charles , Raman Malavika , Lawrence Michael , Chen Wei
Published: June 2021

WNKs (with‐no‐lysine [K]) 1–4 are unique protein‐serine/threonine kinases conserved in multicellular organisms. The ATP‐binding lysine present in all protein kinases is in an atypical position in WNKs. Two of the four WNKs were identified as genes mutated in pseudohypoaldosteronism type II, a genetic syndrome characterized by hypertension and hyperkalemia. All four WNKs contain very large noncatalytic regions with no currently known domain organization that appear to function as scaffolds. WNKs have been implicated not only in regulation of ion balance, but also in cell survival and proliferation, membrane trafficking and embryonic organ development. Mitogen‐activated protein kinases MAPKs transduce signals in a multitude of cellular pathways in response to many ligands and cell stimuli. MAPKs have been implicated in diseases including cancer, inflammatory disease, obesity, and diabetes. The MAPKs ERK1/2 are required for glucose‐regulated insulin gene transcription in pancreatic beta cells. Nutrients and hormones that stimulate or reduce insulin secretion have a parallel effect on ERK1/2 activity in beta cells. ERK1/2 control the function of several transcription factors that mediate glucose‐regulated insulin gene transcription, including at least two factors that are encoded by maturity‐onset diabetes (MODY) genes.

Research supported by grants from the National Institutes of Health, R01‐GM53032, R37‐DK034128, R01‐DK055310 and the Robert A. Welch Foundation I‐1243 and the American Diabetes Association Mentor‐based Postdoctoral Fellowship award.
Discovery of a Widely Distributed Antibiotic Biosynthesis Gene Cluster
Authors: Mitchell Douglas A , Lee Shaun , Markley Andrew L. , Hensler Mary E. , Dixon Jack E.
Published: June 2021

Bacteriocins represent a large family of ribosomally‐produced antibiotics. Here, we describe the discovery of a widely‐conserved prokaryotic bacteriocin biosynthetic operon, encoding a toxin precursor and all of the necessary modifying enzymes for toxin maturation. We present evidence that the cytolytic Streptococcus pyogenes virulence factor, streptolysin S, and analogous toxins from Clostridium botulium and Pyrococcus furiosus are converted to highly active cytolysins by the actions of the modifying enzymes. These enzymes also accept alternate substrates, boosting hopes to exploit this system to create novel antibiotics.
The role of the sodium hydrogen exchanger in stress fiber formation in human lung cancer stimulated with urokinase plasminogen activator, lysophosphatic acid, and phenylephrine
Authors: Canine Jenny , Wallert Mark A , Provost Joseph J
Published: June 2021

One of cancers hallmarks is the ability of cells to spread from the tumor of origin to other areas of the body, a process called metastastis. The Sodium‐Hydrogen Exchanger (NHE) plays a role in pH regulation and cytoskeletal attachment to the plasma membrane. In studies using CCL39 lung fibroblasts, phenylephrine (PE) induced stress fibers, and caused cells to become tumor like. In wound assay studies, PE stimulated stress fiber formation in cells immediately adjacent to the wound area and in migrating cells. These data indicate that stress fiber formation has a direct involvement in cell migration. In this study, we relate data from CCL39 cells to stress fiber formation in human lung cancer. We studied stress fiber formation in H358, H460, and H1299 cells. Our data shows that in H358 cells both 50μM PE and 10nM urokinase‐type plasminogen activator (uPA) significantly increase stress fiber formation. In H460 cells, 3μM lysophosphatidic acid (LPA) and UPA significantly increase stress fiber formation, and in H1299 all three hormones significantly increase stress fiber formation. Our data also shows that using ethylisopropylamiloride to inhibit NHE decreases the level of stress fiber formation in H460 and H1229 cells. These studies indicate that NHE plays a vital role in stress fiber formation and cellular migration, metastasis, in human lung cancer cells.
Inhibition of succinate‐linked mitochondrial respiration by hydrogen peroxide
Authors: Humphries Kenneth M. , Moser Michelle D.
Published: June 2021

