bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of the Hsp70 Protein Quality Control System by Phosphorylation of Chaperones and Co‐chaperones
Authors: Page Richard 
Published: June 2021

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of the co‐chaperones CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Hsp70 can only interact with one of the co‐chaperones at a time, thus modulating interactions of Hsp70 with either CHIP or HOP controls the Hsp70 protein quality control axis by shunting misfolded client proteins to either the degradative pathway or the refolding pathway. Interactions of Hsp70 with CHIP promote CHIP‐mediated degradation via the ubiquitin proteasome pathway. Interactions of Hsp70 with HOP promote protein refolding since HOP connect Hsp70 with the 90 kilodalton heat shock protein Hsp90. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp641 of Hsp70. In particular, phosphorylation of Hsp70 Thr636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Thus, phosphorylation of Hsp70 Thr636 acts as a switch that directs the output and function of cellular protein quality control. Phosphorylation of CHIP Ser20 or CHIP Ser24 also modulates the interactions between CHIP and Hsp70, providing yet another level of regulation for the Hsp70 protein quality control system. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and HOP‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Misfolded Protein Client as a Determinant of Substrate Ubiquitination by the Hsp70/CHIP Complex
Authors: Paththamperuma Chathura , Zhang Huaqun , Page Richard 
Published: June 2021

The E3 ubiquitin ligase CHIP (carboxy terminus of Hsc70 interacting protein) targets misfolded proteins chaperoned by heat shock protein 70 (Hsp70) for ubiquitination and subsequent proteasomal degradation. Hsp70 recruits and attempts to refold misfolded proteins, or “clients”, in iterative, ATP‐dependent cycles. In contrast, binding of Hsp70 to the tetratricopeptide repeat (TPR) domain of CHIP, allows for client proteins to be recruited for ubiquitination by CHIP. Ubiquitination of lysine residues on a client protein allows CHIP/Hsp70 complexes to promote the proteasomal degradation of the client. Thus, the CHIP/Hsp70 complex stands at the crossroads of degradative and refolding pathways of client proteins which is crucial for cellular protein quality control. The current knowledge in the field lacks a clear understanding of how the CHIP/Hsp70 complex executes its triage mechanism that directs misfolded proteins for either refolding or degradation. Recent models of ATP and ADP bound Hsp70 revealed the distances between the substrate binding domain (SBD) and the E2~ubiquitin conjugate active site as ~120 Å and ~60 Å respectively. This implies, with the dynamic conformational changes of Hsp70 complex, the ADP‐bound state of Hsp70 organizes the chaperoned ubiquitination complex by properly orienting the client with respect to CHIP which allows the client protein to span this 60 Å gap and accept the ubiquitin from the bound E2‐ubiquitin conjugate. Therefore, the distance between Hsp70 binding site and the active site of the E2~ubiquitin conjugate bound to CHIP plays a significant role as a determinant in the triage mechanism of CHIP/Hsp70. In this study, we focus on investigating the relationship between key distances within the CHIP/Hsp70/client complex and the ubiquitination activity of the CHIP/Hsp70 complex by utilizing in‐vitro ubiquitination assays of modified substrates.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“The relationship of structural stability to temperature‐dependent activity in a family of thermophilic inteins”
Authors: Exconde Patrick M. , Gomez Alvin V. , Mills Kenneth V. 
Published: June 2021

Protein splicing is a post‐translational process by which a self‐catalyzing intervening sequence called an intein removes itself from a precursor protein and concomitantly ligates the flanking sequences, known as exteins. Our inteins of interest interrupt the DNA Polymerase II from the extreme thermophiles Thermococcus sibiricus (Tsi) and Thermococcus gammatolerans (Tga). We are interested in these inteins because of their sequence similarity to those from the extreme thermophiles Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), all of which are mini‐inteins that lack a homing endonuclease domain. The Pho intein has been found to require higher temperature for splicing than the Pab intein, perhaps due to a shorter, more conformationally‐restricted region that replaces the missing homing endonuclease domain. The Tga and Tsi inteins have loop lengths more similar to the Pho intein. A splicing assay under increasing pressure will determine if there is a difference in activity in the two inteins that vary in the depths at which the parent organisms dwell. A preliminary assay at atmospheric pressure with an optimal isothermal temperature of 52°C showed that the Tga intein splices more efficiently than the Tsi intein. We will measure the temperature dependence of the intrinsic tryptophan fluorescence to link the overall stability of the fold to the difference in activity.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Camille and Henry Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Salt‐Dependent Protein Splicing: In Vitro Enzymology and In vivo Physiological Relevance
Authors: Janton Christopher J. , Gomez Alvin V. , Makkay Andrea M. , Papke R. Thane , Mills Kenneth V. 
Published: June 2021

