bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphorylation regulates apoptotic caspase function through diverse molecular mechanisms
Authors: Hardy Jeanne A , Serrano Banyuhay P , Eron Scott J 
Published: November 2023

Caspases, the cysteine proteases that execute apoptosis, are tightly regulated via phosphorylation by a series of kinases. Although all apoptotic caspases work in concert to promote apoptosis, different kinases regulate individual caspases. Several sites of caspase‐7 and −9 phosphorylation have been reported, but without knowing the molecular details, it has been impossible to exploit or control these complex interactions, which normally prevent unwanted proliferation. This work provides new molecular mechanisms for phosphorylation‐based control of both caspase‐7 and caspase‐9 function.During dysregulation, PAK2 kinase plays an anti‐apoptotic role, phosphorylating caspase‐7 and promoting unfettered cell growth and chemotherapeutic resistance. PAK2 phosphorylates caspase‐7 at two sites, inhibiting activity using two different molecular mechanisms, before and during apoptosis. Phosphorylation of caspase‐7 S30 allosterically obstructs its interaction with caspase‐9, preventing intersubunit linker processing, slowing or preventing caspase‐7 activation. S239 phosphorylation renders active caspase‐7 incapable of binding substrate, blocking later events in apoptosis. In parallel and in response to cAMP, Protein Kinase A (PKA) phosphorylates caspase‐9 at three sites preventing caspase‐9 activation and suppressing apoptosis progression. Phosphorylation of caspase‐9 by PKA at the functionally relevant site, S183, acts as an upstream block of the apoptotic cascade, inactivating caspase‐9 by a two‐stage mechanism. First, S183 phosphorylation prevents caspase‐9 self‐processing and directly blocks substrate binding. In addition, S183 phosphorylation breaks the fundamental interactions within the caspase‐9 core, promoting disassembly of the large and small subunits. This occurs despite S183 being a surface residue distal from the large: small interface. This phosphorylation‐induced disassembly promotes the formation of ordered aggregates around 20 nm in diameter. Similar aggregates of caspase‐9 have not been previously reported. This two‐stage regulatory mechanism for caspase‐9 has not been reported previously but may be conserved across the caspases. Each of these mechanisms of regulation for caspase‐7 and caspase‐7 is novel, representing new opportunities for synergistic control of caspases and their counterpart kinases.Support or Funding InformationThis work was supported by the National Institutes of Health (NIH) GM 080532.
Creating Peptide Hydrazides via Intein Splicing for Native Chemical Ligation and Protein Labeling
Authors: Santoleri Dominic A , Liu Jun , Ekanayake Oshini , Rozovsky Sharon 
Published: November 2023

Peptide hydrazides are powerful surrogates for the thioesters typically used in protein ligation as well as being an effective handle for labeling. Typical peptide hydrazide preparation relies on solid‐phase peptide synthesis, a technique limited by peptide size and solubility that also uses harsh chemicals. Here, a more effective and biochemically‐friendly means of generating these peptide hydrazides has been established involving split PhoRadA inteins, which only regain their splicing activity upon association with each other. This method also avoids several issues involved with full intein splicing, such as in vivo self‐cleavage and low expression levels. C‐Terminal peptide hydrazides are a powerful means of creating protein labels as we have shown them to react with small aldehyde‐containing molecules that can serve as a tag or a marker in analytical studies.Support or Funding InformationFunding for this research project is provided through an NSF CAREER Award.
Role of SUMOylation in Alcohol‐induced Liver Fibrosis
Authors: Tomasi Maria Lauda , Cossu Carla , Ramani Komal 
Published: November 2023