Hydrogen peroxide produced from electron transport chain derived superoxide is a relatively mild oxidant, and as such, the majority of mitochondrial enzyme activities are impervious to physiological concentrations. Previous studies, however, have indicated that succinate dehydrogenase (SDH or complex II), a crossover point between the Krebs cycle and electron transport chain, is sensitive to H2O2 mediated inhibition. As such, SDH represents a potentially critical target for free radical induced mitochondrial dysfunction and/or redox regulation. In the present study, the effects of H2O2 on isolated cardiac mitochondria respiring on succinate were therefore examined. The results presented indicate that H2O2 inhibits state 3 mitochondrial respiration in the absence of rotenone, a complex I inhibitor, but not in its presence. Under normal conditions, SDH is inactivated during state 3 respiration and reactivated during state 4. In the presence of H2O2, SDH is inactivated during state 3 but then fails to fully recover activity when transitioning to state 4. Rotenone is able to completely prevent inactivation of SDH during state 3 in control and H2O2 treated mitochondria. In contrast to SDH, NADH oxidase activity increases during state 3 respiration and decreases during state 4. H2O2 treatment sustains heightened NADH oxidase activity during state 3 which parallels the loss in SDH activity. It is concluded that H2O2 inhibits succinate linked respiration by maintaining SDH in an inactive form via an indirect mechanism and not a result of a direct oxidation of SDH.
Scd1 deficiency causes endoplasmic reticulum stress and metabolic dysfunction in mice fed a very low‐fat diet
Authors: Flowers Matthew T. , Keller Mark P. , Choi YounJeong , Lan Hong , Kendziorski Christina , Ntambi James , Attie Alan
Published: June 2021

We performed microarray analysis of hepatic gene expression in mice deficient in stearoyl‐CoA desaturase‐1 (Scd1) and maintained on a chow or a very low‐fat (VLF) diet for 10 days. We identified an extraordinary number of differentially expressed genes (>4000 probe sets) in the VLF Scd1−/− relative to both VLF Scd1+/+ and chow Scd1−/− mice. Transcript levels were reduced for genes involved in several facets of fatty acid metabolism including biosynthesis, elongation, desaturation, oxidation, transport, and ketogenesis. This pattern is attributable to the decreased mRNA abundance of several key lipid metabolism transcription factors, including LXRα, RXRα, PPARα, PGC‐1β, SREBP1c, and ChREBP. A robust induction of endoplasmic reticulum (ER) stress is indicated by the increased expression of stress‐induced transcription factors, including CHOP and ATF3, and several of their target mRNAs. Activation of the unfolded protein response pathway is further evidenced by increased splicing of XBP1 mRNA. The gene expression profile is also consistent with induction of an acute inflammatory response and macrophage recruitment. These results highlight the importance of monounsaturated fatty acid synthesis for maintaining metabolic homeostasis in the absence of sufficient dietary unsaturated fat and point to a novel cellular nutrient‐sensing mechanism linking fatty acid availability and/or composition to the ER stress response. Supported by NIH grants HL56593, DK58037, and DK066369.
Cyclooxygenase‐2 Induction by trans‐10, cis‐12 Conjugated Linoleic Acid in Adipose Tissue
Authors: Belda Benjamin , Vanden Heuvel John P.
Published: June 2021

trans‐10, cis‐12 conjugated linoleic acid (10e12z) is the isomer linked with weight loss in animal and decreased adipose differentiation in cell culture models. The mechanism of this is still being determined. However, 10e12z induces an inflammatory response in adipose in the form of induction of interleukin (IL)‐6 and activation of NF‐κB. COX‐2 is an inflammatory mediator and is induced in response to NF‐κB. Therefore, we asked if there is a role for COX‐2 in the response of adipocytes to 10e12z. Here we show, in differentiating 3T3‐L1 cells, 10e12z reduces the ability of preadipocytes to differentiate and accumulate lipid, as measured by expression of markers such as aP2, C/EBPα and ADRP as well as oil red‐o staining. Furthermore, these effects of 10e12z are evident 5 days after induction of differentiation. Concurrently, a marked increase in the expression of COX‐2 by 10e12z treatment occurs before increases in IL‐6 expression. These results suggest that COX‐2 induction is adipocyte specific. Consistent with this, we could not induce COX‐2 in 3T3‐L1 preadipocytes. However, COX‐2 mRNA is induced rapidly in mature adipocytes reaching a maximum after 12 hours and declining to baseline after 48 hours. COX‐2 mRNA is also dose responsive, and can be significantly induced with 12.5 uM 10e12z CLA. These studies indicate that the induction of COX‐2 is an important event mediating the metabolic effects of 10e12z in adipocytes.
Spo20p‐GFP, a probe that allows visualization of the dynamic of phosphatidic acid during regulated exocytosis.
Authors: Vitale Nicolas , Zeniou‐Meyer Maria , Bailly Yannick , Bader Marie‐France
Published: June 2021