Protein splicing is a post‐translational process in which two separate polypeptides are produced from a single gene. Splicing involves the excision of an intervening protein sequence, called the intein, and ligation of the flanking exteins. Inteins have been found in halophilic archaea, including Halobacterium salinarum (Hsa), and previous work has demonstrated that in vitro splicing of the Hsa DNA Polymerase II (Polll) intein only occurs under reducing conditions with high salt concentrations. We compared the in vitro activity of the Hsa Polll intein to that of the only other Hsa intein, which interrupts a Cell Division Control protein (Cdc21a). The Hsa Cdc21a intein does not splice in E. coli, but can be induced to splice at similar conditions to the Hsa Polll intein in vitro. Additionally, we used a Cys1Ala mutation to study the extent of the third step of splicing. We are creating intein‐less strains of H. salinarum, and will use these to study the fitness cost of maintaining inteins under varying growth conditions.Support or Funding InformationThis work was supported by NASA (grant NNX15AM09G to RTP), the National Science Foundation (grants MCB1244089 and MCB1517138 to KVM) and the Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endonuclease and splicing activity of a Haloferax volcanii intein
Authors: Cawood Alexandra 
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide called an internal protein, or intein. The intein is located between two flanking polypeptides, called exteins. The intein catalyzes its excision from the extein concomitantly with the ligation of the exteins. The extremophile Haloferax volcanii exists in a high salt environment. We are interested in the halophile because we want to learn how efficiently the intein that has invaded its PolB gene splices as a function of salt concentration. We have shown that we can induce splicing of the intein in vitro at high salt. Collaborators have demonstrated that the intein has an active homing endonuclease domain in vivo, and that the presence of the intein has a high fitness cost for the organism. We are interested in studying the endonuclease activity of the intein in vitro, given that a salt‐dependent conditionally‐active nuclease may have utility in biotechnology applications.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Variable Salt Dependence of Mini‐Inteins from Haloquadratum walsbyi
Authors: Conroy Owen R. , Gomez Alvin V. , Mills Kenneth V. 
Published: June 2021

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the protein (exteins) while simultaneously ligating the two flanking exteins. Inteins can be found in many types of archaebacteria, including halophiles, which thrive in environments with high salt concentrations. Previous results have shown that mini inteins of Haloquadratum walsbyi, such as those interrupting the cdc21 protein, require high concentrations of salt to splice. One intein that interrupts the DNA Gyrase B splices in E. coli, whereas similar inteins have required high salt incubation in vitro for splicing. The Hwa cdc21a, cdc21c, and PolIIb inteins were likely present in the most recent halophile ancestor as they are monophyletic within the halobacteria, the PolIIa and cdc21b inteins are polyphyletic, and the GyrB intein is likely related to inteins from bacteria. It is hypothesized that the GyrB intein has more recently invaded the halobacteria, therefore making the intein less stable to unfolding in the presence of salt when compared to earlier invading inteins, which many have adapted to high salt environments and are dependent on high salt concentrations for activity.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A bacterial flavin‐dependent oxidoreductase that captures carbon dioxide into biomass
Authors: Mattice Jenna , Streit Bennett , Prussia Greg , Peters John , DuBois Jennifer 
Published: June 2021

