bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Protein Translation Initiation by Estrogen
Authors: Holz Marina K
Published: November 2023

Translational control of gene expression is critical for cellular homeostasis. We focus on eukaryotic initiation factor 3 (eIF3), a large translation initiation factor complex composed of 13 subunits (eIF3a‐eIF3m) in human cells. Overexpression of several eIF3 subunits (eIF3a, b, c, h, i, and m) or underexpression of two eIF3 subunits (eIF3e and f) are observed in a variety of malignancies, including breast, prostate, lung, cervical, and gastric cancers.

Estrogens are key hormones regulating the development and function of reproductive organs in all vertebrates, including puberty onset, fertility, and the estrous cycle. Estrogens also play important roles in many non‐reproductive functions in mediating cardiovascular function, bone metabolism, embryonic and fetal brain development, and other critical biological processes. Estrogen signaling is critical for the development and progression of several types of cancer. Estrogen Receptor (ER)a is a nuclear transcription factor that upon binding to its ligand estrogen can promote expression of growth and survival genes, and inhibit expression of transcriptional repressors, antiproliferative and proapoptotic genes.

We hypothesized that in addition its transcriptional role, estrogen‐ERa signaling acts to regulate protein synthesis. We observed that ERa regulates both eIF3 levels and activity, and therefore rearranges the translational machinery to promote the synthesis of specific factors involved in malignant transformation. Our results indicate there exists regulation of eIF3 transcription by ERa. Moreover, estrogen controls the ERa/eIF3 interaction, subsequently directing the assembly of translation initiation complexes and mRNA translation.

In conclusion, estrogen may act as a master regulator of gene expression. In addition to its genomic activity in control of transcription, non‐genomic estrogen signaling regulates protein synthesis, and leads to increased levels of proteins that mediate estrogenic functions.

Support or Funding Information

NIH ‐ R15CA151112; Atol Charitable Trust; American Cancer Society ‐ RSG‐13‐287‐01‐TBE
Mediating Protein Synthesis in Developing Neurons: Netrin Receptor Deleted in Colorectal Cancer (DCC) Binds Eukaryotic Ribosomes to Prevent Translation of Messages Independent of Initiation Mechanism
Authors: Filbin‐Wong Megan E. , Gonen Tamir , Kieft Jeffrey S.
Published: November 2023

Axonal outgrowth and guidance is dictated by a variety of chemotropic signals. Netrin receptors at the growth cones of developing neurons function to attract or repel outgrowth during commissure formation. The dependence receptor Deleted in Colorectal Cancer (DCC) is activated by netrin‐1, and dimerization initiates a variety of responses, including activation of Src tyrosine kinases and focal adhesion kinase. Prior to netrin‐1 activation, the monomeric C‐terminal tail of DCC binds to 80S ribosomes and prevents polysome formation. DCC activation and dimerization releases 80S ribosomes for translation initiation, presumably to promote axonal outgrowth. The C‐terminal tail of DCC is a 35 kDa protein that is composed of three domains (P1–3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). The exact mechanism by which the P1 domain binds rpL5 and inhibits translation initiation is unknown. Our initial goal is to determine whether or not the DCC C‐terminal tail halts 80S ribosomes via a canonical or noncanonical initiation pathway. Titrating the full‐length C‐terminal tail into translation‐competent rabbit reticulocyte, wheat germ, and bacterial lysates, we found that the tail specifically inhibits eukaryotic translation, despite only 57% sequence identity between the rpL5 protein in Arabidopsis thaliana and Homo sapiens. Likewise, the C‐terminal tail inhibits protein synthesis of messages that use both cap‐dependent and internal ribosome entry site translation initiation. Hence, we propose that the DCC C‐terminal tail functions to inhibit translation primarily through binding the ribosome, and not eukaryotic initiation factors. In fact, we have evidence from co‐sedimentation and gel filtration chromatography studies to show that the full‐length tail binds to apo‐80S complexes, potentially inhibiting ribosome‐priming with mRNA in these stalled complexes. Collectively, our results support the idea that the DCC C‐terminal tail inhibits translation independent of the canonical translation initiation pathway, and the mechanism by which it inhibits initiation might be by preventing ribosomes from binding to messages in the first place. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones.

