bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of Eukaryotic CMG Helicase at a Replication Fork and Implications for Replisome Architecture and Origin Initiation
Authors: Yuan Zuanning , Georgescu Roxana , Bai Lin , Santos Ruda , Zhang Dan , Yurieva Olga , O'Donnell Michael , Li Huilin
Published: June 2021

In eukaryotes, DNA replisome is a large protein complex that carries out DNA replication, which contains several enzymatic protein subunits, such as helicase, RNA primase and DNA polymerase. Replisome generates a DNA replication fork and duplicates both the leading and lagging strand of duplex DNA. Previous studies showed the Ctf4 trimer is proposed to be a hub to coordinate the subunits in replisome, Ctf4 trimer may link CMG and Pol a through binding CIP motif of Sld5 and Pol a. By using single‐particle cryo‐EM of an active CMG on the forked DNA with a dual biotin‐streptavidin block, we solved the first structure of helicase in complex with a DNA fork. The DNA duplex stem penetrates into the central channel of the NTD tier and the unwound leading ssDNA traverses the channel through the NTD tier into the CTD motor tier, 5′‐3′ through CMG. Therefore, during translocation the CTD motor is behind the NTD ring that is pushed ahead to face the fork, opposite the currently accepted polarity. The polarity of the NTD ahead of the CTD places the leading Polɛ below CMG and Polα‐primase at the top of CMG, above the unwinding point of the replication fork. The DNA direction requires that the Mcm rings must first pass one another when the Mcm2‐7 double hexamer come apart. Interestingly, the new NTD‐to‐CTD polarity of translocation reveals an unforeseen quality control mechanism that each Mcm2‐7 is fully converted from encircling dsDNA to ssDNA before they can depart from the origin. We propose this architecture of two motors head to head may generate energy that underlies initial melting at the origin.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural basis for teneurin function in circuit‐wiring: A toxin motif at the synapse
Authors: Arac Demet
Published: June 2021

Teneurins (TENs) are cell‐surface adhesion proteins with critical roles in tissue development and axon guidance. Here we report the 3.1‐Å electron cryo‐microscopy structure of the human TEN2 extracellular region (ECR), revealing a striking similarity to bacterial Tc‐toxins. The ECR includes a large β‐barrel that partially encapsulates a C‐terminal domain, which emerges to the solvent through an opening in the mid‐barrel region. An immunoglobulin (Ig)‐like domain seals the bottom of the barrel while a β‐propeller is attached in a perpendicular orientation. We further show that an alternatively spliced region within the β‐propeller acts as a switch to regulate trans‐cellular adhesion of TEN2 to latrophilin (LPHN), a transmembrane receptor known to mediate critical functions in the central nervous system. One splice variant activates trans‐cellular signaling in a LPHN‐dependent manner, whereas the other induces inhibitory postsynaptic differentiation. These results highlight the unusual structural organization of TENs giving rise to their multifarious functions.

Support or Funding Information

NIGMS

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functionally Important Conformational Sub‐States in Human Ribonuclease Family
Authors: Bafna Khushboo , Narayanan Chitra , Bhojane Purva P , Bernard David , Howell Elizabeth E , Doucet Nicolas , Agarwal Pratul K
Published: June 2021

Eight members of the human pancreatic ribonuclease (RNase) family have diverse biological functions including angiogenesis and host defense (cytotoxic and anti‐pathogen activities) but share common structural scaffold and catalyze the hydrolysis of ribonucleic acid (RNA). However, the catalytic efficiency and rate of dynamics among these enzymes differ by more than a million (106) folds. Emerging evidence from other enzyme systems propose that internal motions drive the sampling of short‐lived minor population of conformations (called as sub‐states) that can contain features to promote various steps during the function of an enzyme. Therefore, quantitatively characterizing the conformational sub‐states in the catalytic cycle of enzymes, including substrate binding, structural rearrangements leading to the transition state, product formation and product release will enable us to obtain detailed insights in the role of enzyme dynamics in catalysis by RNases. We have used a combination of theoretical modeling, computer simulations, steady state kinetics and NMR experiments. Our studies indicate that RNases superfamily can be classified into sub‐families with designated biological functions and conserved dynamical behavior. Further, computer simulations show diverse binding preferences across the human RNase family and the conformational sub‐states in various steps of the catalytic cycle differ among the members belonging to each of the sub‐family. Moreover, we observe a ~106 fold difference in the catalytic rates of the human RNases. Additionally, we are using hybrid QM/MM method to model the catalysis of the single stranded RNA that would enable us to identify the distinct conformational sub‐states sampled by each of the members of human RNase family in their respective minimum energy reaction pathway. Results show interesting differences in the conformational sub‐states in the members of human RNases family.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palladin is needed for proper actin‐rich comet tail formation and can functionally replace the Arp2/3 complex during intracellular motility of Listeria bacteria
Authors: Dhanda Arandeep Singh , Vogl A Wayne , Albraiki Sharifah E , Otey Carol A , Beck Moriah R , Guttman Julian A
Published: June 2021

