bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating Contributions of the N and C Terminus to FOXO1 and DNA Interactions
Authors: Stenson Jazmin Marie
Published: November 2023

FOXO1 is a mammalian transcription factor that belongs to the FOXO protein family. FOXO1 regulates glucose and fatty acid metabolism in the liver, and changes in FOXO1 expression have also been identified in a variety of cancer cells. All FOXO proteins bind to two consensus sequences known as the Daf‐16 binding element or DBE (5′‐TT (A/G)TTTAC‐3) and the insulin‐response element or IRE (5′‐TT(G/A)TTT(G/T)(G/T) ‐3′). FOXO1 binding can be enhanced by the presence of multiple copies of these consensus sequences although the mechanism behind this enhancement is not known. Molecular Dynamics Simulation and in vitro assays indicate that Wing 1 of the FOXO1 DNA‐binding domain makes contact with DNA bases outside of the consensus sequence. However, this data does not explain the enhancement in binding observed with multiple copies of the IRE or DBE that extend beyond the potential reach of Wing 1 indicating the need to explore additional mechanisms for this enhancement. We are therefore interested in determining how the N and C terminus of FOXO1 might contribute to the interaction with DNA. Bacterially expressed and purified versions of FOXO1 lacking specific domains were used to determine the effects on DNA binding through in vitro assays. Understanding the contribution of the entire protein structure to DNA binding may allow for a better understanding of how FOXO proteins bind and recognize specific target sequences.

Support or Funding Information

NIH MARC 5T 34GM07716‐37; NIH BUILD RL5GM118966‐02, NIH RCMI 2G12MD007595‐06
Characterization of a novel apolipophorin‐III/apolipoprotein E C‐terminal domain chimera
Authors: Kakutani Leesa Miiko
Published: November 2023

Human apolipoprotein E (apoE) is a two‐domain anti‐atherogenic lipid transport protein that mediates plasma cholesterol homeostasis by serving as a ligand for the low density lipoprotein (LDL) receptor. Of its three isoforms E3, E2, E4, the latter two have been associated with increased risk of cardiovascular and Alzheimer's disease respectively, yet all three contain an identical C‐terminal (CT) domain. The CT domain (residues 201–299) is responsible for tetramerization and has been found to initiate lipid binding that influences critical functional features of apoE. The N‐terminal (NT) domain of apoE has four α‐helices arranged in a bundle similar to apolipophorin III (apoLp‐III), a model insect apolipoprotein containing five α‐helices in one domain. To better understand the role apolipoprotein domains play in structure and function, a novel chimeric apolipoprotein was designed by attaching apoE‐CT to apoLp‐III. A disulfide bond was introduced to apoLp‐III to lock the helix bundle and eliminate its lipid binding. Recombinant apoLp‐III/apoE‐CT, apoE3, apoE‐CT, and apoLp‐III were expressed in bacterial cells, purified by affinity chromatography, and the purity verified by SDS‐PAGE. Western blot analysis using monoclonal apoE‐CT specific antibody confirmed the presence of apoE‐CT in the chimera. Crosslinking studies using dimethylsuberimidate revealed that the apoLp‐III/apoE‐CT chimera formed oligomers similar to apoE, while apoLp‐III was monomeric. Far UV circular dichroism showed increased α‐helical content, and incubation with 1‐anilinonaphthalene‐8‐sulfonic acid showed increased fluorescence intensity when apoE‐CT was added to apoLp‐III, consistent with the presence of water‐shielded dye binding exposed hydrophobic regions. Our results suggest that the chimeric protein has structural similarities to the parent proteins, and that self‐association properties of apoE can be transferred to apoLp‐III through the addition of the CT domain. Future studies will include measuring the ability of the chimera to solubilize phospholipid bilayers and binding to low density lipoproteins. The chimeric approach offers the potential to obtain insight into domain interactions and the structure‐function relationships in apolipoproteins.

Support or Funding Information

This research was supported by the National Institute of General Medical Sciences of the NIH (8UL1GM118979‐02, 8TL4GM118980‐02, 8RL5GM118978‐02, GM089564 and GM105561).
DNA Binding by a HEAT‐Repeat Subunit of Saccharomyces cerevisiae Condensin
Authors: Sarkar Rupa , Rybenkov Valentin V.
Published: November 2023

Condensins play a crucial role in higher order chromosome organization in both prokaryotes and eukaryotes. In budding yeast Saccharomyces cerevisiae, condensin complex consists of five subunits: two evolutionary‐conserved structural maintenance of chromosome (SMC) subunits and three non‐SMC subunits. SMC subunits adopt a V‐shaped structure, in which two globular head domains are connected to a central hinge domain via two long coiled coils. Non‐SMC complex consists of a kleisin, which binds the head domains of SMC, and two HEAT‐repeat subunits Ycs4 and Ycg1. DNA binding and organization primarily resides in the SMC proteins. Non‐SMC subunits contribute to the activity of SMC and regulate the recruitment of holoenzyme to the chromosome. Unlike in bacteria, the non‐SMC complex of yeast S. cerevisiae binds DNA. In this study, we mapped the DNA binding site of non‐SMC complex using mass spectrometry based DNA footprinting. The DNA binding site is located on the C‐terminal domain of Ycs4 next to the HEAT‐repeat cluster. We verified the implicated amino acids using site‐directed mutagenesis and observed a reduction of DNA binding by Ycs4. Furthermore, we found that the mutations affect the cellular growth of S. cerevisiae and the activity of the holoenzyme condensin in vitro. These results show that DNA binding of Ycs4 is essential for the activity of condensin in vitro and in vivo.