Support or Funding Information

HHMI Janelia Farm Research Campus Visiting Scientists Program
Effect of Transient Helicity of cMyb TAD on its Binding Affinity to the Kix Domain of CBP/p300
Authors: Poosapati Anusha , Borcherds Wade , Carbtree Michael D , Shammas Sarah L , Clarke Jane , Daughdrill Gary W
Published: November 2023

Intrinsically disordered proteins (IDPs) are involved in various protein‐protein interactions and they often experience a coupled folding and binding reaction when they bind to their protein partners. Many IDPs, like the cMyb trans activation domain (cMyb TAD), exhibit transient helical structures in solution that resemble their bound structures. We hypothesize that the levels of transient helicity in the free IDP will affect the binding affinity to their protein partners in a predictable way. Previous studies have shown that increasing the transient helicity of intrinsically disordered p53 tumor suppressor increases the binding affinity to MDM2 and had negative effects on cellular function. We aim to study the effect of changing cMyb TAD transient helicity on its binding affinity to the kix domain of CBP/p300.

cMyb is a hematopoietic regulator which is known to play a role in tumorigenesis. Unlike p53 TAD, cMyb TAD has a high residual helicity in solution in the absence of binding partners. Prolines that flank the N and C‐terminus of this helical region were mutated to alanines to determine whether there was a change in helicity that would affect the binding of Kix. Upon mutation, the increase in helicity had almost no effect on the binding affinity of the cMyb‐Kix complex. Further, mutations that reduce the helicity of cMyb‐TAD were designed to test the effect of reducing transient helicity on the binding affinity to Kix. To achieve this goal, conservative mutations of the solvent exposed amino acids in cMyb were made. We are currently measuring the transient helicity and binding affinity of these mutants with the long‐range goal of identifying a general relationship between changes in transient helicity and binding affinity of IDPs.

Support or Funding Information

This research was supported by funding to GWD by National Institutes of Health (2R01CA14124406‐A1 and 1R01GM115556‐01A1). This work was also supported by funding to JC by Wellcome trust (WT 095195MA) and by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship to MDC.
Investigating the Binding Affinity of the Peptide Humanin and its Analogs to Amyloid Beta
Authors: Esckilsen Daniel Edward , Guttikonda Prathyusha , Iwaniec Brandon William Joseph , Evans Hedeel , Milletti Maria C , Heyl‐Clegg Deborah
Published: November 2023

Mitochondrial dysfunction has been proposed as a trigger and an early event in the pathology of Alzheimer's disease. Humanin is a 24‐residue, mitochondrially‐encoded peptide, known to exhibit strong neuro‐ and cyto‐protective effects in Alzheimer's disease models. Amino acid residues 5–15 of humanin have been previously shown to interact with residues 17–28 of amyloid‐β, the main component of amyloid plaques, found as extracellular deposits in the brains of Alzheimer's patients. As both Amyloid β and humanin are known to activate the same G protein‐coupled receptor, FPRL1, it is likely that humanin exhibits its cytoprotective effects by regulating access of Amyloid β to the receptor ultimately modulating the Erk1/2 signaling cascade. In addition to mitochondrially‐encoded humanin, ten different nuclear‐encoded analogs have been identified with little or no known function. Using cloning, molecular dynamics, peptide synthesis, Western blotting, immunoprecipitation, and tissue culture, we investigate the contribution of certain amino acid residues of humanin to its binding affinity to Amyloid β and their neuroprotective effects on amyloid β‐induced toxicity in PC12 cells.