Listeria monocytogenes (Listeria) bacteria are well‐known for their ability to hijack the eukaryotic actin cytoskeleton during infections. To propagate disease, Listeria generate Arp2/3‐based actin‐rich structures called comet tails that move the bacteria within and amongst host cells. The host protein palladin is a key component of actin‐rich structures normally generated during eukaryotic cell motility, however the precise function(s) of palladin during motility remains unclear. Here we tested the hypothesis that palladin is a crucial factor for Listeria motility and simultaneously used Listeria as a model to determine if palladin itself could functionally replace the Arp2/3 complex. Using palladin‐targeting antibodies, we identified palladin at Listeria invasion sites and comet tails. Strikingly, when we depleted cells of palladin, comet tails became shorter and severely misshapen. In cells expressing palladin mutants defective for actin or VASP binding, comet tails began to disintegrate or became progressively thinner as they moved. Ultrastructural examination of these thin comet tails revealed a switch in the comet tail actin network from highly branched arrays to parallel bundles. To test whether palladin could compensate for the Arp2/3 complex during bacterial motility, we overexpressed palladin in cells treated with the potent Arp2/3 inhibitor CK‐666. In these cells Listeria motility was unperturbed. Next we used a cell line depleted of several Arp2/3 complex subunits. As expected, Listeria were non‐motile during infections of these cells, however palladin overexpression in this Arp2/3 functionally null cell line restored the ability of Listeria to generate the actin‐rich structures formed by the bacteria. To definitively demonstrate palladin's ability to compensate for a lack of functional Arp2/3, we used purified protein components in conjunction with Listeria bacteria to show that actin‐rich structures formed by Listeria are generated in a cell‐free system containing palladin in place of the Arp2/3 complex. In conclusion, we show that palladin structurally organizes bacterial actin‐rich structures and importantly, compensates for the Arp2/3 complex without hindrance during bacterial actin‐based motility.

Support or Funding Information

Grant Funding Source: NSERC (grant no. 355316 to J.A.G and grant no. 155397 to A.W.V), NIH (grant no. R15 GM120670 to M.R.B), SFU departmental funds and SFU Multi‐Year Funding (A.S.D)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional genomic analysis of morphogenesis
Authors: Kiger Amy A , Velichkova Michaella , Ribeiro Inês
Published: June 2021

Changes in cell shape are central to development and disease. To systematically dissect the temporal‐spatial control of cellular processes in morphogenetic programs, we are applying functional genomic and in vivo genetic approaches in Drosophila. Specifically, we are investigating developmental mechanisms that spatially regulate specific lipid signals that mediate cellular elongation and underlie human diseases. The localized regulation of interrelated but distinct forms of phosphoinositide phosphates (PIPs) serve as nodes that spatially coordinate cellular functions through the recruitment of proteins to discrete membrane compartments. We are employing systematic RNAi screens for genetic modifiers of specific cell‐based phenotypes to help identify molecular mediators of phosphoinositide signaling pathway function. We are also investigating subcellular localizations of different PIPs in live cells undergoing cell shape changes using a collection of fluorescently‐tagged PIP‐binding proteins and microscopy imaging. The combination of RNAi loss of function and imaging approaches have helped us to identify specific roles for individual members of large conserved families of phosphoinositide regulators, as well as to delineate the functional significance of specific PIP pools important for cellular elongation.
Calmodulin‐like protein enhances myosin‐10 translation
Authors: Strehler Emanuel , Bennett Richard
Published: June 2021

Myosin 10 (Myo10) is an unconventional myosin involved in a wide range of cell processes from filopodial formation and extension to spindle orientation during cell division. Among its multiple functional domains, Myo10 contains three IQ motifs that bind calmodulin and specifically calmodulin‐like protein (CLP) as light chains. We have recently shown that CLP expression up‐regulates Myo10, leading to increased Myo10‐dependent cell motility and filopodial extension (R.D. Bennett et al., J. Biol. Chem. 282, 3205–3212, 2007). CLP‐dependent Myo10 up‐regulation occurs without a concomitant increase in Myo10 mRNA. We followed Myo10 degradation in vivo and in vitro and found that CLP does not affect the decay rate of Myo10. Myo10 decayed rapidly with a half‐life of about 2.5 hours indicating that this myosin participates in signaling processes. Using an in vitro transcription/translation system we determined that CLP increased Myo10 heavy chain translation, resulting in a higher relative accumulation of Myo10 in the presence than in the absence of CLP. Taken together, our data suggest that CLP functions to increase translation of Myo10 possibly by acting as a chaperone for the emerging Myo10 heavy chain protein. Supported in part by the Mayo Graduate School and grant BCTR‐0201979 from the Susan G. Komen Breast Cancer Foundation.
Targeting and inactivation of endoplasmic reticulum chaperones by L‐homocysteine: a pathway to ER stress
Authors: Glushchenko Alla V. , Axhemi Armend A. , Al‐Bayati Hiam , Austin Richard C. , Jacobsen Donald W.
Published: June 2021

Homocysteine may disrupt protein folding within the endoplasmic reticulum (ER) causing ER stress and activating the unfolded protein response. We hypothesize that L‐homocysteine induces ER stress by interacting with and modulating the activity of 78‐kDa glucose‐regulated protein (GRP78) and protein disulfide isomerase (PDI). In vitro S‐homocysteinylation of human recombinant GRP78 (hrGRP78) and rat recombinant PDI (rrPDI) was identified by SDS‐PAGE and phosphorimage analysis. We determined that 0.27 mol of 35S‐L‐homocysteine and 0.38 mol of 35S‐L‐homocysteine react per mol of hrGRP78 and rrPDI, respectively. Evidence for in situ S‐homocysteinylation of GRP78 and PDI was obtained by incubating rat liver microsomes with 35S‐L‐homocysteine, immunoprecipitating with mouse anti‐KDEL monoclonal antibodies, phosphorimaging and LC/MS/MS. Using surface plasmon resonance, we observed weaker interactions between peptide pp38 and S‐homocysteinylated‐hrGRP78, compared to native hrGRP78 protein. In contrast, L‐homocysteine stimulated the activity of PDI in the insulin precipitation assay. We conclude that L‐homocysteine directly targets and alters the function of the ER resident chaperones GRP78 and PDI. This pathway links homocysteine to ER stress, cardiovascular disease and other disorders associated with abnormal protein accumulation. (Supported by NIH, HL52234 (DWJ)).
Oxidative stress suppresses dexamethasone‐induced gene expression through inhibition of glucocorticoid receptor nuclear translocation
Authors: Xu Haishan , Chu Shijian
Published: June 2021

