bluuebunny/crossref_metadata_2025 huggingface dataset:
Stress‐triggered phase separation, tuned by unusual features of an intrinsically disordered region, promotes cellular fitness during stress
Authors: Riback Joshua A , Katanski Chris , Kear‐Scott Jamie L. , Sosnick Tobin L. , Drummond D. Allan
Published: November 2023

In eukaryotic cells, heat shock triggers the accumulation of proteins into stress granules marked by poly(A)‐binding protein, or Pab1 in yeast. Formation of stress granules has been reported to involve a liquid‐liquid phase separation via multivalent interactions promoted by intrinsically disordered regions (IDRs). Here we report that Pab1 autonomously phase separates into a protein‐rich gel in vitro in response to physiological perturbations such as heat and acid. Pab1's highly conserved IDR, the proline‐rich “P domain”, tunes but is not necessary for the phase separation in vivo and in vitro. Evolutionary analysis of the P domain reveals compositional selection for low charge (<10%), a unique selection on its aliphatic residues, and a conserved above‐average hydrophobicity compared to other folded and disordered domains. We find that the domain collapses but is disordered by SAXS, CD and NMR. Collapse is robust, retained upon sequence randomization or substitution of all glycines (14% of residues) to prolines (from 19% to 33%) or alanines (from 12% to 26%). Of the mutations tested, only those to aliphatics or aromatics that lessen the IDR's hydrophobicity perturb its collapse, in contrast to expectations from studies of polar tracts (ie polyQ). Additionally, while previous results suggested that phase separation involves IDR‐IDR interactions between polar, aromatic, and charged residues, we find that hydrophobic interactions from aliphatic residues in the P domain modulate phase separation in vivo and in vitro. Mutations that reduce Pab1's heat‐triggered phase separation also reduce cells' ability to grow at during heat stress, indicating that phase separation is adaptive. Our results indicate that Pab1's heat‐induced phase separation represents a largely autonomous, evolutionarily tuned self‐assembly response to stress.
A role of deubiquitinating enzyme Ubp3 in coping with oxidative stress
Authors: Jin Xin
Published: November 2023

Ubiquitination plays important roles in regulating numerous cellular processes. In response to extracellular and intracellular stimuli, deubiquitinating enzymes (DUBs) cooperate with E3 ubiquitin ligases to control the level and the type of ubiquitination on cellular substrates. Thus, DUBs are emerging as important regulators for cellular processes. Here, we show that Ubp3, a deubiquitinating enzyme in yeast, is required for cell survival under oxidative stress. The ubp3 deletion mutant exhibits increased sensitivity to the treatment of hydrogen peroxide. Notably, the mutant phenotype can be rescued by wild type Ubp3 but not a Ser695Ala mutant form of Ubp3, suggesting a potential role of phosphorylation of Ubp3 by MAPK in this process. Consistent with this model, we find that Mpk1 interacts with Ubp3 and disrupting Mpk1 but not any other MAPK in yeast similarly renders cells more sensitive to hydrogen peroxide treatment. Our results suggest phosphorylation and activation of Ubp3 by Mpk1 as a potential new mechanism for cells to cope with oxidative stress.
Effects of VACM‐1/Cul5 Gene Knockout on Cellular Proliferation Using CRISPR‐Cas9 Approach
Authors: Gager Christopher , Rios Santiago , Cunningham Mark , Burnatowska‐Hledin Maria
Published: November 2023

The VACM‐1 gene codes for the VACM‐1/Cul5 protein, which is a part of the ubiquitin E3 ligase system. This system is utilized to degrade cellular proteins. VACM‐1/Cul5 expression is shown to decrease cellular proliferation. Lack of regulation in this pathway can lead to cancer. We utilized CRISPR‐Cas9 to knockout the VACM‐1 gene. The CRISPR system is a bacterial immune system that functions by targeting specific sequences of DNA. It can be programmed to target genes of interest, enabling specific gene editing in eukaryotic cells. We have used this system to knockout VACM‐1/Cul5 in a human umbilical vein endothelial cell line (HUVEC). Growth assays indicate that VACM‐1/Cul5 knockout allows cells to proliferate at an increased rate. We also explored whether CRISPR‐Cas9 knockout of VACM‐1/Cul5 compromises the antiproliferative effects of resveratrol. Our results indicate that VACM‐1/Cul5 knockout cells do not respond to these drugs in the same manner as normal HUVEC cells, and experienced more rapid growth. We also aim to prove gene knockout via genomic analysis, as well as to use microarrays to explore possible pathways impacted by the knockout.
Molecular Chaperones of the Hsp70 Family Interacting with the Proteasome Assembly Network
Authors: Hammack Lindsay J , Firestone Kyle , Kusmierczyk Andrew R
Published: November 2023

Proteins Ssa1 and Ssa2 are members of the HSP70 family. These nearly identical ATPases are well characterized in maintaining protein homeostasis. Their functions include aiding in protein folding and targeting short‐lived proteins for degradation. These proteins are highly abundant and are routinely detected in mass spectrometry data following affinity purification. However, due to their abundance, these proteins are often assigned to groups of proteins that non‐specifically interact with the purification matrix. Perhaps the best known of these collections is the CRAPome, an online repository of common contaminants found in affinity purification. Even though Ssa1 and Ssa2 are part of the CRAPome, we find that Ssa1/Ssa2 interact with assembling proteasomes, specifically with defined subcomplexes of the core particle. Furthermore, our genetic results also indicate that Ssa1/Ssa2 interact with the with the proteasome assembly network. Proteasome assembly has been characterized as a stepwise process that includes dedicated proteasome assembly chaperones. This work supports an active role for the general molecular chaperones, Ssa1/Ssa2, in this process as well.