Support or Funding Information

OCAST HR 14‐042
L‐Arginine Inhibits a Critical Structural Contact Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β
Authors: Pulugulla Sree H , Rutter Nathan W , Esposito Emilio X , Yang Zhiyong , Madura Jeffry D , Auron Philip E
Published: November 2023

Interleukin 1β (IL‐1β) is a proinflammatory cytokine that is normally expressed and released by monocytes in response to infection and injury. Chronic expression of IL‐1β has been associated with numerous inflammatory diseases. We previously reported chromatin looping for the IL1B gene that is mediated by a mutual DNA‐binding domain (DBD) interaction between two transcription factors, Spi‐1/PU.1 (Spi‐1) at the promoter and C/EBPβ at a distal enhancer. The nature of this critical interaction between the Spi‐1 wHTH domain and the C/EBPβ leucine zipper was investigated by using the existing X‐ray structures of these two proteins bound to DNA as a guide in designing mutants for in vitro protein‐protein interaction studies. This generated surface interaction maps for the two proteins that in concert with computational techniques supported an association mode similar to, but distinct from, the C/EBPβ·c‐Myb X‐ray structure reported by others. The distinction was that, in addition to the C/EBPβ leucine zipper interaction with the c‐Myb wHTH domain, Spi‐1 utilizes an additional pseudo‐symmetrical bidentate interaction between the carboxyl‐tail extensions of each of the C/EBPβ leucine zipper monomers with the two ends of the Spi‐1 DNA recognition helix. This is consistent with our previous report that the carboxyl‐tail of C/EBPβ is critical for Spi‐1 association, suggesting that pocket‐like surfaces at the ends of the Spi‐1 DNA recognition helix might be targets for inhibition. Candidate small molecules were generated using a virtual screening approach. L‐arginine, a known anti‐inflammatory molecule, was identified from this approach as having potential to bind to each end of the Spi‐1 recognition helix, possibly inhibiting IL1B gene expression. Studies in 2.5 h LPS‐treated human THP‐1 macrophages in the presence of L‐arginine revealed a concentration‐dependent blunting of transcription in parallel with a decrease in C/EBPβ association with Spi‐1 on the IL1B gene promoter, while Spi‐1 binding to DNA was unaffected. In the same experiment, there was no significant change in direct binding of C/EBPβ to DNA at a well‐characterized site in the IL6 gene. In conclusion, these studies are consistent with an L‐arginine‐dependent decrease in IL1B gene expression that is due to its ability to inhibit the protein‐protein interaction between C/EBPβ and Spi‐1 in the absence of any effect on direct DNA binding for either factor. This is consistent with the predicted structure of the C/EBPβ·Spi‐1 interaction.
Computational Analysis of CHP‐NHE Protein‐Protein Interaction
Authors: Bell Isaac Walter , Latzer Joachim , Provost Joseph John
Published: November 2023

The Sodium Hydrogen Exchanger (NHE1) is an ATP independent membrane transporter that exchanges an intracellular proton for an extracellular sodium ion. In addition to maintaining intracellular pH homeostasis, NHE1 plays a critical role interacting with proteins on the leading edge of migrating proteins controlling directed cell motility. Regulation of NHE1 is complex. Phosphorylation, lipid binding and protein‐protein interactions all take place at the 300 amino acid carboxyl terminal regulating NHE1 protein exchange and protein interactions. Two NHE1 binding proteins whose roles are not fully clear are the calcineurin homologous protein isoforms 1 and 2 (CHP1 and CHP2). While both CHP1 and CHP2 bind to nearly the same domain of NHE1 (CHP Binding Domain amino acids 503–545; NHE1‐CBD), each has a unique effect on NHE1 function. Because CHP2 is primarily expressed in gut and transformed tumor cells, the interaction between CHP and NHE is a potential anticancer target. In order to quantify the interaction between the CHP1‐NHE and the CHP2‐NHE we created GST fusion protein of NHE1‐CBD with a thrombin cleavage site between fusion proteins as well as 6XHis‐CHP1 and 6XHis‐CHP2. Each protein was expressed and purified from bacterial culture. GST‐NHE1‐CBD was cleaved from its fusion partner on the column. After purification each protein was dialyzed in 10mM K‐ phosphate, 100mM NaCl, and 0.5mM Ca2+, pH of 7.4 Circular Dichroism (CD) wavelength scan of each protein was determined to identify the helical nature of the protein; CHP1 with 51.60% helical structure, CHP2 with a 49.74% helical structure, and NHE1 with a 40.91% helical structure in the CBD. Thermal folding of each protein alone or in combination was determined following CD as a function of temperature. Melting point (Tm) for CHP1 (56.33+/−0.72 °C), and NHE1‐CBD (59.43+/−0.62 °C) alone increased to 59.73 +/−0.21 °C when the two proteins were combined. We determine the van't hoff enthalpy and entropy of unfolding to estimate the binding constant of protein interaction for CHP1‐NHE1 and CHP2‐NHE1. From the experimental data collected, the theoretical temperature at which each construct denatures; for both the CHP2‐NHE construct and CHP1‐NHE construct were inputed into a Large‐scale Atomic/Molecular Massively Parallel Simulator (LAMMPS). Results from the LAMMPS analysis were then inputted into a Weighted Histogram Analysis Method (WHAM) in order to generate specific base‐pair interactions in the binding and unbinding in each of the two CHP isoforms and NHE. WHAM analysis also provides a theoretical depiction of the intermediate steps of binding, helping to determine if the process is one‐stage or a step‐wise process. This data along with pdb files created at set intervals during the LAMMPS simulation will be used to create a visual representation of the binding of the different constructs. This work will help predict the binding mechanism of CHP‐NHE1 interaction to identify potential therapeutic targets blocking CHP2 but not CHP1 from NHE1.
Selecting Medically Relevant In Vitro Protein Characterization Projects with a High Potential For Success
Authors: Zajac Diana , Tucker Brady , Cotsoradis Alicia , Mallmann Clara , Cassidy Christine , Gurnon Daniel
Published: November 2023