Although glucocorticoids are often used for the control of pulmonary edema, asthma and other chronic inflammatory diseases, a portion of patients fail to respond to the treatment. In this study, we hypothesized that oxidative stress, which is often seen in patients with COPD, asthma and pulmonary inflammation, induces steroid resistance. We found that, in lung epithelial cells, expression of α‐ENaC, SP‐B and IkB‐α are upregulated by dexamethasone (DEX) and blocked by the glucocorticoid receptor (GR) antagonist RU‐486. Co‐treatment with H2O2 inhibited DEX‐induced gene expression. Furthermore, we found that in cells pretreated with H2O2, DEX‐induced nuclear GR increase was markedly reduced and cytoplasmic GR was proportionally increased. GR expression however was not affected. Our results demonstrate that 1. GR mediates DEX‐induced α‐ENaC, SP‐B and IkB‐α gene expression; 2. H2O2 attenuates DEX‐induced gene expression; and 3. H2O2 inhibits DEX‐induced GR nuclear translocation but not GR expression. These results suggest that reduced GR nuclear translocation may be partially responsible for oxidative stress associated corticosteroid resistance. This work was supported by a grant from the Department of Veterans Affairs.
Growth Rates of Dosage Suppressed Mutants of Temperature Sensitive Lethal Mutation in sec39 Gene
Authors: Qiu Chenxi
Published: June 2021

The purpose of this project is to understand how genetic interactions give rise to function in Saccharomyces cerevisiae. For this experiment, we focused on SEC39, an essential gene of unknown function, but proposed to be involved in the secretory pathway. Previous screens found that the temperature sensitive mutant sec39 is involved in several dosage suppressor interactions. The goal of this experiment is to test whether the dosage suppression can be maintained in liquid culture, so that microarray experiments can be performed later. We incubated two colonies (one RFC3 and one SPT20) in media containing galactose and raffinose but no uracil, at the restrictive temperature, and both colonies grew. We also incubated RFC3, SPT20, and the parent strain below the restrictive temperature at first, and then raised the temperature at mid‐exponential phase. The parent strain had a low growth rate initially and the cells dwindled at the restrictive temperature, but the cells with plasmids survived. We replica plated the cells with RFC3 in media containing galactose and raffinose but no uracil. The results show that about 26% of the cells retained the plasmid; the remaining cells are suspected to be revertants. So far, it can be concluded that both plasmids rescue the mutant cells and accelerate their growth at high temperature, but the parent strain can only subsist below the restrictive temperature.
Expression, purification and characterization of glutamate cysteine ligase from Saccharomyces cerevisiae
Authors: Biterova Ekaterina , Sommer Brandi , Barycki Joseph J
Published: June 2021

Glutathione is a critical cellular reductant and its homeostasis is required for maintenance of intracellular redox balance. glutamate‐cysteine ligase (GCL) catalyses the ATP‐dependent peptide bond formation between gamma‐carboxylate of glutamate and cysteine, the first step of glutathione biosynthesis. Mammalian GCL are heterodimers consisting of a catalytic subunit and a modifier subunit. The catalytic subunit catalyzes formation of glutamylcysteine and is inhibited by GSH, whereas the modifier subunit enhances the GCL affinity for L‐glutamate and decreases the inhibition by GSH. An examination of the S. cerevisiae genome revealed a single gene with sequence homology to the catalytic GCL subunit. The S. cerevisiae GCL catalytic subunit was cloned, expressed in E. coli and purified to homogeneity. Size exclusion chromatography and DLS techniques were used to examine the oligomeric state of GCL catalytic subunit. The kinetic activity of GCL catalytic subunit was measured using a coupled assay and apparent Km and Vmax values were determined. Kinetic constants were comparable to those reported for other eukaryotic GCL, and inhibition studies indicate that the enzyme is susceptible to competitive inhibition by the ultimate product of the pathway, glutathione. A proteomic method is currently being developed to identify the corresponding modifier subunit of S. cerevisiae GCL.
Time‐lapse imaging of extracellular matrix assembly
Authors: Wagenseil Jessica E , Kozel Beth A , Czirok Andras , Rongish Brenda J , Mecham Robert P
Published: June 2021

The extracellular matrix (ECM) is a composite polymer made up of many different components. The cell takes an active role in ECM assembly through targeted secretion of ECM components and through the active participation of matrix‐organizing binding proteins on the cell surface. To learn more about how the cell organizes ECM, we studied elastic fiber formation using time‐lapse imaging microscopy. Elastic fibers are formed from soluble elastin monomers, tropoelastin, which are crosslinked to form fibers of mature elastin. The initial step in elastic fiber assembly is the formation of tropoelastin aggregates on the cell surface (microassembly). Over several hours, these aggregates coalesce to become larger and are eventually transferred onto microfibrils, which serve to structure the growing fiber in the extracellular space (macroassembly). The assembly of microfibrils also occurs in a defined spatial and temporal sequence. Fibronectin, fibrillin‐1 and MAGP‐1 start as fibers, while fibulin‐5 and tropoelastin start as a combination of fibers and aggregates. Many of the fibulin‐5 aggregates co‐localize with the subsequent tropoelastin aggregates suggesting that fibulin‐5 may play a role in elastic fiber microassembly.
Cell motility versus tissue motion in embryos: Which cells are really moving?
Authors: Little Charles D. , Rongish Brenda J , Czirók András , Zamir Evan A , Cui Cheng
Published: June 2021