Background & AimAlcohol‐induced liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) components, especially collagen in response to chronic hepatocellular damage. If alcohol consumption is continued, fibrosis may progress to cirrhosis which is a major cause of morbidity and mortality. Reactive oxygen species (ROS) generated in hepatocytes by alcohol metabolism plays an important role in producing liver damage and initiating hepatic fibrogenesis. ROS disrupts lipids, proteins and DNA inducing necrosis and apoptosis of hepatocytes and amplifies the inflammatory response via Kupffer cells that directly induces hepatic stellate cells (HSCs) activation, resulting in the initiation of fibrosis. SUMOylation is a post‐translational modification that plays an important role in a wide range of cellular processes. SUMOylation is oxidative stress inducible and involves the covalent attachment of a member of the SUMO (small ubiquitin‐like modifier) to the target protein. We previously demonstrated that the level of Ubiquitin‐conjugation enzyme 9 (Ubc9), a sole E2‐conjugating enzyme essential for SUMOylation, is induced in the livers of intragastric ethanol‐infusion (EI) treated mice and lipopolysaccharide‐induced inflammation promotes Ubc9 phosphorylation and its activity in Kupffer cells. Our aim was to examine whether the dysregulated SUMOylation could regulate the ethanol‐induced liver fibrosis and elucidate the molecular mechanism(s).MethodsStudies were done using in vivo binge ethanol‐fed mice, primary mouse and human HSCs and LX‐2 cells. Oxidative stress markers were measured using commercial kits.ResultsWe found that ethanol treatment for 48 hours (25mM) in LX‐2 and HSCs cells increased ROS production, Ubc9, α‐SMA and COL I mRNA levels, well known as activated HSCs markers, by 1.5‐fold (H2O2), 1.4‐fold (Ubc9), 1.3‐fold (α‐SMA) and 1.5‐fold (COL I), respectively. RNAi‐mediated knockdown of Ubc9 to 75% of control blocked ethanol‐induced ROS production and the increase in α‐SMA and COL I mRNA levels. Interestingly, we found that silencing SUMO‐1 in activated LX‐2 apoptosis was induced by 50%, while quiescent LX‐2 cells in matrigel were not affected. This finding confirmed that sumoylation is playing a key role on stellate cells activation. In addition, isolated mouse stellate cells from binge ethanol‐fed mice overexpressed high level of Ubc9 mRNA and protein levels compared with control mice.ConclusionThis data is strongly suggesting that ethanol is inducing HSCs activation in human and mouse liver involving the oxidative stress‐induced sumoylation machinery. Identifying the molecular mechanisms will require knowing which proteins are affected and what the functional outcomes of a change in sumoylation status is on those proteins, which will pave the way for future exploring.Support or Funding InformationSupported by NIH grant 5 K01 AA022372‐04, maria lauda tomasi
Thermodynamics of the Gamma B Crystallin Protein Demonstrated by T1/T2 NMR Experiments
Authors: Umphred‐Wilson Katharine , Fadden Aaron , Zanet John , Mathews Kaylee , Thurston George , Mills Jeffrey , Michel Lea Vacca 
Published: November 2023

Gamma B Crystallin proteins are soluble proteins found in the eye lens which help maintain its transparency; however, over time their structure may mutate due to UV radiation and other environmental stresses causing the proteins to aggregate and develop into cataracts. Using light scattering, nuclear magnetic resonance (NMR) spectroscopy and other protein modeling techniques, our team is attempting to understand the intermolecular interactions of gamma B crystallin proteins that are responsible for this aggregation. We expect to learn the structural and chemical properties that dictate how the proteins interact, as well as the effect of the thermodynamic properties in the fluid mixtures of the eye lens on the crystallin interactions. We expressed the gamma B crystallins in Escherichia coli and isolated them using size exclusion and ion exchange chromatography. The protein was analyzed using various types of NMR experiments, including T1/T2 experiments. The Tau C values, which were derived from these experiments, correlate with a decreased tumbling time; this means that lower Tau C values indicate increased aggregation. Our preliminary results suggest that cold temperatures and high concentrations of our protein increase aggregation levels and the number of attractive intermolecular interactions.Support or Funding InformationNIH EY018249 (G. Thurston)
Probing solvation environments in Tt H‐NOX by site‐specific incorporation of 4‐cyano‐L‐phenylalanine
Authors: Kearney Caroline , Olenginski Lukasz T. , Tariq Daniyal , Hirn Trexler D. , Brewer Scott H. , Phillips‐Piro Christine M. 
Published: November 2023