Atmospheric carbon dioxide is used as a carbon source for building biomass in plants and photosynthetic microbes. Non‐photosynthetic processes that also fix carbon dioxide have more recently been discovered. This research focuses on a microbial mechanism for coupling acetone to CO2 to make a central metabolite, acetoacetate. The key reaction is catalyzed by NADPH‐2‐ketopropyl‐coenzyme M oxidoreductase/carboxylase (2‐KPCC), a bacterial enzyme that is part of the flavin and cysteine‐disulfide containing oxidoreductase family (DSORs) which are best known for reducing metallic or disulfide substrates. Our research asks: how has nature repurposed a DSOR to, uniquely, break a C‐S bond and then trap and fix CO2? 2‐KPCC lacks a conserved, catalytically essential acid‐base histidine possessed by all other DSOR enzymes, having instead a phenylalanine (F501) at the same position. Mutagenesis showed that a F501H mutant has a similar rate of catalytic turnover; however, the product is acetone instead of acetoacetate (Figure 1). We hypothesized that F501 is important for both the reductive half reaction – which generates the reactive, C‐S bond breaking form of the active site – and for the oxidative half, in which an enolacetone intermediate reacts with either the correct (CO2) or incorrect (H+) electrophile. In this study, we used real‐time and spectroscopic methods to examine the reductive half reaction. In typical DSORs, this reaction generates a Cys/FAD charge transfer species. However, we showed that 2‐KPCC generates an electronically unique form of the active site, in which the flavin is oxidized and a pair of active site histidines are reduced and protonated. We hypothesize that this form of the active site generates the substrate‐reactive Cys in a more nucleophilic form, where it is capable of cleaving relatively strong C‐S bonds. The resulting enolacetone carbanion is an extremely potent nucleophile that is capable of directly attacking CO2. Research on 2‐KPCC and other biological CO2 fixation methods adds to our arsenal of strategies for carbon dioxide capture and use.Support or Funding InformationDOE:DE‐FG02‐04ER15563This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrostatic interactions in natural enzymes: What can we learn for enzyme design?
Authors: Ondrechen Mary Jo , Coulther Timothy A. , Ngu Lisa , Beuning Penny J. 
Published: June 2021

The goal of this work is to understand the intrinsic properties that give natural enzymes their catalytic power and to learn how to build these properties in silico, for the design and expression of artificial enzymes that catalyze reactions that do not occur in nature. Partial Order Optimum Likelihood (POOL) is a machine learning method developed by us to predict residues important for function, using the 3D structure of the query protein. The input features to POOL are based on computed electrostatic and chemical properties from THEMATICS. These input features are effectively measures of the strength of coupling between protonation events. POOL is used to characterize the properties of natural enzymes that are necessary for efficient catalysis. Catalytic sites in proteins are characterized by networks of strongly coupled protonation states; these networks impart the necessary electrostatic and proton‐transfer properties to the active residues in the first layer around the reacting substrate molecule(s). Typically these networks include first‐, second‐, and sometimes third‐layer residues. POOL‐predicted, multi‐layer active sites with significant participation by distal residues have been verified experimentally by single‐point site‐directed mutagenesis and kinetics assays for Ps. putida nitrile hydratase, human phosphoglucose isomerase, E. coli replicative DNA polymerase Pol III, E. coli Y family DNA polymerase DinB, and E. coli ornithine transcarbamoylase. In designed enzymes, such as retroaldolases, the residue‐specific input features to POOL – measures of the strength of coupling between protonation equilibria – rise as the enzymes evolve to higher rates of catalytic turnover. An approach to build these properties into the initial designs is proposed.Support or Funding InformationNSF MCB‐1517290 (MJO & PJB) and a National Institute of Justice Fellowship (TAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Influence of Pressure on the Activity of Enzymes from Deep‐sea Extremophiles
Authors: Ramsoomair Christian Kieran , Chavez Angel , Ruiz Mercedes Anessa , Gomez Alvin V. , Mills Kenneth Vincent 
Published: June 2021

Protein splicing is a post‐translational process facilitated by an intervening polypeptide, or intein. We studied inteins that interrupt the DNA Polymerase II from three different extremophiles, H. salinarum (Hsa), Pyrococcus abyssi (Pab), and Pyrococcus horikoshii (Pho). Intriguingly, all three of these inteins splice conditionally based on environmental cues. We hypothesized that since deep‐sea extremophiles such as P. abyssi and P. horikoshii live under moderately high pressures (150–200 atm), splicing will be unaffected or perhaps even enhanced under pressure. We also hypothesized that inteins from surface dwelling extremophiles like Hsa will not function as efficiently at moderate pressure. Lastly, we hypothesized high pressure (2000 atm and above), regardless of the native environment, will destabilize, unfold, and inactivate the activity. Results suggest that moderate pressures (200 atm and below) do not affect the splicing efficiency of inteins from either deep‐sea extremophile. While similarly unaffected at moderate pressure (100 atm), the function of Hsa inteins appear to be significantly diminished by higher pressures (1000 atm and above). In contrast, higher pressures appear to have no negative effect on the splicing of inteins from the deep‐seas extremophiles, and in some cases can even increase the splicing efficiency.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) (KVM), the Camille & Henry Dreyfus Foundation, and by Kim and Wendell P. Weeks P15 to the Holy Cross Alumni/Parent Summer Research Scholarship Fund (CKR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipid abnormalities and hepatic dysfunction associated with Yersinia pestis infection in non‐human primate model
Authors: Gautam Aarti , Muhie Seid , Chakraborty Nabarun , Hoke Allison , Donohue Duncan , Miller Stacy Ann , Hammamieh Rasha , Jett Marti 
Published: June 2021