As of November 2016, well over one‐third of the more than 170,000 unique variation records in ClinVar are of unknown importance; these variants are either (a) characterized as variants of unknown significance (VUS), (b) have no clinical significance provided, or (c) list conflicting reports of clinical significance. The vast number of ambiguous entries in ClinVar offer an opportunity for researchers seeking new projects in the area of in vitro protein characterization; not only are there many variants to choose from, the size of the dataset provides potential to select targets that are a good fit for the capabilities of the lab. The opportunity to preselect medically relevant projects with a higher degree of success is especially intriguing for researchers at undergraduate institutions, who do not have the resources of larger labs.

We describe the development of a selection algorithm for choosing an in vitro protein characterization project that is amenable to undergraduate research. Our approach combines information from ClinVar and the Protein Databank to select enzymes with published crystal structures of the human protein, that have been previously expressed and purified in e. coli, and that feature at least one missense VUS entry in ClinVar.

We also describe progress on the characterization of four variants of unknown significance in Glucose‐6‐Phosphate Dehydrogenase, an enzyme associated with hemolytic anemia.

Support or Funding Information

Support provided by DePauw University and the Howard Burkett Scholar Fund.
Effects of Endoplasmic Reticulum Stress on Protein Quality Control
Authors: Buchanan Bryce William , Rubenstein Eric M
Published: November 2023

Protein quality control is critical for cellular health. The Endoplasmic Reticulum (ER)‐Associated Degradation (ERAD) protein quality control system degrades aberrant proteins at the ER membrane. ERAD can be divided into multiple pathways, including ERAD‐L (lumen), ‐M (membrane), ‐T (translocon), ‐C (cytosol), and ‐RA (ribosome associated). In general, each ERAD pathway degrades proteins with aberrations in specific positions relative to the ER membrane. In ERAD‐L and ‐M, the ubiquitin ligase Hrd1 recognizes proteins with aberrations in ER luminal or transmembrane segments. Hrd1 also functions in ERAD‐T, in which proteins that persistently or aberrantly associate with the translocon are targeted for destruction. In ERAD‐C, the ubiquitin ligase Doa10 recognizes soluble or transmembrane proteins with cytosolic aberrations. In ERAD‐RA, the ubiquitin ligase Rkr1/Ltn1 recognizes translationally stalled ER‐targeted proteins. ER stress occurs when misfolded proteins accumulate in the ER. Multiple human diseases, including Parkinson's disease and diabetes, are characterized by elevated levels of ER stress. The extent to which ER stress contributes to pathology is not clear. In this study, we systematically evaluate the effect of ER stress on ERAD in the model organism Saccharomyces cerevisiae by comparing the abundance and rate of degradation of a panel of model substrate proteins. Our data suggest that the ERAD pathways are differentially affected by ER stress. A better understanding of the effects of ER stress on protein quality control pathways may contribute to an improved understanding of the consequences of ER stress on cellular physiology and facilitate the discovery of therapeutic targets for diseases characterized by increased levels of ER stress.

Support or Funding Information

National Institutes of Health Grant R15 GM111713 (to EMR).

Funds from the Ball State University Provost's Office and Department of Biology (to EMR).

Ball State University ASPiRE research awards (to BWB).
A neuronal‐specific surface‐exposed membrane proteasome complex modulates neuronal signaling through extracellular signaling peptides
Authors: Ramachandran Kapil V , Margolis Seth S
Published: November 2023

Proteasome‐dependent proteolysis is a tightly regulated process that is essential for life in all archaeal and eukaryotic cells. In the nervous system, proteasome function regulates a wide variety of cellular processes that are critical for proper neuronal function. Largely, the roles of proteasomes in the nervous system have been attributed to modulating the levels of proteins critical for particular cellular processes. However, certain rapidly‐occurring processes such as neuronal transmission, vesicle recycling, and calcium signaling are also affected by acute inhibition of proteasome function. It remains unclear how proteasomes can acutely regulate a cellular process within seconds to minutes, as this remains inconsistent with their canonical role in proteostasis. Here, we made the discovery of a nervous system‐specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is tightly associated with the neuronal plasma membrane, exposed to the extracellular space, and is catalytically active. We have determined this complex largely to be made up of 20S proteasome subunits, with little to no 19S cap. Analysis of this membrane proteasome complex by mass spectrometry revealed nervous‐system specific auxiliary membrane glycoproteins which were sufficient for membrane proteasome expression. Selective inhibition of this membrane proteasome complex by a cell‐impermeable proteasome inhibitor blocked extracellular peptide production and attenuated neuronal activity‐induced calcium signaling within seconds. Moreover, membrane proteasome‐derived peptides are sufficient to robustly and rapidly induce neuronal calcium signaling, in part by acting through NMDA receptors. The nature of proteasomal membrane attachment, association with auxiliary transmembrane proteins, exposure to the extracellular space, and capability to signal across the membrane are characteristic of a transmembrane complex. Our discoveries challenge the prevailing notion that proteasomes primarily function to maintain protein homeostasis, and highlight the existence of a form of rapid neuronal communication through a novel transmembrane proteasome complex.