Heme Nitric Oxide and/or Oxygen (H‐NOX) binding proteins are a diverse group of hemoproteins that bind diatomic gases like nitric oxide (NO), carbon monoxide (CO), and/or oxygen (O2). H‐NOX proteins, which can be found in both prokaryotic and eukaryotic cells, contain two distinct domains: a heme‐containing sensor domain that binds gaseous ligands and an effector domain that generates a cascade of signals for cells to function. Here local protein environments in Thermoanaerobacter tengcongensis H‐NOX (Tt H‐NOX) were investigated using the vibrational reporter unnatural amino acid (UAA) 4‐cyano‐L‐phenylalanine (pCNF). This UAA was genetically site‐specifically incorporated into Tt H‐NOX at multiple positions individually including near the heme pocket using amber codon suppression technology. Infrared analysis of the constructs probing the position of the nitrile symmetric stretch of pCNF revealed a myriad of local environments in the protein. These results highlighting solvated or buried positions in the protein will be presented.Support or Funding InformationNIH R15GM1219842CMPP and the Fred A. Snavely Research Award
Labilization of the Cobalt‐carbon Bond in Vitamin B12 Bound to Adenosyltransferase
Authors: Campanello Gregory Cook , Twahir Umar , Brunold Thomas , Warncke Kurt , Banerjee Ruma 
Published: November 2023

Vitamin B12 is an essential, scarce, and reactive cofactor that is acquired by mammals from the diet. Studies on patients with inborn errors of cobalamin metabolism have led to the discovery of nine genes involved in the tailoring, chaperoning, and utilization of cobalamin as a cofactor. One of these genes encodes adenosyltransferase (ATR), a bifunctional enzyme that is responsible for the synthesis of 5′‐deoxyadenosylcobalamin (AdoCbl) and for its delivery to the client enzyme, methylmalonyl CoA mutase (MCM). Mutations in ATR and MCM result in methylmalonic aciduria, a devastating metabolic disorder, which can result in infant mortality. ATR catalyzes the synthesis of AdoCbl via transfer of the adenosyl group of ATP to the supernucleophile cob(I)alamin, resulting in the release of inorganic tripolyphosphate (PPPi). PPPi binds to the ATR•AdoCbl complex with an apparent Kd≤ 1 μM. Binding of PPPi to ATR•AdoCbl under anaerobic conditions induces a unique UV‐visible spectroscopic signature with an absorption maximum at 439 nm. Exposure of this complex to O2 results in cleavage of the cobalt‐carbon bond and formation of an ATR‐bound 4‐coordinate cob(II)alamin species with a λmax of 464 nm. The resulting spectrum is virtually identical to that of neopentylcobalamin, a synthetic B12 derivative with a weak cobalt‐carbon bond. Under aerobic conditions, neopentylcobalamin also undergoes spontaneous decomposition to cob(II)alamin (1). We are using multiple spectroscopic approaches to characterize this unusual reaction and to place it in a functional context. We hypothesize that binding of PPPi to ATR‐bound AdoCbl results in weakening of the cobalt‐carbon leading to the formation of tightly bound cob(II)alamin, thus prioritizing sequestration of the cofactor under conditions where the acceptor MCM, might be limiting.Support or Funding InformationWe are very grateful for NIH grants awarded to G.C.C. (5 F32 GM113405‐02) and R.B. (DK45776) which supported this research.
Structural Changes in the Nickel‐Responsive Regulator NikR from Helicobacter pylori
Authors: Baksh Karina Amelia , Wong Jeremy , Pham Tamara , Zamble Deborah Beth 
Published: November 2023

Helicobacter pylori is a pathogenic bacterium estimated to infect 50% of the global population and causes peptic ulcers, gastritis, and gastric cancer. H. pylori is able to survive the fluctuating pH levels of the stomach mainly due to expression of two enzymes – urease and [NiFe]‐hydrogenase – that require nickel as an essential cofactor. Therefore, regulation of nickel concentration is crucial in order to avoid toxicity while maintaining an adequate supply. This is achieved by the nickel‐responsive transcription factor NikR (HpNikR), which maintains intracellular nickel homeostasis through binding the promoters of various genes, including those encoding nickel storage and import factors, and activating or repressing their transcription. HpNikR also plays a role in acid acclimation and there is evidence that its DNA‐binding activity can be modulated by changes in pH. It is therefore likely that there are promoter specific conformations exhibited by HpNikR that are sensitive to both nickel concentration and pH. However, the mechanisms by which nickel and pH activate DNA binding remain unclear. In this work, a series of mutants at key secondary structural elements were created as targets for labeling with a 19F probe and 19F‐NMR was used to uncover how nickel and pH influence the HpNikR conformational changes required for binding to different promoters.Support or Funding InformationThis work was supported in part by funding from the Canadian Institutes of Health Research.
New ABCG2 homology model reveals importance of C‐terminus in protein trafficking
Authors: Cui Jinming , Sayed Hiba , Huang Jonathan , Woodward Owen M. 
Published: November 2023