Support or Funding Information

This work was funded in part by a Research Support Funds Grant from IUPUI (to A.R.K.).
This is SPrTAC: A New Method to Distinguish Multisubunit Complexes with Highly Similar Compositions In Vivo
Authors: Tomko Robert J , Peterson Anna K.
Published: November 2023

Large multisubunit complexes conduct the vast majority of essential cellular processes in eukaryotes. These complexes are often constructed of dozens or even hundreds of highly similar but non‐equivalent subunits. Further, the exact subunit composition of these complexes is frequently altered to fine‐tune their biological activities. For example, changes to the subunit composition of the proteasome promote antigen processing, suppress self‐recognition by immune cells, and lead to chemotherapy resistance in some cancers. Thus, a difficult and pervasive challenge in deciphering the biology of multisubunit machines is distinguishing complexes with non‐identical but extremely similar compositions from one another in vivo. To address this important challenge, we have developed a new approach, called Split Protein‐based Tracking of Assembly or Composition (SPrTAC). This method exploits split protein complementation to produce growth‐based, or potentially, fluorescence‐based readouts for particular subunit configurations, subcomplexes, or the presence of accessory factors within a given multisubunit complex in vivo. We demonstrate the utility of SPrTAC to detect a rare proteasome isoform containing two α4 subunits and whose production is driven by several known oncogenes in humans. Further, we use this approach to show that disruption of subunit expression stoichiometry compromises normal proteasome biogenesis in vivo. This methodology can be applied to any multisubunit complex for which a basic subunit architecture is known, and can be used to monitor the composition and abundance of such complexes with minimal perturbation to cell function.
ScMC2 is a Calcium‐Dependent Metacaspase Cleaving Peptides after Arg Residues
Authors: Scharbach Stephanie N , Fox Kristin
Published: November 2023

Programmed cell death (PCD) is a terminal pathway important in the maintenance of cell homeostasis. Recent studies have shown that PCD can also happen in unicellular organisms. Like all pathways in the cell, PCD must be controlled. Metacaspases have been implicated in the cell death pathway in yeast and are present only in protozoa, fungi and plants. Metacaspases are proteases which are cysteine‐dependent and distantly related to the caspases found in metazoans. Some metacaspases require the presence of calcium for activation. Most caspases have a prodomain that must be cleaved to produce active enzyme.

This project focuses on characterizing Schizophyllum commune metacaspase 2 (ScMC2). To clone ScMC2 with and without the prodomain, the genes were amplified using PCR and fused to small ubiquitin modifier (SUMO) in a vector via Gibson Assembly. ScMC2 and ScMC2Δpro were then expressed and purified using nickel affinity chromatography.

ScMC2 and ScMC2Δpro expressed using these plasmids were found to be active from the activity studies using the substrate Z‐GRR‐AMC. The optimal concentration of calcium in enzyme assays for MC2 was determined to be 25 mM. This is higher than the optimal concentration for the other S. commune metacaspases. ScMC2 and ScMC2Δpro were successfully cloned and when expressed produced an active metacaspase.
Molecular Dynamics and Distance‐from‐Average‐Structure Characteristics of Human Cytochrome CYP2D6
Authors: Colwell Quinton E , Furge Laura
Published: November 2023

CYP2D6 is a major enzyme involved in metabolism of at least 10% of all chemicals including ~12% of pharmaceutical drugs. Understanding of how CYP2D6, and CYPs in general, are able to metabolize such a wide‐range of chemicals despite having a buried active site is partly achieved through understanding the motion of structural regions of the protein relative to one another. However, these conformational dynamics of CYP2D6 are poorly defined in part because structure‐determining techniques (e.g. X‐ray crystallography) are limited to a single conformation per protein crystal structure. The current study uses molecular dynamics in conjunction with analytical methods to overcome this limitation. In particular, distance‐from‐average‐structure measurements were made as a means to understanding the conformational dynamics of ligand unbound structures of CYP2D6 in a dilute, aqueous, simulated in vitro environment. Our results show that the protein conformation is constantly changing in statistically significant ways that support regional movements and an overall “breathing” motion of the protein that may allow for ligand movement to and from the active site. Also, specific secondary structural elements were found to have greater ranges in motion than others. These differences followed the commonsensical trend of loop regions being more dynamic than helical regions, although there was significant variability between similar structural elements. Furthermore, significant intra‐regional variability in the ranges of motion existed for all structural elements. Though more analyses will need to be done to understand the significance of these dynamic differences, overall the conclusions from the analysis support the validity of our molecular dynamic simulations approach.