Support or Funding Information

This work was funded by institutional funding and the following grants to S.S.M. (R01 MH102364‐02). K.V.R. was supported by a training grant T32 GM007445 and NSF Graduate Research Fellowship DGE‐1232825.
Repeat expanded Ataxin‐1 mRNA and protein is co‐regulated at PML bodies
Authors: Fanslow Danielle , Cogswell Alexis , Strojny Chelsee , Garza‐Gongora Arturo , Smith Erica , Kosak Steven
Published: November 2023

The trinucleotide repeat disorder Spinocerebellar Ataxia‐1 (SCA‐1) is a neurodegenerative disease characterized by progressive motor neuron deterioration. The disease is caused by an expansion of the CAG tract in the gene ATXN1, resulting in highly structured ATXN1 mRNAs and misfolded Ataxin‐1 protein aggregates. It is becoming clear that aggregation‐prone expanded mutant Ataxin‐1 proteins are degraded through a nuclear protein homeostasis mechanism. However, the regulation of expanded mutant ATXN1 mRNA remains unclear. In the current study we use a mutant Ataxin‐1 with an expanded polyglutamine tract (85Q‐Atxn1) as a model of altered mRNA structure and proteotoxic stress. Our group hypothesizes that nuclear polypeptide synthesis is integrated with a cellular response to stress, specifically responding to the presence of aberrant mRNA secondary structures in the nucleus. Interestingly, we detect nascent polypeptides labeled by puromycin (puro) associated with 85Q‐Atxn1 inclusion bodies as well as ATXN1 mRNA. 85Q‐Atxn1 with an associated puro signal significantly localizes to promyelocytic leukemia (PML) nuclear bodies. PML bodies are multifunctional subnuclear organelles consisting of PML protein and a heterogeneous composition of protein binding partners as well as nucleic acids. PML body composition dynamically changes in response to Atxn1 expression, as shown by a proximity labeling proteomics analysis. Altered PML expression changes puro signal intensity as well as 85Q‐Atxn1 accumulation, suggesting a protective role of PML during expression of 85Q‐Atxn1. Additionally, ribosomal stalk proteins significantly associate with PML and 85Q‐Atxn1. The No‐go decay (NGD) mRNA surveillance mechanism is necessary for the protection against the accumulation of 85Q‐Atxn1 and correlates with PML localized puro accumulation. Overall, our analysis has uncovered a novel regulatory phenomenon in which aberrant mRNAs, protein synthesis, and protein degradation are functionally integrated at PML bodies.

Support or Funding Information

Funding by CMBD Training Grant‐NIH T32 GM08061, NIH DP2 OD008717 to S.K, NIH F32CA189413 to A.A.C, and CBC PDR‐003 to S.K and A.A.C.
Molecular mechanism of JNK attenuation by parkin
Authors: Davis Cameron Wayne , Zhan Xuanzhi
Published: November 2023

Parkinson's disease (PD) is characterized by the disappearing pigmentation, accumulation of lewy bodies, and the ultimate degeneration of neurons in the substantia nigra pars compacta region of the brain. The most prevalent indication of hereditary PD is a mutation in the PARKIN gene. Parkin facilitates the proteasomal degradation of target proteins through its ubiquitination‐catalyzing activity. The E3 ubiquitin ligase has been further implicated in attenuating the activation of the c‐jun N‐terminal kinase (JNK) pathway, a pro‐apoptotic mitogen‐activated protein kinase (MAPK) signaling cascade, implying neuroprotective characteristics. However, the mechanism whereby parkin regulates the JNK pathway remains obscure. Utilizing in vitro pull‐down assays in tandem with co‐immunoprecipitation methods, we have identified a novel interaction between parkin and apoptosis signal‐regulating kinase (ASK‐1), an upstream kinase in the JNK pathway. This interaction was characterized through co‐overexpression in HEK293T cells, which indicated the parkin‐induced degradation of ASK1. In vitro ubiquitination assays showed an enhanced activation of parkin in the presence of ASK1. Our results suggest the interaction with ASK1 is able to disrupt the intrinsic auto‐inhibition of parkin, enhancing its activation to carry out the ubiquitin‐signaled degradation of ASK1.