It is axiomatic that during embryogenesis new cellular patterns form within constantly expanding tissue scaffolds. For example, primordial endothelial cells, in bird and mammalian embryos, assemble early vascular patterns in the context of continuous tissue expansion. Our novel computational time‐lapse software and algorithms are designed to quantify large‐scale tissue drift and deformations, in warm‐blooded embryos. The computer code also enables recording of fluorescent objects, i.e., cells and ECM fibers. Computation of tissue displacement fields using the motion of ECM fibrils as in situ markers for tissue drift — while simultaneously tracking individual cells in the same tissue— allows calculation of active cell‐autonomous motility versus passive convective tissue motion. The data demonstrate that passive tissue motion contributes significantly to apparent embryonic cell trajectories. In other words, tissue drift significantly impacts the de novo assembly of vascular networks. Previous notions regarding “cell migration” in warm‐blooded embryos will have to be revisited in light of these data. The work has profound implications for understanding widely accepted embryonic cell guidance mechanisms, such as cellular chemotaxis in response to gradients of soluble molecules.
Live‐cell Imaging of Endothelial Cell Tube Formation: Inhibition by Chondrostatin
Authors: Sandell Linda J , Wang Zhepeng , Franz Carl , Bryan Jennifer , Siegel Adam , Mecham Robert , Wagensail Jessica , Ell Brian , Rapraeger Alan
Published: June 2021

Cartilage tissue is avascular and resistant to tumor invasion. The basis for these properties has been actively sought for the last 35 years. Based on the assumption that cartilage contains inhibitors of angiogenesis, previous studies have focused on isolating potential vascular inhibitors from cartilage. Here we report that the NH2‐propeptide of type IIB procollagen (chondrostatin), a product of collagen biosynthesis, is capable of inhibiting angiogenesis mediated by the integrins, αVß3 or αVß5 present on endothelial cells. Chondrostatin inhibits tube formation by human umbilical vein cells (HUVEC) and inhibits endogenous endothelial cell outgrowth in a rat aortic ring angiogenesis bioassay. The kinetics of formation of vessel tubes by HUVEC cells was investigated by imaging cells as they formed tubes in a collagen gel. As αVß3 and αVß5 integrins are expressed primarily on endothelial cells, cancer cells and osteoclasts, but not on normal chondrocytes, we propose that cartilage chondrostatin protects cartilage by targeting endothelial cells during chondrogenesis, thus inhibiting angiogenesis and rendering the tissue avascular. We show that chondrostatin also binds to chondrosarcoma, breast and cervical cancer cells via the integrins αVß3 and αVß5 and inhibits migration, eventually inducing cell death.

Supported by NIH R0136994
The actin binding protein cortactin behaves as an intrinsically unstructured protein
Authors: Kruchten Anne E
Published: June 2021

Cortactin is an actin binding protein known to be involved in several cancer metastases. The experimentally derived weight of 80/85 kDa is in direct contradiction to its theoretically derived molecular weight of approximately 60 kDa. Previously it was reported that cortactin has a larger Stokes radius than predicted for a protein of its size. These data taken together suggest that cortactin does not behave as a typical globular protein but rather as an intrinsically unstructured protein (IUP). Here it is reported that based on the results of computations by computer based algorithms and size exclusion chromatography, cortactin is predicted to have a number of unfolded regions. Phosphorylation of cortactin affects its role in cellular metastases, and it was hypothesized that this may be via modulations of the protein structure, similar to other IUPs. Stokes radii of phosphorylation mutants were analyzed by size exclusion chromatography and compared to wild type. In the future, cortactin structure will be analyzed by biophysical methods such as circular dichroism spectroscopy. This work is supported by a Faculty Start‐Up award from the M.J. Murdock Charitable Trust to A. E. Kruchten.
Designing a Fluorescence Resonance Energy Transfer (FRET) Tool to Analyze the Intramolecular Folding Properties of Cortactin
Authors: Braden Chris , Kruchten Anne
Published: June 2021

Evidence indicates that the actin‐binding protein and Src‐substrate cortactin may be a member of a class of proteins termed “intrinsically unstructured proteins.” In contrast to globular proteins with defined secondary and tertiary structure, proteins in this class are not natively folded, and their lack of structure may serve an important functional role. As serine and tyrosine phosphorylation on cortactin serves a regulatory function, we hypothesize that this phosphorylation may influence cortactin's folding. The present project will use single‐molecule FRET to address the question of whether cortactin is intrinsically unstructured, and if so, whether phosphorylation can induce folding. We are presently cloning the yellow fluorescent protein portion of pEYFP, a rat cortactin sequence, and the cyan fluorescent protein portion of pECFP into one vector creating a single molecule FRET reporter system that will detect changes in cortactin structure. Future work will analyze cortactin folding both in vitro and in situ by following FRET signal in response to phosphorylation states of the protein. This work is supported by a Faculty Start‐Up award from the M.J. Murdock Charitable Trust to A. E. Kruchten.
C‐terminal membrane spanning region of human Heme Oxygenase‐1 mediates a time‐dependent complex formation with NADPH cytochrome P450 reductase
Authors: Huber Warren J , Reed James R , Backes Wayne L
Published: June 2021