Support or Funding Information

Support: NIH 1R15‐GM086767‐02 and the F. W. and Elsie L. Heyl, Roger F. and Harried G. Varney, and Hutchcroft Endowments of Kalamazoo College.
Amino Acid Changes in Allelic Variants of CYP2D6 Alter Kinetics and Susceptibility to Inactivation
Authors: Cho Christi W. , Glass Sarah M. , Osorio Victoria M. , Sunden Kyle F. , Fujiwara Rina , Furge Laura Lowe
Published: November 2023

Cytochrome P450s (CYPs) are a superfamily of heme‐containing enzymes that are responsible for the metabolism of many xenobiotics and other small endogenous substrates. CYP2D6 is a major P450 enzyme studied during drug development due to its ability to metabolize ~20% of all pharmaceuticals. Moreover, there are over a 100 allelic variants of CYP2D6 resulting in a wide‐range of drug responses among individuals including cases of adverse drug events. The kinetics and susceptibility to inactivation of four allelic variants ‐ three with a series of distal mutations (*34, *17‐2, *17‐3) and one ultra‐metabolizer (*53), and one active‐site mutant (CYP2D6‐T309A), were investigated in the current study. The variants were expressed in E. coli and characterized with the prototypical substrates bufuralol and dextromethorphan. Decreased activity was observed for *34, *17‐2, and *17‐3 while the increased activity of *53 confirmed it as an ultra‐metabolizer. CYP2D6‐T309A displayed similar kinetic values as reference CYP2D6*1. A new abundant metabolite of dextromethorphan formed by *53 was identified using mass spectrometry. The inactivation of 2D6 variants by a known‐mechanism based inactivator, SCH66712, was investigated in a time‐ and concentration‐dependent manner for *1, *53, and T309A. Though *1 was inactivated by SCH66712, *53 and T309A were not susceptible to inactivation. These findings support the designation of T309A as the nucleophilic target for inactivation of CYP2D6 by SCH66712. The greatly decreased rate of inactivation of *53 suggests that inactivation may be dependent on time and positioning of inactivator in the active site. Computational methods using Molecular Dynamics and AMBER tools were used to gain insights into how individual amino acid changes cause alterations in structural stability and plasticity, and may lead to observed differences in reaction rates and susceptibilities to inactivation.

Support or Funding Information

Support: NIH 1R15‐GM086767‐02 and the F.W. and Elsie L. Heyl, Hutchroft, and Roger F. and Harriet G. Varney Endowments of Kalamazoo College.
Deciphering the Resistance Profile of Cancerous Thymidylate Synthase
Authors: Islam Zahidul , Gurevic Ilya , Saeed Muhammad , Rasool Sobia , Kohen Amnon
Published: November 2023

Thymidylate Synthase (TSase) is an enzyme that converts 2′‐deoxyuridinemonophosphate (dUMP) to thymidylate (dTMP), a base for DNA. Since there is no other de novo route to dTMP in cells, TSase is critical to cell replication and survival. 5F‐dUMP – produced intracellularly from the chemotherapeutic pro‐drug 5‐fluorouracil (5‐FU) – inhibits TSase, thus halting the supply of the DNA base thymidylate, causing cessation of DNA synthesis and leading to thymine‐less cell death. While it is very common for pathogenic organisms to become resistant to certain antibiotic drugs, cancerous cells have also developed some mechanisms to cope with the cytotoxic effect of chemotherapeutic drugs. 1

A variant of human (hs) TSase (Y33H) derived from a primary culture of human colonic tumor was reportedly found to confer resistance to 5‐FU.2 Crystal structures of hs TSase indicate Y33 is a remote residue that does not make direct contact either with the inhibitor or the residues interacting with it. Nevertheless, mutation at Y33 affects the affinity of the inhibitor for the enzyme. To resolve this enigma, we aim to investigate the molecular origin behind the resistance due to this remote mutation. Recombinant WT hs TSase and the mutant Y33H were expressed and purified in E. coli host systems. We structurally and kinetically interrogated WT and the mutant. Our studies revealed that the mutation at Y33 causes subtle changes across the protein that affect the chemical transformations at the active site. This change in protein could account for the low affinity of the mutant for the drug 5‐FdUMP.
Identification of Molecular Interactions within Histone Deacetylase Isozymes for the Development of Selective HDAC2 Inhibitors for the Design of Targeted Cancer Therapies
Authors: May Xavier , Watkins Davita , Stoddard Shana V.
Published: November 2023

Histone deacetylase inhibitors (HDACi) are necessary for the therapeutic intervention of various cancers. HDACi decrease the function of a class of isozymes called histone deacetylases (HDAC). HDAC's are involved in the regulation of gene expression. Various members of this HDAC family are being targeted for therapeutic design. HDAC2 is seen to have elevated levels of expression with colorectal cancer. Our two part docking study compared our nine potential synthetic targets and known HDACi's Belinostat, Mocetinostat, Vorinostat, and Panobinostat within 11 HDAC isozymes to determine which candidates would be the best for synthesis. Additionally, we looked to determine if there are any molecular interactions that can be taken advantage of for the design of a more selective HDACi. ETS4 shows promising signs of in silico selectivity. HDAC2 and HDAC11 both experience binding score values that are 100 times higher than the other receptors. We observed pi‐pi stacking interaction with F155 and F 210 within the core of the receptor, as well as metal ion coordination and multiple hydrogen bonds throughout the receptor for HDAC2. Interestingly, HDAC11 does not have this pi‐pi stacking interaction occur, yet still has a high binding score. The work from this study will help to develop more selective HDAC2 inhibitors for targeted cancer therapies.