Support or Funding Information

Tennessee Technological University Chemistry Department Student Research Grant
Which polyubiquitin polymer(s) is responsible for mitophagy in yeast cells?
Authors: Hoskins Tyler Dayton , Cooper Eric
Published: November 2023

Families have been identified in which individuals develop Parkinson's disease at very early ages (~ 21–40 years old). Genetic studies determined that these patients possess defective versions of a gene named Parkin, which encodes an enzyme that targets damaged mitochondria for degradation via a process termed mitophagy. Specifically, Parkin assembles a polymer, or chain, of a protein called ubiquitin on specific proteins lining the surface of mitochondria, which then “flags” it to be engulfed into a vacuole and digested. Ubiquitin can be assembled into seven different types of polymers, and we are using yeast, a simple and commonly used laboratory organism, to determine which one(s) are necessary for mitophagy induction. To do so, we are adapting an established yeast mitophagy assay (), which requires us to introduce a fluorescent version of the mitochondrial gene OM45 into yeast strains incapable of assembling each different type of ubiquitin polymer. We are monitoring mitophagy in the yeast cells using a common analytical technique called Western Blotting, and anticipate that if we can determine the ubiquitin polymer responsible for mitophagy in yeast, we can extrapolate our results to mitophagy induction in human cells.
Proteasome Activation via a Functional Switch of the Rpt6 C‐terminal Tail Following Chaperone‐dependent Assembly
Authors: Park Soyeon , Li Frances , Sokolova Vladyslava
Published: November 2023

In the proteasome, the proteolytic 20S core particle (CP) associates with the 19S regulatory particle (RP) to degrade polyubiquitinated proteins. Six ATPases (Rpt1‐Rpt6) of the RP form a hexameric Rpt ring and interact with the heptameric α ring (α1–α7) of the CP via the Rpt C‐terminal tails individually binding to the α subunits. Importantly, the Rpt6 tail has been suggested to be crucial for RP assembly. Here, we show that the interaction of the CP and Rpt6 tail promotes a CP‐Rpt3 tail interaction, and that they jointly mediate proteasome activation via opening the CP gate for substrate entry. The Rpt6 tail forms a novel relationship with the Nas6 chaperone, which binds to Rpt3 and regulates the CP‐Rpt3 tail interaction, critically influencing cell growth and turnover of polyubiquitinated proteins. CP‐Rpt6 tail binding promotes the release of Nas6 from the proteasome. Based on disulfide crosslinking that detects cognate α3‐Rpt6 tail and α2‐Rpt3 tail interactions in the proteasome, decreased α3‐Rpt6 tail interaction facilitates robust α2‐Rpt3 tail interaction that is also strongly ATP‐dependent. Together, our data support the reported role of Rpt6 during proteasome assembly, and suggest that its function switches from anchoring for RP assembly into promoting Rpt3‐dependent activation of the mature proteasome.

Support or Funding Information

Boettcher Foundation
Nitrogen Starvation and Rapamycin both Induce Autophagic Degradation of Proteasome Complexes
Authors: Roelofs Jeroen , Waite Kenrick A , Vontz Gabrielle , De La Mota‐Peynado Alina
Published: November 2023

Under conditions of nitrogen starvation yeast cells need to degrade proteins to obtain nutrients that can be dedicated to the synthesis of proteins involved in crucial cellular processes. Two systems in the cell are responsible for the degradation of most cellular proteins, the ubiquitin‐proteasome system and the lysosome/vacuole, and thus could contribute to this process. The proteasome is a 2.5 MDa complex that consists of a core particle (CP) that harbors the proteolytic active sites within a cylindrical structure, and one or two regulatory particles (RPs), that control substrate binding, unfolding, and entry of substrates into the CP. We reported recently that proteasomes themselves are degraded upon prolonged nitrogen starvation. Using GFP‐tagged proteasome subunits we showed proteasomes are targeted to the vacuole and that this depends on the core autophagy machinery. This degradation, however, does not simply reflect general bulk autophagy of cytosolic proteasomes, because we observed that nuclear proteasomes are exported out of the nucleus upon nitrogen starvation. Furthermore, starvation induces a dissociation of proteasomes into CP and RP. Both subcomplexes are targeted to the vacuole, but depend on different factors; only CP autophagy required the deubiquitinating enzyme Ubp3. Ubp3 is not required for bulk autophagy, further confirming the model that proteasomes are specifically targeted to the vacuole. The next goal of our lab is to understand the signaling pathways and mechanisms that are responsible for the nuclear export and vacuolar targeting of proteasomes. One major signaling pathway inhibited upon nitrogen starvation is the target of rapamycin complex 1 (TORC1) pathway. Surprisingly, it was recently reported by Rousseau and Bertolotti (Nature, 2016) that an inhibitor of this pathway, rapamycin, actually induces an upregulation of proteasomes. Similar to their data, we observe an initial upregulation of proteasome levels and activity upon rapamycin treatment. However, prolonged exposure results in vacuolar targeting and degradation of proteasomes similar to what we reported upon nitrogen starvation. We observed several differences between nitrogen starvation and rapamycin treatment, indicating the autophagic degradation of proteasomes upon nitrogen starvation does not solely depend on the mTORC1 pathway.

Our work demonstrates that in addition to the well‐characterized transcriptional upregulation of genes encoding proteasome subunits, cells are also capable of down regulating cellular levels of proteasomes by targeting them for autophagy. Characterizing the pathways responsible for degradation of proteasomes upon nitrogen starvation or under conditions of superfluous, damaged, or misassembled proteasomes will be important to understand how cells regulate proteolytic capacity and protein homeostasis in the cell.