Recent genetic studies have implicated ABCG transporters in human disease and identified potential causal variants. However interpretation of the resulting molecular defect is often limited by the lack of high‐resolution crystal structures. This has been the case for ABCG2. Here we present a new homology model of ABCG2 using a recent ABCG5/G8 crystal structure (PDB:5do7) as a template in an effort to better understanding the implications of human ABCG2 gout causing mutations. Our homology model is composed of the ABCG2 NBD domains (residues 29–288) and TMD domains (residues 289–655) and was built using the Swiss‐Model server. To avoid forcing the ABCG2 into a heterodimer, the ABCG5 served as template for both ABCG2 copies because its NBD structure is closer to the canonical NBD structure. The loop regions of the model were further refined by GalaxyLoop. The final model complied with the modular multi‐domain architecture of ABC proteins, and had RMSD values with ABCG5 NBD, ABCG5 TMD, ABCG8 NBD, ABCG8 TMD of 0.2Å, 0.5Å, 1.7Å, and 2.4Å, respectively. The new ABCG2 homology model allows for the visualization of parts of the protein previously undefined. One intriguing finding was that the loop between the first and second β‐strands of the NBD (residues 42–63) is in close proximity with the TMD C‐terminus S655. Notably, the corresponding loop (residues 47–65) and the C‐terminus R651 were not resolved in the ABCG5/G8 crystal structure, suggesting a dynamic interaction in this region. We explored the significance of the C‐terminal tail (resides 652–655; KKYS) on protein abundance and trafficking. The addition of a C‐terminal Flag‐tag or the deletion of the last 4 residues (651X) led to a profound decrease in protein abundance. The 651X protein is not glycosylated and localizes to the ER only. Interestingly, a YS653AA (KKAA) substitution is less deleterious and results in protein that reaches the Golgi compartment, highlighting the dilysine motif, a potential COPI target, as key in trafficking ABCG2. Finally, we found the S655A substitution (KKYA) results is significantly greater protein abundance than wild type and partially rescues the abundance of the Q141K human gout mutation, implicating the Y654 as a key trafficking residue. In conclusion, our new ABCG2 homology model suggests the C‐terminus is in close proximity to the NBD and may be a critical feature in regulating the trafficking of the ABCG2 protein.Support or Funding InformationAmerican Heart Association 14SDG18060004
Structure of hRpn13 at the Proteasome
Authors: Lu Xiuxiu , Liu Fen , Nowicka Urszula , Sridharan Vinidhra , Dyba Marzena , Tarasov Sergey G. , Walters Kylie J. 
Published: November 2023

The proteasome has a 20S core particle (CP) that is capped at either end by a 19S regulatory particle (RP). An ATPase ring from the RP directly contacts the CP and extends out to three ubiquitin receptors (Rpn13/Adrm1, Rpn10/S5a and Rpn1/S2/PSMD2) that recognize ubiquitin and ubiquitin folds of substrate shuttle factors. Here, we solve the solution structure of the ubiquitin‐binding hRpn13 Pru (Pleckstrin‐like receptor for ubiquitin) domain with the C‐terminal 14 amino acids of hRpn2 that is used to dock hRpn13 into the proteasome by NMR. We find that the hRpn13:hRpn2 interaction surface is proline rich, with hRpn2 extended across a shallow canyon of hRpn13 Pru domain. Our structure imposes an orientation to hRpn13 in available cryoEM density maps of the yeast 26S proteasome, and provides atomic level details for how hRpn13 assembles into the proteasome. Moreover, hRpn13 has been reported to be a promising therapeutic target. Cell permeable bis‐benzylidine piperidone RA190 restricts cancer growth in mice xenograft models of multiple myeloma and ovarian cancer and conjugates to hRpn13. We present structure‐guided biochemical assays that provide novel insights into the mechanism of action for RA190.Support or Funding InformationIntramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research
Structural Insights into Oxalate Biosynthetic Component
Authors: Rhee Sangkee , Oh Juntaek , Kim Sanghoon 
Published: November 2023