Support or Funding Information

William Randolph Hearst Foundation and Rhodes College Department of Chemistry
Inhibition of Cholinesterases by β‐Carboline Derivatives
Authors: Soule Jessica , Horton William , Peerannawar Swarada , Kugyela Nandor , Kulkarni Aditya , Török Béla , Török Marianna
Published: November 2023

Multiple factors contribute to the development of Alzheimer's disease (AD). The cholinergic hypothesis of AD attributes its symptoms to low concentrations of acetylcholine, a neurotransmitter. Concentrations of acetylcholine can be increased through inhibition of the enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Novel compounds with β‐carboline core structure were synthesized with an environmentally friendly method and subjected to Ellman's colorimetric enzyme activity assay. The potency of these molecules as cholinesterase inhibitors were compared to galantamine, a drug currently on the market. In general, the tested β‐carbolines showed negligible effect on the activity of AChE, however, they were highly active in the inhibition of BuChE; over 60% of the studied compounds appeared to be a better inhibitor of BuChE than galantamine. Compounds that inhibited BuChE more than 50% in the preliminary screening at the concentration of 10 mM were further assayed in order to determine their IC50 values. Molecular docking of the most effective compounds in the active site of BuChE, and for comparison of AChE, was also carried out using the Glide module of the Schrodinger package. A few compounds with IC50 in the nanomolar to low micromolar range have emerged as promising candidates for further lead development.
Development and Mechanism of Small‐Molecule SIRT6 Activators
Authors: Klein Mark , Liu Can , Camacho Beatriz , Tang Weiping , Denu John M.
Published: November 2023

Sirtuin 6 (SIRT6) is a histone deacetylase that regulates numerous biological functions including transcriptional repression, DNA repair, and telomere maintenance. SIRT6 imparts its function primarily through deacetylation of histone 3 at lysine 9 and 56 (H3K9 and H3K56) however also functions as a non‐histone deacetylase with targets including GCN5 and CtIP. In recent years our understanding of the complexities of SIRT6 enzymatic and biological functions has expanded and its protective roles against carcinogenesis, non‐alcoholic fatty liver disease, and obesity are now commonly accepted. This mounting evidence implicates SIRT6 as an untapped, yet valuable, therapeutic target in combating numerous human diseases. In the work described here our lab has identified a number of SIRT6 activating compounds through targeted in vitro screening. Subsequent characterization of these molecules suggests that activation of SIRT6 deacetylation occurs through increased affinity for acetylated substrate. In collaboration with Professor Weiping Tang, we performed structure‐activity relationship studies to further develop an identified activator and demonstrated its capacity to activate SIRT6 deacetylation on extracted mammalian histones. Current and ongoing work of this project aims to provide a detailed mechanistic understanding of SIRT6 activation by small‐molecules, evaluate our compounds in cell‐based disease models, and identify endogenous SIRT6 activating compounds.

Support or Funding Information

National Institutes of Health (NIH)

Chemistry Biology Interface Training Program (CBITP)

University of Wisconsin Institute for Clinical and Translational Research (ICTR)
Expression of Keratin in Pichia pastoris for Biopolymer Applications
Authors: Ward Michael A. , Schoepke Andrew , Haverhals Luke M. , Fry Michelle R.
Published: November 2023

Alpha‐keratin is a fibrous, structural protein that can be found in the skin, hair, nails, and horns of mammals. The coiled‐coil structure of α‐keratin affords strength and resiliency to friction and physical stress, making this protein a prime biopolymer candidate for use in the biomedical field. To obtain sufficient quantities of α‐keratin for analysis of this biopolymer and its potential use in the preparation of functional composites, we cloned KRT genes encoding recombinant GFP‐tagged human α‐keratin proteins into a pPICZαC vector for expression in Pichia pastoris. The KRT2‐containing pPICZαC vector has been verified by restriction digest and transformed into GS115, a wild type P. pastoris strain, by electroporation. The resultant transformants are being screened and optimized for expression of the recombinant α‐keratin.
Rational Design of Epitope Binding Monobodies: New Tools for Autoimmune Therapy
Authors: Zaravar AliReza , May Xavier , Stoddard Shana
Published: November 2023

Autoimmune Disease is the misguided targeting of self‐proteins or tissues by immune cells and is the number two cause of chronic long‐term disease. Many therapeutic options for autoimmune disease involve the use of immunosuppressive medicines that reduce the immune system's abnormal response. This approach is not specific to the individual autoimmune disorder and as a result these drugs weaken the entire immune system leaving the patient more prone to infection. Therefore, more targeted approaches to treatment of autoimmune disorders is needed. In our work we are developing an antigen specific approach to autoimmune therapy by hiding the epitope (specific auto‐antibody binding site) on the antigen to which an antibody is binding. Using the model system, idiopathic membranous nephropathy (IMN), we have developed epitope binding nanobodies which cover up the epitope region. Twenty‐five epitope binding nanobodies were designed and docked to the epitope region. Binding scores to the epitope regions of the IMN antigen phospholipase A2 receptor were evaluated. Our results show that several prominent interactions lead to improved binding scores compared to the starting nanobody template. Gel analysis of the protein expression of the ySMB, SH13‐M001, and SH13‐M003 epitope binding nanobody candidates, demonstrates successful protein expression and purification. Future studies underway are ELISA assays to confirm binding of epitope binding nanobodies and their ability to inhibit autoantibody binding in vitro.

Support or Funding Information

William Randolph Hearst Foundation and Rhodes College department of Chemistry
Prolonged Half‐life of Recombinant Immunotoxins in Mice by Adding an Albumin Binding Domain
Authors: Onda Mansanori , Pastan Ira
Published: November 2023

Background

Reconbinant immunotoxins (RITs) are potent anti‐cancer agents, that are now in clinical trials in tumors that express CD25, CD22 or Mesothelin. The entry of antibodies and antibody‐based therapeutics into tumors is affected by their half‐life in the circulation and the size of the protein. A longer half‐life should increase tumor penetration and efficacy.