Support or Funding Information

NIH‐NIGMS 1R15GM112142 and R01GM118660
Inhibition of Eurygaster Integriceps Puton Prolyl Endoprotease (spPEP) and Human Prolyl Endopeptidase (hPEP) using αS1 ‐ Casein Peptide Inhibitors
Authors: Kadakova Petra
Published: November 2023

Prolyl endopeptidases (PEPs) are widely distributed among all domains of life (Di Cera., 2009). The focus of this study is mainly on prolyl endoprotease originated from the salivary glands of Erygaster integriceps Puton (Sunn pest) identified as spPEP by Darkoh (2008, 2010). The widely spread Eurygaster genus represents the most damaging and highly adaptable wheat pest in Northern Africa, Eastern Europe, Middle East with increasing concerns in Australia and United States. The natural substrate for spPEP is gluten from wheat grains, making spPEP the first known eukaryotic PEP capable of degrading substrates longer than 30 amino acid residues. To study the binding mechanism of this unique enzyme, recombinant peptide inhibitors were generated from αS1‐casein containing the inhibitory sequence LNENLLRFFVAPFPEV (Juillerat‐Jeanneret et al., 2011) varying in the length of the peptide and the positioning of the inhibitory sequence. All peptides were constructed with an N‐terminal 6x His Tag for purification as well as a Thrombin protease site for His Tag cleavage, ligated into pET15b vector and transformed into BL21(DE3)pLysS expression host (Vishram.,2016). The expression and purification of the peptides, including thrombin digestion and HPLC for final purification and quantification are shown. Enzyme kinetics for each of the recombinant inhibitory peptides in the presence of GPpNA as substrate is compared between spPEP and human prolyl endopeptidase (hPEP). This is the first study to show the hPEP to bind a peptide longer than 30 amino acid residues as well as inhibition of spPEP with large peptides.
Enhancing efficacy of protease drugs through site‐directed mutagenesis
Authors: Amorello Angela Nicole , Batt Anna , Baird Teaster
Published: November 2023

A modern approach to drug development draws from nature to treat disease. Proteases, historically targeted for the development of inhibitors, show therapeutic potential. Development of protease therapeutics is complicated by the presence of endogenous inhibitors, which result in reduced serum half‐lives of these agents. Determining which residues influence inhibitor binding can inform the design of efficacious protease therapeutics that have less favorable interactions with inhibitors through mutagenesis. Trypsin‐fold serine proteases account for the majority of commercially available protease therapeutics. Consequently, substitutions that confer inhibitor resistance without compromising activity in trypsin may be applied to prospective serine protease drugs. Examination of crystal structures of trypsin and its macromolecular inhibitors have shown a conserved hydrogen bond interaction between Tyr 39, Lys 60 and inhibitor. Using PCR mutagenesis, three trypsin variants were produced with amino acid substitutions at position 39 (Y39E, Y39S, and Y39L). We have demonstrated that certain Tyr 39 substitutions confer weaker enzyme‐inhibitor interactions. Y39A trypsin had a lower association rate (4.1 × 10‐3 μM‐1 s‐1) and higher dissociation rate (3.3 × 10‐3 s‐1) compared to wild type (5.5 × 10‐3 μM‐1 s‐1 and 2.5 × 10‐3 s‐1) respectively with bovine pancreatic trypsin inhibitor and also demonstrated catalytic activity similar to that of wild type. Substitutions at position 39 in trypsin show promise to retain catalysis and reduce inhibition.
Identifying a serine protease network involved in ovarian cancer progression using activity‐based protein profiling (ABPP)
Authors: Mehner Christine , Hockla Alexandra , Radisky Derek C , Radisky Evette S
Published: November 2023

Ovarian cancer remains a devastating disease with 5 year survival rate in advanced stage of only 27%. Advanced stage tumors metastasize through continuous shedding of tumor cells into the peritoneal fluid, aided by the resistance of these cells to anoikis, a form of apoptosis normally triggered by loss of cell‐matrix interactions. We have previously shown that the serine protease inhibitor SPINK1 activates anoikis resistance in ovarian cancer cells via its protease inhibitory function; however, the specific relevant protease target(s) remain unknown. To find these targets, we have synthesized an activity‐based protein probe with a diphenylphosphonate warhead that covalently modifies serine proteases, with greatest specificity towards trypsin‐like proteases. We have used this probe to identify ovarian cancer cell secreted and membrane‐bound serine proteases possessing differential activity in the presence versus absence of SPINK1, using a proteomics approach. We are evaluating candidate proteases for involvement in anoikis resistance in ovarian cancer models. Our studies implicate a serine protease network in regulation of anoikis in ovarian cancer cells. By identifying critical proteases that control anchorage‐independent viability in ovarian cancer metastasis, we aim to elucidate important downstream pathways and ultimately develop therapeutic approaches that can intervene in mechanisms of anoikis resistance.

Support or Funding Information

Mayo Clinic Graduate School, Rochester MN
Structural and Biochemical Insights into the Activation and Substrate Selectivity of Clostripain‐like Proteases Secreted From Commensal Gut Bacteria
Authors: Roncase Emily J , O'Donoghue Anthony J , Wolan Dennis W
Published: November 2023