Heme oxygenase‐1 (HO‐1) catalyzes the oxidative degradation of heme to biliverdin, carbon monoxide, and free iron in a reaction requiring the interaction of HO‐1 with NADPH‐cytochrome P450 reductase (CPR). Full‐length HO‐1 (flHO1) is bound to the endoplasmic reticulum by 23 C‐terminal amino acids, however a soluble HO‐1 (sHO‐1) lacking this membrane spanning region has been extensively studied. The goal of this project was to characterize the effect of the C‐terminal hydrophobic domain on formation of the flHO‐1/CPR complex. In previous experiments, we demonstrated a catalytic difference between flHO‐1 and sHO‐1; indicating that the C‐terminal region influences HO‐1 binding to CPR. Recent kinetic data proposed that the increased specific activity of flHO‐1 may be attributable to a time dependent formation of a high affinity flHO‐1/CPR complex that was not seen with sHO1. Gel filtration analysis confirmed the formation of multiple high molecular weight complexes in the presence and absence of the synthetic lipid dilauroylphosphatidylcholine (DLPC). However, the largest complex appeared following a two hour incubation of flHO‐1 and CPR in DLPC, suggesting that C‐terminal region was required for flHO‐1/CPR complex formation and membrane incorporation. These data displayed that the C‐terminal region influenced flHO‐1 complex formation and ultimately its affinity for CPR. (Supported by NIEHS ES004344)
PKA Type IIa Holoenzyme Structure Reveals Isoform Diversity for Inhibition of Catalysis
Authors: Wu Jian , Brown Simon HJ , von Daake Sventja , Taylor Susan S
Published: June 2021

Isoform diversity of the regulatory (R) subunits, RI and RII, of cAMP‐dependent protein kinase (PKA) is a primary mechanism for achieving specificity. The RII subunits are substrates as well as inhibitors and do not require ATP to form holoenzyme, which distinguishes them from RI subunits. To understand isoform diversity, we solved the crystal structure of an RIIα holoenzyme complex. The deletion mutant, RIIα (90–400), contains the unphosphorylated inhibitor site and two cAMP‐binding domains (CNB‐A and CNB‐B). The inhibitor site with a P‐site Ser docks to the large lobe but does not engage the small lobe or the C‐terminal tail. The C‐subunit is in an open conformation. The CNB‐A domain, which dominates the interface, extends from the αG helix to the Activation Loop. The CNB‐B domain, which is essential for high affinity binding of RII, but not RI‐subunit, docks to a novel AGC‐specific insert in the αH‐αI loop. The helical subdomains of RIIα undergo major conformational changes upon binding to C. The hallmark of this conformational reorganization is that the two CNB domains are splayed apart by the fusion of the αB/αC helix in CNB‐A while the αC helix of CNB‐B assumes a helix‐turn‐helix motif. Many of the multivalent contacts between C and R are made by residues that were solvent exposed in the cAMP‐bound conformation. This structure also provides a new paradigm for designing isoform‐specific activators or antagonists for PKA.
Regulation of Creatine Kinase Activity by Phosphorylation of Serine‐199 by AMP‐Activated Kinase
Authors: Palmer Allyson K , Fraga Dean , Edmiston Paul L
Published: June 2021

Creatine kinase (CK) catalyzes the reversible phosphorylation of creatine by MgATP to form phosphocreatine and MgADP. An accessible reservoir of high energy phosphate in the form of phosphocreatine is useful in excitable tissues (i.e. brain, heart, or muscle) which require large energy fluxes. Thus, CK is important to energy homeostasis in cells. Previous work has suggested that AMP‐activated kinase may regulate the activity of CK by phosphorylation, presumably at a serine residue (Ponticos, et al. EMBO J. 17:1688.). To confirm this hypothesis, a set of serines in CK were changed to glutamate by site‐directed mutagenesis to assess if any of these residues were possible phosphorylation sites. Notably, the S199E variant of CK showed a 200‐fold reduction in the catalytic rate constant (phosphocreatine formation) compared to wild‐type suggesting that phosphorylation of S199 is likely involved in regulation of CK. The possible means by which phosphorylation alters the active site will be discussed. These results point to a higher degree of cellular control of ATP concentration by regulating the activity of CK. Such a regulatory mechanism may have implications on better understanding energy flux within a cell.
NMR characterization of substrate induced changes in structure and dynamics of human phosphomevalonate kinase
Authors: Olson Andrew L , Yao Huili , Cai Shen , Herdendorf Tim , Miziorko Henry M , Sem Daniel S
Published: June 2021

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, involving the transfer of a phosphoryl group from ATP to mevalonate‐5‐phosphate (M5P), producing mevalonate‐5‐diphosphate. This is the sole pathway for biosynthesis of steroids and isoprenoids in mammals. Despite the importance of this pathway in human cardiovascular disease, the mechanism of this enzyme is not well characterized. Here we have titrated both ligands to the protein, first M5P to free PMK then ADP. Then, titration of ADP to free PMK followed by M5P, monitoring chemical shift changes upon addition of these ligands using 1H‐15N HSQC‐NMR. The dynamic psec‐nsec timescale changes that occur in PMK with all complexes were characterized with NMR relaxation experiments (S2). Following chemical shifts during ligand titration, we found they all bind with similar dissociation constants: 6 +/− 3 μM for M5P binding to free PMK and 56 +/− 16 uM for subsequent ADP binding; 19 +/− 9 μM for ADP binding to free PMK and <20 μM for subsequent binding of M5P. Chemical shift changes mapped onto a PMK structural model indicate that upon addition of Mg‐ADP the majority of changes are clustered around the binding pocket of ADP, however the binding of M5P causes changes throughout the whole protein. The first ligand bound induces the major conformational change, as there are few chemical shift changes in the ternary complex for both situations.
Biochemical and structural basis for feedback inhibition of mevalonate kinase
Authors: Miziorko Henry M. , Fu Zhuji , Voynova Natalia E. , Kim Jung‐Ja P.
Published: June 2021