Goal

Because RITs have a relatively short half‐life in the circulation, we have explored the possibility of adding albumin‐binding domains (ABDs) to change their pharmacokinetics and increase their half‐life in the circulation.

Approach

We have inserted two different ABDs into a recombinant immunotoxin targeting CD25. These modifications can be done genetically, and require no additional step in the modified immunotoxins for the clinical use. We have measured their cytotoxic activities in vitro, half‐life in the circulation and anti‐tumor activity in mice.

Results

Two different expression plasmids were constructed with 2 types of ABDs. These RITs were refolded from the inclusion bodies expressed in E. coli according to the standard procedure used for other immunotoxins in clinical use. M1(dsFv)‐PE38, targeting CD25, was used as the parental molecule. The ABDs were inserted near the C‐treminus of domain III, but before the REDLK sequence essential for toxin activity. The pahamacokinetics experiments in mice showed the prolonged retention of the modified immunotoxins as expected by 3‐fold of the half‐life. Also specific cytotoxic activities of the ABD‐RITs in vitro were 2‐fold less comparing to parental unmodified RIT. Moreover, these ABD‐RITs showed similar level of nonspecific acute in mice in spite of the prolonged retention time. Antitumor experiments indicated that no significant improvement was observed by the ABD addition.

Conclusion

Increasing the half‐life of an immunotoxin by the insertion of an ABD does not increase its anti‐tumor activity. Insertion of an ABD into regions of the molecule that are not necessary for cytotoxicity may overcome this loss of activity.

Support or Funding Information

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Conductive Gel Polymers as an Extracellular Matrix Mimic and Cell Vehicle for Cardiac Tissue Engineering
Authors: Maldonado Marcos A , Bonham Andrew J
Published: November 2023

Cardiomyopathies, diseases of the heart, are one of the major causes of death in the United States, and thus research into preventing and treating these complications is imperative. To do this, further improvements in culturing human tissues/tissue‐derived cells and development of new techniques in tissue engineering are needed, particularly in finding a suitable extracellular environment in which cardiac cells such as cardiomyocytes can be developed and maintained. As such, a great deal of effort has gone into producing polymer mimics of the natural cell environment in properties such as binding sites, stiffness, reactivity, and hydration. Here, we present work on the facile fabrication of high purity gold nanorods with carboxylate functionalization to aid in the development and characterization of conductive polymer scaffolds for cardiac tissue support. Such conductive polymers have been shown to have key roles in properly accommodating the needs of cardiac cells to build functional cardiac tissue constructs and further improve cell retention, spreading, homogenous distribution of cardiac specific markers, cell‐cell coupling and synchronized beating behavior at tissue level. We illustrate high purity gold nanorod synthesis and purification that yields high aspect ratio (> 10:1), stable gold nanorods. These were crosslinked into functional polyethylene glycol‐based scaffolds for cardiomyocyte differentiation from human induced pluripotent stem cells. This conductive extracellular matrix mimic shows great promise in improvement in control of cellular differentiation for cardiac tissue engineering.

Support or Funding Information

Support for this work was provided by the Metropolitan State University of Denver Provost's and LAS Dean's offices.
Translational Top Down Proteomics of KRAS‐driven Cancers and Models Thereof
Authors: Ntai Ioanna , Fornelli Luca , Hutton Josiah , LeDuc Richard , Fellers Ryan , Haverland Nicole , Compton Philip , Whiteley Gordon , Boja Emily , Rodriguez Henry , Kelleher Neil
Published: November 2023

KRAS is a frequently mutated oncogene and activating missense mutations have been shown to have a causative role in tumor formation. More than a third of human colon cancers are driven by KRAS mutations. Here, we integrated genomic, transcriptomic and proteomic information to study isogenic cell lines and primary tumor samples with defined KRAS genotypes. We combined RNA‐seq data with top‐down and bottom‐up proteomic analyses to measure differential expression within the proteome, and KRAS proteoforms specifically. Integration of these three complementary omics datasets provided us with a list of molecular entities showing differential expression in response to the mutational status of KRAS. Overall, the results show a strong enrichment for kinases, consistent with the established effect of KRAS mutations in constitutive activation of the MAPK signaling pathway. In addition, proteins associated with cytoskeleton reorganization seemed to be upregulated in the presence of the KRAS G13D mutant allele. This could be involved in increasing cell motility and lead to a more invasive phenotype. Furthermore, we developed a targeted top‐down assay for the detection and quantitation of endogenous KRAS proteoforms to measure allele‐specific modification patterns. Such quantitative measurements (both targeted and untargeted) provide new insights into colorectal cancer phenotypes through the measurement of cancer‐specific protein variants.