Commensal gut bacteria secrete a broad variety of biological and chemical signals to influence intestinal homeostasis and modulate immune responses, including active proteases. Enteric cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria and they play a crucial role in intestinal inflammation as they cleave host proteins, activate cell surface receptors, and disrupt epithelial barrier integrity. Recently, members of the clostripain‐like (C11) family of cysteine proteases were shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. We have structurally and functionally characterized two additional members of this family from the predominant gut symbiont Bacteroides thetaiotaomicron: BT1308 and BT0727. These secreted proteases are predicted to be packaged into outer membrane vesicles capable of interacting with host epithelial cells and are known to be activated through internal cleavage events. We verified the necessity of self‐cleavage for maturation, showed this cleavage event occurs in trans, and identified the sites of cleavage following designated arginine residues. Using Multiplex Substrate Profiling by Mass Spectrometry we identified a unique cleavage motif of X‐X‐T/S‐K/R common among C11 proteases and determined protease‐specific differences in P3–P4 recognition. To supplement this biochemical data, we solved the structures of both active enzymes in complex with a tetrapeptide inhibitor at under 2.4Å resolution, and these structures highlighted subtle variations in active site architecture that confer selectivity in substrate recognition. Furthermore, comparing our structures to an inactive mutant of a C11 protease from Parabacteroides distasonis revealed that proteolytic activity could be dependent on a conformational change that reorients the active site histidine upon self‐cleavage. Our findings provide insight into the mechanism and specificity of C11 protease activity and will aid in the rational design of protease‐specific probes that can be used to interrogate the role of C11 proteases in inflammation and bacterial fitness.

Support or Funding Information

Boehringer Ingelheim, National Science Foundation
The effect of disrupting a distal hydrogen bond network on serine protease function
Authors: Villa Rodolfo , Baird Teaster
Published: November 2023

Engineered serine proteases constitute a distinct therapeutic class with a potential for diverse clinical applications. One main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. However, if a protease is to be effective as a therapeutic agent, it needs to display resistance towards inhibition. Given that the majority of protease therapeutics currently used are of the trypsin fold, trypsin itself serves as an ideal structure‐function relationship model to address this issue. Previous studies, using small synthetic peptides, bovine pancreatic trypsin inhibitor (BPTI) and an ecotin variant (M84R ecotin) with engineered trypsin variants have shown that prime side interactions can affect association with macromolecular inhibitors through interaction with residues at positions 39 and 60. The K60A trypsin variant (K60A‐Tn) had the lowest amount of free enzyme in equilibrium inhibition studies while using BPTI, but the opposite was observed with M84R ecotin. There is evidence that increased mobility of Y39 caused the K60 substitution to impact P4′ interactions and P2′ hydrogen bond, resulting in altered trypsin:inhibitor interactions. Specifically, replacing lysine‐60 with alanine does not allow the K60 and Y39 charged hydrogen bond interaction regardless of the P4′ hydrogen bond presence, suggesting that a polymorphism at position 60 may influence interaction between Y39 and inhibitors. However, given that BPTI and ecotin are also proteins, their interaction with trypsin likely reflects the interaction that serine proteases will have with natural protein substrates. Therefore, a desired weakened interaction between the engineered trypsin and the inhibitor may also suggest a similar weakened interaction between the engineered trypsin and protein substrates which cannot be observed with the small peptide substrates that were used previously. Our focus in this study is to use a protein as a substrate, anticipating insight on the enzyme‐substrate interaction via catalytic activity of K60A‐Tn compared to wild‐type trypsin. Without the K60 and Y39 charged hydrogen bond interaction, we predict to see weakened substrate affinities, causing lowered catalytic efficiency.

Support or Funding Information

Dr. Teaster Baird National Institute of Health (NIH)
Site‐Directed Mutagenesis of Recombinant Aedes aegypti Trypsin‐like Serine Proteases II and IV (AaSPII and AaSPIV)
Authors: Eilerts Diane F , Rascon Alberto A
Published: November 2023

Aedes aegypti mosquitoes act as vectors of Dengue fever, Zika, yellow fever, and Chikungunya viruses. The female Ae. aegypti mosquito uses blood meal proteins to obtain nutrients required for the egg development process. Inhibition of specific proteases responsible for blood meal digestion may allow for novel methods of vector control. Characterization of newly identified Ae. aegypti serine proteases such as AaSPII and AaSPIV is necessary to gain insight into the complex mechanism of mosquito blood meal digestion. Recombinant expression of Ae. aegypti trypsin‐like serine proteases II and IV (AaSPII and AaSPIV) was achieved in preparation for protein purification. Preliminary results obtained from growth experiments indicate that these proteases are likely autocatalyzing and autodigesting. Site‐directed mutagenesis was successfully performed in order to introduce a point mutation at the active site serine residue to an alanine. This change should yield an inactive protease and help determine if our hypothesis of autocatalytic activation is true. Future studies will focus on purification of inactive recombinant enzyme and structural studies.

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SC3 Research Grant from NIH
Overexpression and Characterization of the rquA Gene Product Involved in the Biosynthesis of Rhodoquinone
Authors: Zander Alison , Shepherd Jennifer
Published: November 2023

Infections caused by parasitic helminths are sometimes referred to as “the great neglected tropical diseases.” Endemic to many countries, including the United States, parasitic helminths are at the origin of the diseases most commonly diagnosed in developing countries. Affecting both humans and livestock, these helminths are the target of extensive research as their genome is further elucidated. The drug products that currently exist to combat infections are subject to parasitic resistance; therefore, the race to discover different drug‐targets specific to these parasites is essential. In an effort to develop an answer to this problem, we have been researching the rhodoquinone (RQ) biosynthetic pathway. RQ is the final electron transporter in the anaerobic fumarate reductase pathway, which occurs in the mitochondrial and plasma membranes. Although its biosynthetic pathway is still largely unknown, it has been discovered that the gene known as rquA is required for RQ biosynthesis in the model organism, Rhodospirillum rubrum. Expression and purification of the gene product of rquA (RquA) could provide a target for inhibition of RQ biosynthesis. Additionally, determining the crystalline structure and any proteins that are in a complex with RquA could further information in development of a novel drug. After isolation and purification of RquA from overexpression in E. coli, a pull‐down experiment was performed using nickel‐coated magnetic beads to bind the protein (containing a hexahistidine tag). Following this, the bead protein complex was combined with lysate collected from solubilized wild‐type R. rubrum membranes. Providing these conditions for the protein allowed RquA to complex with any other proteins that it may interact with in its normal state. Upon SDS‐PAGE gel electrophoresis, and staining with Coomassie blue, there was evidence of at least 4 proteins that appear to be associated with RquA.