Mevalonate kinase (MK) catalyzes a key reaction in polyisoprenoid and sterol metabolism. Both farnesyl diphosphate and farnesyl thiodiphosphate (FSPP) compete with substrate ATP to produce MK feedback inhibition. Structures of human MK and a binary complex formed by incubation of rat MK with FSPP are reported. ATP and FSPP binding sites overlap, with the α‐phosphate of FSPP nearly superimposed on ATP's β‐phosphate and FSPP's polyisoprenoid chain overlapping ATP's adenosine moiety. The functional importance of observed interactions between amino acid side chains and the FSPP polyisoprenoid chain has been evaluated in human MK mutagenesis experiments. Single and double mutations at T104 and I196 produce inflation of the Ki for FSPP (∼40 fold for T104A/I196A). The effect persists when Ki values are normalized for effects on Km for ATP. Comparison of animal MK protein alignments and structures with those of S. pneumoniae MK indicates that sequence differences between N‐ and C‐terminal domains correlate with differences in interdomain angles. Bacterial MKs may exhibit more solvent exposure of feedback inhibitor sites and, consequently, weaker binding of these inhibitors. DK53766
Crystallization and preliminary X‐ray analysis of the secreted VirA effector protein from Shigella
Authors: Davis Jamaine S , Zhang Di , Cherry Scott , Tropea Joseph , Waugh David , Wlodawer Alexander
Published: June 2021

VirA is an effector protein secreted by the type III secretion system of Shigella. VirA functions as a cysteine protease, with its catalytic cysteine residue identified by mutation to alanine. In the study of Yoshida et al. (Science. 2006; 314 (5801):985), the ability of the enzyme to degrade microtubules was tested. These authors illustrated that VirA plays a role in the intra‐ and intercellular spreading of the bacterium by selectively degrading α‐tubulin. How VirA recognizes and degrades α‐tubulin over β‐tubulin substrates remains unclear.

Bacterial invasion is an essential step in the pathogenesis of shigellosis. It is suggested that VirA is essential in creating a path to enable the bacteria to move through the dense, organized cytoplasmic network of the host cell. Shigella mutants that lack virA are unable to move through the cytoplasm and the invasiveness of these virA mutants was attenuated, suggesting that VirA is essential for Shigella virulence.

We report here progress on determination of the crystal structure of VirA. The enzyme was expressed in E. coli, purified to homogeneity, and crystallized using vapor‐diffusion technique. The crystals belong to the monoclinic space group C2, with unit‐cell parameters a=150Å, b=171Å, c=46Å,β =105°, and diffract to 3.0 Å resolution. With the presence of two molecules in the asymmetric unit, the Matthews coefficient (Vm) is approximately 3.2, and corresponds to a solvent content of about 61%. Determination of the crystal structure using SAD data collected from a selenomethionine derivative is in progress.
UDP‐galactopyranose mutase: an unexpected ligand‐binding mode leads to model of substrate binding
Authors: Gruber Todd D , Borrok M Jack , Westler William M , Forest Katrina T , Kiessling Laura L
Published: June 2021

Galactofuranose residues are key components of the cell walls of numerous pathogenic bacteria. UDP‐galactofuranose (UDP‐Galf), a biosynthetic precursor of Galf containing glycoconjugates, is produced from UDP‐galactopyranose (UDP‐Galp) by the flavoenzyme UDP‐galactopyranose mutase (UGM). We have accumulated evidence that the enzymatic reaction proceeds through an iminium intermediate formed between flavin and the substrate. To understand this unique mode of catalysis, we determined the ligand‐bound structure of UGM. We report the structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP‐glucose (UDP‐Glc) at 2.45 Å resolution, in which the crystallographic asymmetric unit contains the UGM dimer in distinct closed and open modes. In the closed monomer, a short loop restructures into an α‐helix, orienting a conserved arginine residue toward the phosphoryl groups of the nucleotide sugar, and positioning the Glc moiety near the flavin cofactor. The uracil moiety stacks with a conserved tyrosine residue, rather than the previously proposed Trp160. Despite its similarity to the natural substrate, UDP‐Glc does not participate in iminium formation upon binding to the enzyme. This observation can be rationalized by the structure; the glucose ring of the nucleotide sugar appears to be oriented improperly for catalysis. From the structure, we propose a model for the UGM‐substrate complex.