Support or Funding Information

This work was supported by Federal Funds from the National Cancer Institute (Office of Cancer Clinical Proteomics Research), National Institutes of Health, under Contract No. HHSN261200800001E, and was carried out in collaboration with the National Resource for Translational and Developmental Proteomics under Grant P41 GM108569 from the National Institute of General Medical Sciences, National Institutes of Health.
Analysis of proteins and protein interactions by size exclusion chromatography – high resolution mass spectrometry
Authors: Busch Florian M , Sahasrabuddhe Aniruddha , vanAernum Zac , Rivera Brian , Wysocki Vicki H
Published: November 2023

Size exclusion chromatography (SEC) is the most commonly used technique for the determination of protein heterogeneity derived from the presence of multiple biomolecules and their interaction via non‐covalent interactions. On‐line detection of eluting species by native mass spectrometry (MS) provides a clear advantage over the traditionally used UV‐spectroscopic detection as it allows for the direct and unambiguous molecular weight measurement of the eluting species. Here, we demonstrate that the combination of sub 2μm particle SEC separation with high‐resolution mass detection is suitable for the analysis of a wide range of proteins. The possibility to detect as little as 100 ng protein makes it possible to analyze complex mixtures at low samples consumption. Non‐ideal interactions of proteins with the stationary phase as indicated by a non‐linear relationship between the logarithm of the molecular weight and the elution time within the column separation range make it challenging to find standard calibration proteins. We show that a mixture of commercially available proteins can be used to assess and monitor the column performance and reproducibility under MS‐compatible mobile phase conditions. The ability to resolve slight molecular weight changes by high resolution MS makes it possible to detect a variety of biomolecular interactions, which can be further studied by surface induced dissociation (SID) and collision induced dissociation (CID). The suitability of this technique to monitor a protein‐ligand interaction in a protein mixture is demonstrated in the case of the biotin‐streptavidin interaction.

Support or Funding Information

We thank the National Institutes of Health (NIH) for funding [grant R01 GM113658].
Cytotoxicity of Urban Dust and Diesel Exhaust Particulates in Murine Astrocyte Cells
Authors: Fortuna Tyler R , Price Peyton C , Lopez Kevin J , Schwader Andrew J , Mazzer Paula
Published: November 2023

Alzheimer's and related neurodegenerative diseases affect as many as 24 million individuals world‐wide—a number which will increase with our increasing global population. In a recent survey of epidemiological evidence, a panel of experts concluded that the existing data suggest a lower incidence of age‐related dementia in developing regions. A potential link between urban environments and neurodegenerative diseases was found by a series of studies conducted in dogs and humans in Mexico City, Mexico, which found markers of amyloid‐β accumulation and oxidative stress in their brains. Dogs and humans from rural areas did not show these neuropathological findings. These findings suggest that the difference may be due to the heavy airborne particular matter in Mexico City.

We studied the ability of urban dust (NIST SRM 1649a) and diesel exhaust particulate matter (NIST SRM 2975) to induce cytotoxicity and oxidative stress in murine astrocyte cells (ATCC C8‐D1A). Diesel exhaust particulate matter (DEP) and urban dust (UD) have very different organic components, with UD containing a higher concentration of oxidized aromatic compounds. Therefore we hypothesized that these two airborne particulate types would produce different toxicological effects in our astrocyte cells.

LC50 determinations showed DEP to be more toxic with an LC50 of 35μg/mL versus the LC50 of UD at 52μg/mL. A caspase 3 activity assay showed that the enzyme was activated by DEP but not by UD. These data suggest that DEP is inducing apoptotic cell death while UD is inducing necrotic cell death. The difference in cell death suggests that UD could potentially be more harmful in vivo due to the release of intracellular components caused by the rupture of the cell membrane. The necrotic cell death could lead to the oxidative stress found in previous studies of brains form polluted environments.

Support or Funding Information

This work was sponsored by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103443.
Shoc2 Mediates Hematopoietic Signals of the ERK1/2 Pathway
Authors: JANG HYEIN , Jang Eun Ryoung , Morris Ann , Forbes‐Osborne Marrie , Galperin Emilia
Published: November 2023

Activity of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) pathway plays a critical role in normal human development. Aberrant ERK1/2 signaling caused by germline mutations in genes of the Ras‐mediated ERK1/2 pathway results in developmental disorders cumulatively called ‘RASopathies’. Shoc2, the scaffold protein in the ERK1/2 pathway, accelerates ERK1/2 activity by bringing the signaling components of the pathway, Ras and Raf‐1, to close proximity. Noonan‐like RASopathy causing Shoc2 mutations emphasize the significance of ERK1/2 signaling transmitted through the Shoc2 scaffolding module. However, the physiological role of Shoc2 and the specificity of the signals transduced through the Shoc2 scaffolding complex remain largely unknown.

In our work, we took advantage of the vertebrate zebrafish model that is ideally suited for developmental genetic studies due to its optical transparency and ex vivo development. To delineate the developmental ERK1/2 signals mediated through the Shoc2 complex, a translation‐blocking shoc2 morpholino (MO) was injected into 1–2 cell stage zebrafish embryos. The outcomes of the Shoc2 depletion were examined using biochemical methods such as Western blot analysis, RT‐PCR and whole embryo staining/imaging. We found that Shoc2 depletion in zebrafish embryos results in decreased ERK1/2 activity. Shoc2 MO‐injected embryos also possessed a larger yolk, kinked tail, and an enlarged hindbrain. Importantly, Shoc2 morphants displayed dramatically reduced numbers of circulating blood cells and loss of erythropoietic and myeloid lineages. To further understand the role of Shoc2 in differentiation of hematopoietic stem cells, we utilized CRISPR/Cas9 gene editing to generate the Shoc2 zebrafish knockout. 12 germ‐line transmitted alleles were recovered from the mutagenesis. All alleles had lesions in similar locations, causing frame‐shifts around amino acid 225 and introducing aberrant stop codons (TAA or TAG) prior to termination.

Our data demonstrate that Shoc2 depletion affects both waves of hematopoiesis: primitive and definitive. Shoc2 CRISPR zebrafish knockout will now be utilized to examine quantitative changes in the hematopoiesis‐specific ERK1/2 transcriptional program.