Support or Funding Information

Provost Research Support Award, The Chemistry Department Sellers Fund, Eastern Michigan University
Biophysical and structural characterization of antigen recognition by the alloreactive HCV1406 TCR
Authors: Wang Yuan M , Baker Brian M.
Published: November 2023

Alloreactivity occurs when organs are transplanted across MHC barriers, and results from T cell recognition of “foreign” MHC proteins. Although alloreactivity has been studied for decades, the underlying molecular mechanisms remain elusive. Previous research identified the capacity to generate allospecific T cell responses via liver transplantation across MHC barriers: the HCV1406 TCR, specific for the NS31406‐1415 epitope from the Hepatitis C virus (HCV) presented by HLA‐A2, expanded when an HLA‐A2−/− host received an HCV‐infected HLA‐A2+ liver. This system provides a model for understanding both general features of TCR‐pMHC specificity as well as the underpinnings of alloreactivity, and the role of the peptide vs MHC polymorphisms in driving alloreactivity. Here, a combination of protein biophysics and structural biology are used to investigate the HCV1406 TCR specificity and allogeneic TCR‐pMHC engagement.
The localization to PML nuclear bodies and stability of TRAIP/RNF206 are controlled by SUMOylation
Authors: Han Yegi
Published: November 2023

TRAIP (TNF Receptor Associated factor Interacting Protein), also known as RNF206 (RING Finger protein 206), is an E3‐ubiquitin‐ligase protein participated in DNA damage signaling, DNA repair pathway and cell cycle progression. Post‐translational modifications of protein are important for stability control, subcellular localization and protein‐protein interaction. SUMO (Small‐ubiquitin‐like modifier) is one of the post‐translational protein modifiers and SUMOylation regulates diverse cellular processes including protein stability control, nuclear‐cytosolic transport transcriptional regulation, cell cycle progression and DNA damage response. In this study, I demonstrated that TRAIP is a new target protein of SUMOylation by western blotting, in vitro SUMOylation assay and immunofluorescence (IF). Using SUMO plot™ and SUMOylation Sites Prediction (SUMOsp) tools, I found putative SUMOylated residues of TRAIP and identified five SUMOylated sites of TRAIP using in vitro SUMOylation assay. I also discovered SUMOylation of TRAIP was crucial for localization to nucleus and PML nuclear bodies by IF. Additionally, SUMOylation of TRAIP prevented ubiquitylation and enhanced its protein stability in MG132 and cyclohexicmide (CHX) treatment experiments. To sum it up, these results demonstrates that SUMOylation regulates the subcellular localization and degradation of TRAIP. Recent studies show that TRAIP may play a role as a tumor suppressor. These findings improve the knowledge and clinical application of TRAIP for cancer therapy.

Support or Funding Information

This research was supported by Global PH.D Fellowship Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF‐2016H1A2A1909739) and by the Korean government (MSIP)(No. 2011‐0030043).
Role of sumoylated SOD2 in alcoholic liver disease and liver cancer
Authors: Spissu Ylenia , Cossu Carla , Floris Andrea , Tomasi Maria lauda
Published: November 2023

Background

Manganese superoxide dismutase (MnSOD, SOD2) is the most important mitochondrial enzyme involved in the defense against reactive oxygen species (ROS) and it is well demonstrated to be a key protein in hepatocellular carcinoma (HCC) progression and in other several cancers development as colon adenocarcinoma. Alcohol abuse is one of the main causes of alcoholic liver disease (ALD) and represents the major risk factor of HCC development. Alcohol abuse generates high level of ROS as a consequence of increased enzymatic activity during the ethanol metabolism. SOD2 plays a crucial role in the build‐up of tolerance to ethanol catalyzing the dismutation of O2•− in H2O2. SUMOylation is a post‐translational modification that regulates a wide range of cellular processes involving the covalent attachment of a member of the SUMO (small ubiquitin‐like modifier; SUMO‐1, SUMO‐2 and SUMO‐3) family of proteins to lysine residues in specific target proteins via an enzymatic cascade. Increased ROS production enhances the global protein sumoylation profile. Our aim was to examine whether sumoylation could regulate SOD2 function in ALD and in HCC elucidating the molecular mechanism(s).