Supported by NIH AI063596
The Effect of SOD‐1 Deficiency and Manganese Supplementation on Oxidative Stress Resistance in Caenorhabditis elegans
Authors: Sprowl Stephanie Ann , Zou Yani , Kaplan Rachel Deanna , Huang Ting‐Ting , Srinivasan Chandra
Published: June 2021

Oxygen free radicals (ROS) are by‐products of normal metabolism in oxygen utilizing organisms. ROS can cause damage to living cells if not removed quickly. Superoxide dismutases (SODs) are antioxidant enzymes that form the first line of defense against ROS. Not much is known about the five forms of SOD in the roundworm Caenorhabditis elegans (C. elegans). The goals of this study are to determine which SODs are essential for oxidative stress resistance and to know if manganese (Mn) supplementation could enhance resistance against oxidative stress. Wild type and individual SOD mutant worms were cultured in growth medium containing various concentrations of paraquat (a ROS generator), and survival was scored twenty hours later. Our results show that only sod‐1 (encodes SOD‐1, a CuZn‐containing SOD) mutant worms are significantly more sensitive to paraquat than the wild type controls. This is in line with our finding that SOD‐1 is the major SOD in C. elegans. Since we have shown Mn to have an antioxidant‐like property, we examined the effect of Mn on paraquat toxicity. Mn supplementation (1 mM) in the culture medium increases resistance, as measured by LD50 by at least 3‐fold in sod‐1 mutant worms. Increased paraquat resistance is also observed in wild type worms treated with Mn; however, the extent of increase is only around 2‐fold. These results lend further support to the antioxidant potential of Mn.

Funding provided by NIH.
Phosphorylation of CtBP1 by PKA Induces CYP17 by Promoting CtBP Heterooligomerization
Authors: Dammer Eric B , Sewer Marion B
Published: June 2021

In the human adrenal cortex, the peptide hormone adrenocorticotropin (ACTH) directs cortisol and adrenal androgen biosynthesis by activating a cAMP/protein kinase A (PKA) pathway. Carboxy terminal binding protein 1 (CtBP1) is a corepressor that regulates transcription of the CYP17 gene by periodically interacting with steroidogenic factor‐1 (SF‐1) in response to ACTH signaling. Given that CtBP1 function is regulated by NADH binding, we hypothesized that ACTH‐stimulated changes in cellular pyridine nucleotide concentrations modulate the ability of CtBP1 to repress CYP17 transcription. Further, we postulated that PKA evokes changes in the phosphorylation status of CtBP1 that control the ability of the protein to bind to SF‐1 and repress CYP17 gene expression. We show that ACTH alters pyridine nucleotide redox state and identify amino acid residues in CtBP1 that are targeted by PKA and PAK6. Both ACTH/cAMP signaling and NADH/NAD+ ratio stimulate nuclear‐cytoplasmic oscillation of both CtBP proteins. We provide evidence that PKA 1) induces metabolic changes in the adrenal cortex and 2) phosphorylates CtBP proteins, particularly CtBP1 at T144, resulting in ACTH‐dependent CYP17 transcription.
Mitochondrial functionality and calcium uptake during hypertension in rat brain mitochondria
Authors: Calderón‐Cortés Elizabeth , Cortés‐Rojo Christian , Esquivel‐Gutiérrez Edgar , Noriega‐Cisneros Ruth , Clemente‐Guerrero Mónica , Manzo‐Ávalos Salvador , Saavedra‐Molina Alfredo
Published: June 2021

Mitochondria contribute to intracellular redox status and Ca2+ homeostasis. Intramitochondrial Ca2+ concentration are regulated via an electrophoretic uniporter that is driven by the transmembrane potential. Several abnormalities have been observed in the Ca2+ metabolism of spontaneously hypertensive rats (SHR). We studied whether mitochondrial membrane potential (mΔΨ), complex IV activity, and Ca2+ metabolism were altered in brain mitochondria of 1‐month and 7‐month SHR. We used safranine probe to determinate membrane potential. Complex IV activity was determinated measuring oxygen consumption with a Clark‐type electrode. Ca2+ levels and uptake were determinated using Fluo‐3AM and Arsenazo III. Ca2+ uptake was decreased in SHR compared to normotensive rats (WKY) of 1 month old. mΔΨ was lower in SHR than WKY 1‐mo rats, while mΔΨ decreased in both 7‐mo groups of rats. Complex IV activity decreased by 35% and 100% in 1‐mo WKY and SHR rats, respectively, in the presence of 480 nmols Ca2+. Mitochondrial oxygen consumption decreased after addition of 480 nmols Ca2+ in 7‐month SHR. mΔΨ decreased in the presence of 640 and 480 nmol Ca2+ in WKY and SHR, respectively. These data show an age‐dependent decrease on complex IV activity of SHR activity parallel to a diminishing in mΔΨ, which impair [Ca2+]m and Ca2+‐dependent mtNOS activity. Supported by Fondos Mixtos CONACYT‐Gob. Edo. de Michoacán (64277, 64308), COECYT and CIC‐UMSNH (2.16) grants.
Radiation Effects on Levels of Total Mitochondrial DNA and DNA Bearing the Common Deletion in a Pearson's Syndrome Pedigree
Authors: Vrooman Scott , Pogozelski Wendy
Published: June 2021

Mutations in mitochondrial DNA (mtDNA) correlate with aging and oxidative stress and they appear in a host of degenerative diseases. Little is known about how ionizing radiation might contribute to the appearance of these mutations, however. Here we quantify a specific deletion of 4977 base pairs (known as the “common deletion”) as a function of X‐ray dose using real‐time PCR. We grew lymphoblast cells derived from a patient with Pearson's Marrow/Pancreas syndrome (known to contain elevated levels of the common deletion) as well as normal cells derived from the patient's mother and sister. We irradiated the cells with doses of 0.5–5 Gy of gamma radiation. Total (nuclear and mitochondrial) and mitochondrial DNA extracts were then analyzed along with cloned plasmids that served as quantification controls. The deletion was only measurable in the Pearson's Syndrome cells and results showed elevated levels with radiation dose. The total mtDNA remained relatively constant in these cells. In contrast, the levels of total mtDNA increased in the normal control cells derived from the patient's mother and sister.