Aerosolized Yersinia pestis causes pneumonic plague and is a highly virulent form of the infection. When left untreated, the fatality rate is very high, and survivors can suffer from complex clinical consequences. The exact mechanisms of disease progression are presently unclear. In this study, we used metabolite profiling to gain mechanistic insight into the progression of the disease. African green monkeys (Chlorocebus sabaeus) were challenged with the highly virulent Y. pestis strain CO92 by the aerosol route, plasma samples were obtained, and untargeted metabolomics profiling of plasma was performed using both liquid and gas chromatography with mass spectrometry. At early time points post‐exposure (6–18 h), we found increases in the abundance of certain polyunsaturated fatty acids, long chain fatty acids, carnitines, and dicarboxylate fatty acids in exposed animals when compared to their time‐matched control samples. The metabolites from lysolipids and bile acid metabolism pathways were lower in exposed animals when compared to their time‐matched controls during the entire course of infection. Many of the metabolites from amino acid pathways were perturbed, and a few from the leucine, isoleucine, and valine pathways were elevated during the course of infection, whereas metabolites from the urea cycle, arginine, and proline pathways were lower. The presence of branched chain amino acids in plasma, in addition to mobilization of fatty acids for subsequent β‐oxidation, indicated an increased energy demand. The involvement of several lipid pathways post‐infection suggested that there was activation of biological pathways linked to inflammation and oxidative stress. Enrichment analysis indicated perturbations in the bile acid pathway during the entire course of infection along with multiple metabolites that may have involvement in liver function indicating potential hepatic dysfunction. Integration of metabolomics and transcriptomics data from blood identified linoleate as a core metabolite with cross‐talk with multiple genes from various time points. Collectively, the data from this study provided new insights into the mechanisms of Y. pestis pathogenesis and into the host‐pathogen interactions that shape the outcome of the infection.Disclaimers: Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.Support or Funding InformationFunding Support from DTRA is highly acknowledgedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atypical Protein Kinase C‐specific Activity Reporter Reveals Novel Activation Mechanism of Atypical Protein Kinase C by Sphingosine 1‐phosphate
Authors: Kajimoto Taketoshi , Caliman Alisha D. , Tobias Irene S. , Okada Taro , McCammon J. Andrew , Nakamura Shun‐ichi , Newton Alexandra C. 
Published: June 2021

The protein kinase C (PKC) family comprises 9 members; conventional PKC isozymes (α, β γ), novel PKC isozymes (δ, ɛ, θ, η), and atypical PKC isozymes (ζ, ι/λ). It is well established that conventional and novel PKC isozymes are activated by second messengers, diacylglycerol and, for the conventional PKC isozymes, calcium ion. However the activation mechanism and physiological function of atypical PKC isozymes are less well understood. Here we show that a lipid mediator, sphingosine 1‐phosphate (S1P) controls the cellular activity of atypical PKC isozymes. First, we generated a genetically encoded reporter with the same modular structure as the original C kinase activity reporter (CKAR) but with a unique substrate sequence that allows specific visualization of atypical PKC activity in cells. Using the atypical PKC‐specific CKAR (aCKAR) we found that intracellular S1P induces the activation of atypical PKC in an S1P receptor‐independent manner. Biochemical studies reveal that S1P directly binds to the kinase domain of atypical PKC isozymes, relieving autoinhibitory constraints to activate the enzyme. In silico docking studies were used to identify potential binding sites for aPKC, one of which was validated biochemically. Our results are consistent with a model in which S1P binds the kinase domain of atypical PKC isozymes, an event that releases autoinhibitory constraints to lock the PKC in an active conformation. Our results provide a new molecular mechanism for controlling the cellular activity of atypical PKC.Support or Funding InformationThis study was supported by NIH to A.C.N., NIH, HHMI, NBCR, NSF to J.A.M., JSPS KAKENHI, Kobe University Grant for Japan‐US Collaboration, Nakatani Foundation Grant for Technology Development Research to T.K., JSPS KAKENHI to S.N. A.D.C. was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.