Support or Funding Information

Funding sources: National Institute of General Medical Siences, American Cancer Society, American Heart Association
The Response of Histone H3.3 to Chromosomal Misalignment and Missegregation: a Novel Biochemical Pathway Correlated to Cell Cycle Arrest
Authors: Fadness Sela , Day Charles A. , Langfald Alyssa , Hinchcliffe Edward H.
Published: November 2023

The spindle assembly checkpoint monitors proper chromosome alignment before cells can proceed into anaphase. Aneuploidy, a common feature of tumor cells, results from incorrect alignment and attachment of chromosomes passing through the spindle assembly checkpoint and generates missegregation. Missegregation is the gain or loss of chromosomes during mitosis and is caused by either defects in sister chromatid cohesion or kinetochore‐microtubule attachments. The mechanism of how cells monitor and prevent aneuploidy is not entirely understood. The goal of this work is to investigate chromosome misalignment and missegregation during anaphase in mitosis. Our results have led us to identify a biochemical pathway that directly monitors chromosome missegregation and induces cell cycle arrest in response to aneuploidy. Histone H3.3, a variant of canonical Histone H3.1, has five key amino acid substitutions whose mutations are correlated with multiple cancers. Two prevalent mutations in H3.3 cause K27M and G34R amino acid substitutions, both of which flank Ser31. Phosphorylation of Ser31 in H3.3 is required for proper segregation during anaphase in mitosis. This phosphorylation typically occurs at pericentromeric regions (Hake et al., 2005). Chromosomes that lag during anaphase accumulate Ser31 phosphorylation along their arms. This phosphorylation continues after anaphase in both the micronucleus and aneuploid daughter nuclei. In our work presented here, Chk1 kinase phosphorylates Ser31 and is followed by Repoman PP1ϒ dephosphorylation of Ser31. Monitoring H3.3 Ser31 phosphorylation will contribute to our understanding of how aneuploidy is normally monitored and suppressed and will lead to further understanding of chromosome missegregation steps.
Phosphorylated Astrin regulated kinetochore function mediated by TRAIP in mitosis
Authors: Chung HeeJin , Park JiEun , Lee JooRak , Kim Hongtae
Published: November 2023

Protein‐protein interaction post‐translational modification and protein degradation are important for the mitotic progression such as chromosome segregation to opposite spindle poles and the stable attachment of spindle microtubules (MTs) to kinetochores (KTs). Error‐free chromosome segregation required precisely timed search and capture of chromosomes by spindles in early mitosis. However, the molecular mechanism underlying conversions from labile to stable attachment are poorly understood. Here, I show that the sequential action of the kinase Cdk1 (Cyclin‐Dependent Kinase 1) or Plk1 (Polo‐Like Kinase 1), TRAIP (TRAF‐interacting protein), and the phosphatase CDC14A (Cell Division Cycle 14A) controls the spindle attachment of Astrin to KTs.

In previously study, the mitotic spindle protein SPAG5/Astrin phosphorylated during mitosis. However, unfortunately there's no evidence which site of Astrin phosphorylated. This study contains that Astrin is transported into centrosomes by Kinastrin and I identified phosphorylation sites of Astrin at S135 and S249 using PhosphoSite program (http://www.phosphosite.org), and it's phosphorylated by Cdk1 or Plk1. Somehow, what protein loaded Astrin onto the spindle and targeted to KTs is unknown. I found TRAIP as a new Astrin interacting protein by using yeast two hybrid screening and confirm that TRAIP binds and co‐localizes with Astrin during early mitotic progression by immunoprecipitation and immunofluorescence assay. Depletion of TRAIP resulted in hyper‐stabilized and multipolar spindles, accompanied by aberrant chromosomal alignment measured by phosphor‐Histone H3 (p‐Histone H3) staining for Fluorescence‐Activated Cell Sorting (FACS), Live cell imaging observation for time‐lapse microscopy and Fluorescence Loss on Photobleaching (FLIP) experiment. Mechanistically, TRAIP regulates Astrin phosphorylation and targets phospho‐Astrin to KTs, leading to the attachment of spindles to KTs. I also demonstrate that CDC14A dephosphorylates Astrin and therefore, overexpression of CDC14A sequestrates Astrin in the centrosome and results in aberrant chromosome alignment. These comprehensive findings uncover a regulatory circuit for protein targeting to KTs that controls the change of KT composition for stable spindle attachment and chromosome integrity.

In summary, this study reveals a new agent involved in search and capture, the signal messenger phospho‐Astrin, which is generated by Cdk1 and targeted to KTs by TRAIP through spindle MTs, and is involved in stable KT attachment to spindle. It elucidates a key mechanism in mitotic progression. This study provides additional information about both the mechanism underlying bi‐oriented spindle formation and chromosome instability in cancer cells.