Bacterial quorum sensing (QS), a cell‐to‐cell communication process in many Proteobacteria, controls the gene expression for bacterial population‐wide characteristics, including bioluminescence, motility, and virulence‐related factors. Recently, QS has been recognized to provide further benefits at the population level by regulating the production of public goods, the function of which could be beneficial to all members of the group. In Burkholderia species, oxalic acid was recently identified as an excreted public good for the QS‐dependent growth. In these species, QS‐mediated oxalogenesis via the oxalate biosynthetic component (obc) is a cellular event indispensable for the survival of bacteria in the stationary phase. In B. glumae, obc consists of two genes encoding ObcA and ObcB for coordinating the production of oxalic acid, as well as acetoacetate and CoA, by using oxaloacetate and acetyl‐CoA as substrates. Unlike B. glumae, there is a bi‐functional enzyme Obc1 from B. thailandensis. Sequence analysis indicates that Obc1 has an ObcA‐like N‐terminal domain and shows ObcB activity in its C‐terminal domain despite no sequence homology with ObcB. We will present our recent progress on structural and functional analyses of ObcA and Obc1. In ObcA, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr322 as a general base, which then attacks the thioester carbonyl carbon of acetyl‐CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal‐coordination shell and absence of general acid(s) likely produces an unusual tetrahedral CoA adduct as a stable product. In Obc1, the C‐terminal domain has an α/β hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggested that His‐934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis.Support or Funding InformationThis work was supported by Next Generation BioGreen 21 program of Rural Development Administration (Plant Molecular Breeding Center) of Republic of KOREA.
Heat Shock Protein 90 is Critical for Plasmodium Parasite Liver Stage Development
Authors: Keim Allison I , Posfai Dora , Haystead Timothy A , Derbyshire Emily R 
Published: November 2023

Malaria remains a severe global health burden, and there is an urgent need to develop effective strategies to combat the Plasmodium parasites responsible for the disease. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood stage Plasmodium parasites, but its role during malaria's elusive liver stages remain unclear. Studies on protein function throughout the infection are challenging as Hsp90 is highly conserved between the parasite and human host. To address this issue, we used target‐based screens to identify compounds that bind to Plasmodium (Pf) and human Hsp90 with varying species selectivity and used them as chemical probes. All identified Hsp90‐binding compounds were potent blood and liver stage Plasmodium inhibitors in cell‐based malaria assays. Importantly, Hsp90 inhibitors synergistically reduced liver stage parasite viability when used in combination with phosphatidylinositol 3‐kinase inhibitors. Most notably, liver stage parasite inhibition strongly correlated with binding to PfHsp90, but not the human host protein. Analysis of Hsp90 protein and gene expression throughout the liver stage coupled with time course inhibition studies further support a critical role of Plasmodium Hsp90 in early liver stage parasite development. Our results suggest that PfHsp90, but not human Hsp90, is essential to malaria's liver stage and highlight the potential to selectively inhibit PfHsp90 in combination therapies for malaria treatment and prevention.Support or Funding InformationThis work was supported by the NIH for laboratory support (GM099796 to E.R.D.) and the Duke Pharmacological Sciences Training Program for fellowship support (2T32GM007105‐41 to A.I.K.).
New Insights from High‐throughput Biophysical Screening of Protein‐sequence and Coding‐sequence Libraries
Authors: Allen Benjamin 
Published: November 2023

The convergence of next‐generation sequencing, large‐scale gene synthesis, and high‐throughput screening has enabled unprecedented, large‐scale characterization of the sequence and contextual factors that govern protein‐assembly outcomes. We have leveraged existing fluorescent readouts for protein solubility, protein aggregation, and enzyme function to evaluate up to 1 million defined coding‐sequence variants of individual protein scaffolds in living bacterial cells. In one example, we targeted diversity to the hydrophobic core of a small, stable protein domain. Preliminary analysis suggests that there are divergent routes to solubility in this system: both minimally hydrophobic and highly hydrophobic sequences can assemble into soluble forms, while soluble assembly may be more exceptional for sequences with intermediate hydrophobicity like the wild type. In another example, we built a library of synonymous coding sequences with either a common or a rare codon at each degenerate site. While this library showed variation in soluble and total protein production, the observed variation could not be explained by simple models of translation initiation or elongation. Contrary to expectation, many highly expressed constructs contained a large number of low‐abundance codons, suggesting that factors other than tRNA availability may be rate limiting in the production of soluble protein. These findings suggest alternative approaches to the engineering of protein solubility and the optimization of coding sequences for high expression.Support or Funding InformationThe Department of Biochemistry and Molecular Biology and the Huck Institutes of the Life Sciences, the Pennsylvania State University