Support or Funding Information

National Institutes of Health (1R15GM096398‐01) and Howard Hughes Medical Institute (award to Gonzaga University)
The Structure and Function of Sulfite Reductase Flavoprotein: Three Faces of a Dynamic Enzyme
Authors: Askenasy Isabel , Andrews Rachel , Stroupe M. Elizabeth
Published: November 2023

In enterobacteria like Escherichia coli, NADPH‐dependent assimilatory Sulfite Reductase (SiR) is the central enzyme in the sulfur assimilation pathway. SiR performs the six‐electron reduction of sulfite (SO3−2) to sulfide (S−2). SiR is a soluble multimeric enzyme with two subunits that combine in a 2:1 ratio to form a stable dodecameric complex. The α subunit (SiRFP) is a cytochrome P450 oxidoreductase (CYPOR) homolog. The β subunit (SiRHP) is a monomeric metalloenzyme with a Fe4S4 cluster and a porphyrinoid group called siroheme that forms the active site for the complex. SiRFP has the prototypical, three‐domain architecture of CYPOR that transfers electrons from the cellular donor NADPH to its FAD and FMN cofactors sequentially, ultimately delivering them to the binding partner, in this case SiRHP. However, SiRFP differs from other CYPORs in several ways: 1) SiRFP is a soluble protein that forms a homo‐octamer when expressed in the absence of SiRHP and 2) SiRFP forms a stable complex with SiRHP. The main aim of our research is to understand the electron transfer between the subunits in the context of the unique stoichiometry and structure of the complex. We identified two regions of SiRFP whose solvent accessibility changes upon assembly with SiRHP. The two regions of the structure are on different faces of the protein: one is in the N‐terminal FMN‐binding domain and the other is in the C‐terminal NADPH binding domain. Electrons are known to pass from the FMN binding domain to SiRHP. Interestingly, the interface that is part of the NADPH binding domain is sufficient to form a tight complex with SiRHP, suggesting the interface within the FMN binding domain is transient. To test this hypothesis, we synthesized three point variants: Y101A, Q191K, and V500D. Y101 is part of the FMN binding domain. Q191 plays a role in the conformational change that is known to govern electron transfer in CYPOR. V500 is part of the SiRFP region that interacts with SiRHP. Then, we used complementation assays and isothermal titration calorimetry (ITC) to probe the effect of these mutations. The three combined mutations has a synergic effect, resulting in cells that were unable to grow in minimal media. The ITC data showed that only the SiRFP V500D and variants combined with it had a reduced affinity for SiRHP respect to the wild‐type enzyme. From these results we conclude that SiR complex formation is driven by hydrophobic interactions in the C‐terminus of SiRFP, far from the site of electron transfer to its SiRHP partner. In contrast, transient interactions involving Y101 are important for electron transfer from the FMN cofactor to the metallic cofactors in SiRHP.

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MCB1149763
Cofactor Analysis of Nicotinate Dehydrogenase and 6‐Hydroxynicotinate Dehydrogenase in Bacillus niacini
Authors: Sherer Laura Ashley , Snider Mark Ashley , Pandelia Maria‐Eirini , Rowlett Roger
Published: November 2023

Prevalent in many pharmaceuticals, dyes, and herbicides, N‐heterocyclic aromatic compounds have recently been recognized as environmental contaminants of emerging concern. Understanding microbial degradation of N‐heteroaromatics may inform bioremediation efforts to remove or neutralize these contaminants. Nicotinic acid (NA, vitamin B3) is considered a model compound, and consequently, studying bacterial catabolism of NA may help elucidate the degradation of N‐heteroaromatics as a whole. In this study, we investigated nicotinate dehydrogenase (NDH) and 6‐hydroxynicotinate dehydrogenase (6‐HDH) from Bacillus niacini, an aerobic soil bacterium. These enzymes hydroxylate NA and 6‐hydroxynicotinic acid (6‐HNA), respectively, catalyzing the first two steps of NA catabolism. To accomplish this chemistry, computational predictions suggest that NDH and 6‐HDH each contain two [2Fe‐2S] clusters, a molydopterin cytosine dinucleotide (MCD), and a FAD coenzyme. The subunits of these enzymes were purified from E. coli and analyzed for their respective cofactors. The [2Fe‐2S] cluster subunit was found to contain 3.9 mol of iron, and the FAD subunit was visibly yellow and found to contain FAD as predicted. In the purified molybdenum subunits, preliminary investigations suggest a 1:10 occupancy of MCD. In order to increase this occupancy, ongoing studies are using Pseudomonas strains as the recombinant host.

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Acknowledgments: Whitmore‐Williams Science Scholarship, the Clare Boothe Luce Program, and the Department of Chemistry at The College of Wooster