Support or Funding Information

This work was supported by National Research Foundation of Korea grants funded by the Korean government (MSIP)(No. 2011‐0030043)
Arsenic and Line‐1 Disrupt Developmental Epithelial to Mesenchymal Transition: Implications for Cardiac Morphogenesis
Authors: Perrera Alec Bennett , Huang Tianfang , Bojang Pasano , Tavera‐Garcia Marco , Reyes‐Reyes Elsa M. , Ramos Kenneth S. , Camenisch Todd D.
Published: November 2023

Chronic exposure to arsenic is linked to increased risk for cancers of the lung, kidney, bladder, and liver. Non‐cancerous disease endpoints such as atherosclerosis, birth defects, metabolic syndrome and hypertension are also associated with arsenic exposure. Many of the organ systems impacted by arsenic require epithelial to mesenchymal cell transition (EMT) to establish and maintain tissue homeostasis. For example, developmental EMT contributes to lung, neural tube and heart morphogenesis. As such, disruption of EMT programming by arsenic can result in birth defects and predispose to diseases later in life. The mechanisms by which arsenic disrupts EMT is not known. Long interspersed element 1 (LINE‐1 or L1) is a retrotransposon of the human and mouse genomes, and recently linked to conotruncal heart defects. Such defects arise from malformation of the outflow tract of the embryonic heart through altered EMT. In the present study, we discovered induction of Line‐1 by arsenic and investigated whether Line‐1 impacts cardiac developmental EMT. An increase in ORF‐1 and ORF‐2 Line‐1 mRNAs was detected, starting 2 hr after exposure to arsenic and peaking at 8 hr. Line‐1 protein is also increased following arsenic exposure with increased dose‐dependent localization of Line‐1 in the nuclear compartment. Furthermore, the transcriptional activity of Line‐1 was induced by arsenic in a time and dose dependent manner. Lastly, using a unique mouse genetic model we found that ectopic Line‐1 expression functionally attenuated cardiac EMT in an ex vivo collagen gel invasion assay. On the bases of these findings we conclude that arsenic and Line‐1 block developmental EMT.

Support or Funding Information

(NIH. ES04940; ES06694; SWEHSC P30ES006694; NIEHS R25ES025494)
Inhibition of microRNA‐17 Promotes the Progression of Thyroid Cancer in a Xenograft Murine Cancer Model
Authors: Yan Yan , Eliason Steve , Ries Ryan , Hong Liu , Cao Huojun , Amendt Brad A.
Published: November 2023

MicroRNAs (miRs) are small non‐coding RNAs that bind to target mRNAs and negatively regulate their expression. The expression of miRs are altered in many cancers and cancer cell types. We previously generated a miR‐17‐92 knockdown mouse model using the Plasmid based MicroRNA Inhibitor System (PMIS). With this new PMIS system we found that thyroid development was abnormal or absence in these mice. Thus, thyroid development requires miR‐17 for normal development. We investigated the role of miR‐17 in SW579 thyroid cancer cells, which are derived from human thyroid tissue. PMIS‐miR‐17, specifically targeting miR‐17‐5p completely inhibited the activity of miR‐17‐5p in SW579 cells. However, inhibiting miR‐17‐5p activity in SW579 thyroid cancer cells also promotes tumor growth in a nude mouse model for thyroid cancer. The tumor formation was due to increase cell proliferation and aberrant gene expression. RNA‐seq data shows knocking down miR‐17 in thyroid cancer cells induces large‐scale changes in miR‐17‐5p target gene expression, including PDGFR and MMP2, which may play oncogenic roles in tumorigenesis. Bioinformatics analyses has identified several new gene expression networks regulated by miR‐17 in thyroid cancer. We have identified several new gene expression mechanisms for miR‐17 and more importantly regulation of miR‐17‐92 expression and processing of the miR‐17‐92 cluster during thyroid development and thyroid cancer.

Support or Funding Information

Iowa Institute for Oral Health Research, College of Dentistry, The University of Iowa, Iowa City, IA
Synthesis of a Mini‐Reporter Construct to Test Gene Transfer of RNA Therapeutics.
Authors: Muralidharan Koushik , Khan Arbaz M , Fuoco Kerriane , Patel Hemangi , Hicks Martin J
Published: November 2023

Glioblastoma multiforme (GBM), a grade IV tumor of the central nervous system, is the most common malignant primary brain tumor in adults, with a median survival of only 14 months. This poor survival rate is due to a lack of efficacy in current therapies, including chemotherapy and radiation, which are limited by the blood‐brain barrier. GBM survival depends on tumor cells to generate new vasculature, which is essential for the exchange of wastes and nutrients. Endothelial cells connect with each other and form the walls of new blood vessels, bridging the gap between the growing tumor mass and the established vasculature of the circulatory system. The membrane receptor that activates this growth is vascular endothelial growth factor receptor 2 (VEGFR2). In our lab, we are developing a novel therapy to alter the VEGFR2 receptor so that it cannot form a bridge with its cognate ligand, vascular endothelial growth factor. Alteration in VEGFR2 so that it cannot form this bridge, blocks its activation and inhibits the development of new blood vessels. We have designed therapies to deliver the genetic sequences of anti‐sense RNA therapy molecules to alter the splicing pattern and expression of the VEGFR2 transcript, creating a soluble VEGFR2 decoy. We have designed and cloned a mini‐reporter‐system that contains the regulatory elements of VEGFR2 splicing. This system measures the efficacy of RNA anti‐sense therapeutics to alter the splicing of the VEGFR2 transcript. The visual marker, eukaryotic green fluorescent protein is used to mimic the natural splicing product, whereas the red fluorescent protein, mCherry detects changes in the the efficacy of our RNA anti‐sense therapy. We use a GBM tissue culture model to measure mini‐reporter and therapy expression. In this manner, the mini‐reporter construct provides a quick and visually measurable test to optimize RNA anti‐sense therapies directed against VEGFR2.

Support or Funding Information

Bristol‐Myers Squibb