Methods

Chromatography by SUMO binding columns was performed to purify sumoylated proteins form total livers of Binge ethanol fed mouse model. Total liver, primary mouse hepatocytes, AML12, HepG2 cells (HCC cell line) and Huh7 cells were used to perform in vitro experiments. Protein and mRNA levels were measured using Western Blotting and RT‐PCR, respectively. SOD2 activity was measured by tetrazolium salt commercial kit and by native gel modified method.

Results

Using sumoylated purified‐proteins, we found that SOD2 is sumoylated in Binge ethanol fed mouse liver. In vitro experiments showed a different trend: ethanol treatment lowers SOD2 protein, mRNA levels and activity in total liver and primary mouse hepatocytes. In HepG2 cells SOD2 activity increases after ethanol treatment. Interestingly, SUMO‐3 silencing prevents ethanol stress‐damage increasing the SOD2 enzymatic activity raising protein stability in primary hepatocytes. In contrast, knockdown of SUMO‐1 lowers SOD2 enzymatic activity and protein level indicating that SUMO‐1 may plays an important role in the oxidative stress prevention.

Conclusion

We report for the first time that ethanol increases sumoylation of SOD2. Furthermore, in AML12 we demonstrated that sumoylation of SOD2 may control ethanol‐induced ROS induction. In addition, SUMO‐1 and SUMO‐3 control SOD2 protein stability and enzymatic activity in mouse hepatocytes and HepG2 cells. This new finding opens a new area of investigation examining the importance of SOD2 sumoylation in ALD and HCC, maybe suggesting SOD2 as potential therapeutic target to protect against induced‐oxidative stress liver damage.

Support or Funding Information

Supported by NIH grant 5 K01 AA022372‐04(Maria Lauda Tomasi)
Inhibition of Sodium Hydrogen Exchanger 1 Palmitoylation is Associated with Suppression of Cell Migration
Authors: Hovde Moriah J , Kooiker Amanda J , Rastedt Danielle E , Provost Joseph J , Vaughan Roxanne A , Wallert Mark A , Foster James D
Published: November 2023

The sodium hydrogen exchanger isoform 1 (NHE1) is involved in many cellular processes including regulation of intracellular pH, coordination of cell migration, control of cell volume, and anchoring protein interactions at the cell membrane. NHE1 is integral plasma membrane protein with 12 transmembrane spanning domains that exchanges an intracellular proton for an extracellular sodium ion. NHE1 is implicated in neoplastic transformation leading to tumorgenesis where the exchanger localizes to the leading edge of migrating cells initiating forward movement of cells indicating a potential role for NHE1 in cancer progression. NHE1 is regulated by many factors including multiple protein‐protein interactions and a number of phosphorylation events. Recent reports indicate that many proteins identified to play key roles in cancer are regulated by palmitoylation. S‐palmitoylation is a dynamic and reversible posttranslational modification, in which the protein undergoes cycles of palmitoylation and depalmitoylation, catalyzed by palmitoyl acyltransferases (PATs) and acyl protein thioesterases (APTs), respectively. Palmitoylation is known to regulate protein trafficking, membrane microlocalization and protein‐protein interactions. These findings prompted us to investigate the presence of this modification on NHE1 using acyl‐biotinyl exchange (ABE), an in vitro assay for post‐hoc detection of endogenous palmitoylation, which revealed that NHE1 is palmitoylated. To further evaluate the nature of NHE1 palmitoylation we treated Chinese hamster lung fibroblasts expressing human NHE1 (PSN) with the irreversible PAT inhibitor 2‐bromopalmitate (2BP). We found 2BP suppresses NHE1 palmitoylation in both a time and dose dependent manner. Additionally, PSN cell migration assessed using electric cell‐substrate impedance sensing was inhibited by 2BP in a manner consistent with the degree of inhibition of NHE1 palmitoylation. These results suggest that palmitoylation may regulate NHE1 function and its role in modulating cell migration.