Several reports have recently proposed phospholipase D (PLD) as a component of the molecular machinery underlying calcium‐regulated exocytosis. In consequence it is expected that PLD should be activated and phosphatidic acid (PA) produced at the site of exocytosis, which would be at the plasma membrane and/or on the membrane of arriving secretory granules. Among bioactive lipids PA is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To assess the dynamic location and actual function of PA in secretory cells, we attempted to visualize the intracellular location of PA production during the process of secretion in neuroendocrine cells, using a Green Fluorescent Protein (GFP) sensor that specifically binds to PA. PC12 cells were transfected with a chimeric protein consisting of EGFP‐fused to the PA‐binding domain (PABD) of Spo20p, a yeast homolog of SNAP25. We were able to show a transient accumulation of the PA sensor at the cell periphery during exocytosis. Using a silencing RNA strategy, we further demonstrate that PLD1 is responsible for PA accumulation at the plasma membrane in secretagogue‐stimulated cells. Finally using immuno‐gold electron microscopy, we observed PA in‐between peripheral granules and the plasma membrane in stimulated cells, suggesting that PA is generated at the granule docking sites.
ERK and p38 MAPK‐regulated peroxiredoxin 6 localization to lamellar bodies in MLE 12 cells
Authors: Sorokina Elena M , Wu Yongzheng , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid turnover and protects lung epithelium against oxidative stress. Prdx6 has been localized by immunocytochemistry and sub‐cellular fractionation to cytosol and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization determines the balance between the 2 activities. Using a GFP‐Prdx6 fusion protein, we show that ERK and p38 MAPK regulate Prdx6 localization to organellar structures compatible with lamellar bodies in the MLE12 mouse type II alveolar epithelial cell line. Treatment of GFP‐Prdx6 transfected MLE12 cells with PD98059 and SB202190, inhibitors of ERK and p38 MAPK, respectively, abolished Prdx6 organellar targeting; a JNK kinase inhibitor had no effect. Using mutagenesis, we show that Prdx6 organellar localization is determined by the 31–40 amino acid region which contains a phospholipid binding sequence GDSWG. Serine to alanine (S32A) mutation within this sequence abolished targeting and decreased protein binding to phospholipids as determined by FRET analysis. These studies suggest that Prdx6 targeting to acidic organelles and its consequent PLA2 activity may be determined by MAPK‐regulated lipid binding to a specific Prdx6 amino acid sequence. [HL019737]
O‐GlcNAc modification of CaMKIV
Authors: Dias Wagner Barbosa , Cheung Win , Wang Zihao , Hart Gerald
Published: June 2021

Similar to phosphorylation, O‐GlcNAcylation (or simply GlcNAcylation) is an abundant, dynamic, and inducible post‐translational modification. In some cases, GlcNAcylation and phosphorylation occur at the same or adjacent sites, modulating each other. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription, among others. GlcNAcylation affects protein‐protein interactions, activity, stability, and expression. Calcium (Ca2+) is a central secondary messenger involved in regulating cytoskeletal proteins, ion pumps, and activities of enzymes, including Ca2+/calmodulin‐dependent protein kinases (CaMKs). CaMKIV is a multifunctional, serine‐threonine protein kinase that is activated in the presence of increased intracellular Ca2+, acting as a potent mediator of Ca2+‐induced gene expression. Here, we show that CaMKIV is highly GlcNAcylated in vitro and in vivo. In addition, we identify five sites of GlcNAcylation on CaMKIV. Site‐directed mutagenesis of these residues and their influence on activity and function are currently under investigation.

This work is supported by NIH grants CA42486 and HD13563. G.W. H. receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHU.
Post‐translational modifications of Deformed Epidermal Autoregulatory Factor‐1
Authors: Jensik Philip J. , Tolley Luke T. , Yau Peter M. , Collard Michael W. , Huggenvik Jodi I.
Published: June 2021

Deformed Epidermal Autoregulatory Factor 1 (DEAF‐1) is a transcriptional regulator that is important for embryonic development in mice, and is linked to depression and cancer in humans. The localization of DEAF‐1 and its interaction with other proteins have been shown to affect DEAF‐1 functions. To identify post‐translational modifications of DEAF‐1 that may affect function, purified proteins from HEK293T cells were subjected to liquid chromatography‐mass spectrometry. Three phosphorylated amino acids and a methylated arginine within the DNA binding domain were identified. In vitro mutagenesis and functional assays were performed to determine if these sites could influence known activities of DEAF‐1, including: DNA binding, transcriptional regulation, localization, apoptosis, and protein‐protein interactions. In vitro assays were used to confirm cyclin dependent kinase 5 (CDK5) as the modifying enzyme of at least two of the phosphorylation sites and protein arginine methyltransferase 6 and 8 (PRMT6 and PRMT8) were found to methylate DEAF‐1. How these protein modifications of DEAF‐1 regulate its functions are being explored.