This research is supported by a Henry Dreyfus Teacher‐Scholar Award.
PPAR delta regulates adipose tissue macrophage activation and insulin sensitivity. Critical roles of PPAR delta in macrophage‐adipocyte crosstalk and insulin sensitivity
Authors: Kang Kihwa , Karabacak Volkan , Reilly Shannon M , Fitzgerald Kelly , Hatano Ben , Lee Chih‐Hao
Published: June 2021

Obesity‐induced insulin resistance is closely associated with chronic low‐grade inflammation. Obesity is characterized by macrophage accumulation in white adipose tissue, and these infiltrated macrophages interfere with adipocyte function. Macrophages can be functionally polarized to classical or alternative activation, and produce pro‐inflammatory and anti‐inflammatory cytokines, respectively. The balance between these two populations thus plays an important role in adipocyte functions and insulin sensitivity. However, the mechanism by which ATM activation is regulated remains unclear. Here we report that peroxisome proliferators‐activated receptor δ (PPARδ) is required for controlling ATM activation toward anti‐inflammatory phenotype. PPARδ ablation renders the transition of macrophages from the anti‐ to pro‐inflammatory phenotype when macrophages are co‐cultured with adipocytes. Reciprocally adipocytes co‐cultured with PPARδ−/− macrophages exhibit elevated levels of inflammatory markers and dysregulated glucose metabolism. In accordance with in vitro data, myeloid specific PPARδ−/− mice have increased infiltrated macrophages showing pro‐inflammatory property and develop adipocyte dysfunction, together with severe liver steatosis and systemic insulin resistance. These results demonstrate an important role for PPARδ in regulating insulin sensitivity by macrophages, and show a molecular basis to understand the cross‐talk between adipocytes and ATM activation
A multi‐compartment mitochondrial model of brain metabolism shows that network topology supports theories in neurochemistry
Authors: Lewis Nathan E , Cheng Jeff , Messersmith Michael Paul , Palsson Bernhard O
Published: June 2021

We present a network reconstruction of brain mitochondria metabolism built using mass‐spectrometric data, gene expression data, and an extensive literature search to fill in network gaps, determine reaction directionality and to curate reaction presence and properties. Reactions were further parsed into cellular compartments allowing for view of metabolite transfer between cell types. Using constraint based methods to analyze the model, we demonstrate that application of known constraints leads to a network topology that supports experimental results of brain energetics, and theories involving glutamate recycling and the astrocyte‐neuron lactate shuttle. This demonstrates the ability of constraint based methods to predict biological function in systems biology. Funded by NIH Training Grant HL‐07089
Systems biology of the mammalian Unfolded Protein Response
Authors: van Riel Natal A , Tiemann Christian A , Jeneson Jeroen A , Hilbers Peter A
Published: June 2021

The Unfolded Protein Response (UPR) is a coordinated endoplasmic reticulum (ER) stress response program. UPR is initiated whenever protein folding in the ER is reduced and accumulation of malfolded proteins takes place. ER stress is associated with diseases, including type 2 diabetes. However, the dynamics and regulation of the underlying signal transduction network are largely unknown.

A first mathematical model for the UPR in mammalian cells was proposed including the PERK and ATF6 pathways. The differential equation model was parameterized from Western blot time course data of phosphorylated PERK and cleaved ATF6 after exposing cells to dithiothreitol (DTT), which prevents the formation of disulfide bonds (DuRose et al. Mol. Biol. Cell, 17: 3095–3107, 2006).

Sensitivity analysis showed that only a few kinetic parameters were critical for the predicted p‐PERK and ATF6 time responses. Interestingly, variations in these parameters could move the UPR system towards different new steady‐states. In certain cases the system returned to its original steady‐state while the cells were still exposed to DTT (so‐called perfect adaptation). In other cases, the stress sensors remained active. However, the difference in endpoints only emerged after 5 hours, when no experimental data was available. This model‐based hypothesis is open for experimental testing.
Joint analysis of a compendium gene expression data and 5′‐untranslated mRNA regions points to a common cis‐regulatory region under epigenetic control
Authors: Mrowka Ralf , Blüthgen Nils , Fähling Michael , Steege Andreas , Martinka Peter , Brinkmeier Thomas , Thiele Bernd J , Persson Pontus B , Patzak Andreas , Holste Dirk
Published: June 2021

Untranslated regions located at the 5′ terminus (5′‐UTRs) of eukaryotic messenger RNAs (mRNAs) are often encoding functional cis‐regulatory sequence elements or structural features to modulate the rate of transcription, the stability of mRNAs or the translational initiation for protein synthesis. Elements that influence transcription are difficult to infer and have largely remained hidden. In an attempt to uncover candidate transcriptional regulatory elements that influence the level of gene expression, we systematically compared sequence similarities of 5′‐UTRs with the experimentally measured co‐expression of human genes across more than 1,200 publicly available microarray data. To this end, we employed a non‐heuristic algorithm for efficient exact matching of local sequence similarities, which is based on the mathematical Fourier transform. The algorithm was used to test oligonucleotides as candidates for transcriptional regulation of 5′‐UTRs. The most pronounced signal was localized at about 30 to 40 nucleotides downstream of the mRNA transcription start site, which were subsequently searched for overrepresented sequence motifs. Statistically significant enrichment of GC‐box motifs suggests a prominent role of DNA methylation, as one possible underlying epigenetic mechanism explaining the observed relationship between sequence similarity and co‐expression.