Support or Funding Information

National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)
Protein Succination‐A Biomarker of Mitochondrial Stress in Adipose Tissue Inhibits Protein Function and is Regulated by Nutrient Balance
Authors: Manuel Allison Marie , Walla Michael , Piroli Gerardo , Frizzell Norma
Published: November 2023

S‐(2‐succino)cysteine (2SC) is an irreversible post‐translational modification formed by the reaction of fumarate with protein cysteines, also known as protein succination. 2SC is a biomarker of glucotoxicity driven mitochondrial stress in the adipocyte. Here, we investigated if 2SC is a mechanistic mediator of adipocyte stress via modification of the critical chaperone protein disulfide isomerase (PDI) and examined if 2SC levels could be regulated by caloric restriction.

Fumarase knockdown or control 3T3‐L1 adipocytes were matured in normal (5 mM) or high (30 mM) glucose for 8 days, or in 30 mM glucose for 4 days and then switched to 5 mM glucose for 4 days. Adipocyte lysates and adipose tissue protein from control and diabetic mice were analyzed by immunoblotting with an anti‐2SC antibody. The fluorescent PDI specific substrate DiEosinGSSG was utilized to assess PDI enzymatic activity in adipocytes. LC‐MS/MS mass spectrometry was utilized to identify the specific site of recombinant PDI succination.

2SC significantly increases in adipocytes matured in high glucose and in the adipose tissue of diabetic mice. Succination significantly lowers PDI reductase activity in fumarase knockdown adipocytes and in the adipose tissue of diabetic mice versus control. MS/MS analysis identified an important PDI active site cysteine as a target of succination. 2SC is turned over in adipocytes when maturation medium is returned to normal glucose.

2SC inhibits PDI reductase activity facilitating adipocyte stress as PDI is no longer able to re‐fold proteins within the adipocyte. The turnover of succinated proteins when nutrient balance is restored illustrates the importance of a balanced diet in preventing obesity and diabetes‐related dysfunction in adipose tissue. Our data demonstrates a novel biochemical mechanism mediating nutrient induced stress in the adipocyte, and that this stress is ameliorated when reasonable caloric restriction is employed, ensuring healthy functional adipose tissue.

Support or Funding Information

National Institute of Health 1F31DK108559‐01(AMM), 1R56DK105087‐01(NF) and 1‐11‐JF‐13 from the American Diabetes Association (NF).
Phosphorylation of a Master Transcription Factor in Human Organogenesis Regulates Developmental Fate
Authors: Chen Yen‐Shan , Nelson B
Published: November 2023

A seeming paradox is posed by metazoan gene‐regulatory networks (GRNs) that are robust yet evolvable. Insight may be obtained through studies of bistable genetic circuits mediating developmental decisions and in particular transcriptional thresholds governing cell‐fate decisions. We illustrate a general strategy to measure such thresholds by focusing on the genetic boundary between organization and dysgenesis. A model is provided by testis determination in therian mammals, which is initiated by SRY, a Y‐encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to development of an XY female with primary amenorrhea (Swyer syndrome). Our study has focused on an inherited mutation in human SRY (R30I) previously identified in a fertile XY father and sterile XY daughter (inherited Swyer syndrome). We have demonstrated that this mutation markedly impaired phosphorylation of SRY within a PKA site N‐terminal to the HMG box. On transient transfection of human and rodent cell lines, the variant SRY exhibited reduced binding to the testis‐specific enhancer of principal target gene SOX9; gene‐regulatory activity was reduced by twofold relative to wild‐type SRY. To our knowledge, this is the first report of a human birth defect due to mutation of a PKA site within a lineage‐specific transcription factor. Our results and their clinical correlation provide novel evidence that the robustness of human testis determination requires regulated serine phosphorylation of SRY. Further, the twofold attenuation of transcriptional activity observed here is characteristic of diverse mutations in SRY associated with inherited Swyer syndrome—irrespective of molecular mechanism—and so defines the narrow transcriptional threshold of male gonadogenesis in human development. We speculate that such a tenuous sex‐specific genetic switch has evolutionary advantages via multi‐level selection as envisaged through the framework of sociobiology.
Novel Physiological Targets of Fic‐mediated Adenylylation/AMPylation
Authors: Mattoo Seema , Sanyal Anwesha
Published: November 2023

Post‐translational modification (PTM) of proteins is a common theme in signal transduction. We previously discovered a family of enzymes, defined by a ‘Fic’ domain, that covalently attach an adenosine monophosphate (AMP) to their targets to alter cellular signaling. This PTM, called Fic‐mediated adenylylation/AMPylation is a strategy used by some bacterial pathogens to adenylylate and inactivate host Rho GTPases, thus evading host defense mechanisms. Recently we demonstrated that the sole human Fic, HYPE, adenylylates the ER molecular chaperone, BiP, thereby functioning as a key regulator of ER homeostasis. Accordingly, we showed that HYPE localizes to the ER lumen to modify BiP and alter the unfolded protein response (UPR) to stress. Using electron tomography and biochemical analyses, here we report that HYPE localizes to cellular compartments other than the ER, and have identified new physiologically relevant protein targets for HYPE. Validation of these targets is discussed.

Support or Funding Information

This work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Core Pilot Grant, 2) a Showalter Research Trust Award, 3) an American Cancer Society (ACS) Institutional Grant, and 4) the National Institute Of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.
Unique Solutions to a Common Problem: Understanding How I‐CreI Homologs Recognize the Same DNA Target Sequence
Authors: Polkinghorn Caley Ray , Kaiser Brett , Wylie Nathan , Tang Malu , Ruff Ray
Published: November 2023

Homing endonucleases (or “meganucleases”) are DNA cleaving enzymes that are widely present in microbial genomes. They are known as “selfish elements” for their ability to cleave their DNA target site within related species and insert themselves into the genome via homologous recombination. We studied a family of related LAGLIDADG homing endonucleases (“I‐Cre1 homologs”) which have been identified within the large ribosomal subunit gene of the chloroplast genome of numerous unicellular green algae. Despite significant divergence at the amino acid level (40–60% identity), these enzymes have retained the ability to cleave nearly identical DNA targets. For this reason, this enzyme family is a great case study to understand protein‐DNA interactions. Our goal was to investigate the unique ways in which a subset of I‐Cre1 homologs cleave their DNA targets. We used X‐ray crystallography to solve the molecular structures of three homologs bound to DNA then constructed contact maps for all of the individual protein‐DNA contacts being made. We also generated cleavage specificity profiles by performing DNA cleavage assays using one‐off mutated substrates for four enzymes (Col2593c, Sob2593c, Cre2593c and Hla2593c). The specificity profiles and DNA‐protein contact maps for the enzymes were analyzed alongside a multiple sequence alignment to identify how key contacts confer regions of specificity and flexibility. These conclusions allow for a detailed analysis of the unique ways each protein recognizes and cleaves their common DNA target.

Support or Funding Information

Murdock Charitable Trust (research)
SAXS: A Versatile Tool to Study Biological Macromolecules in Solution
Authors: Ma Weikang , Chakravarthy Srinivas , Shang Weifeng , Irving Thomas
Published: November 2023

The Biophysics Collaborative Access Team (BioCAT), which operates the high‐brilliance undulator Beamline 18ID at the Advanced Photon Source, Argonne National Laboratory, is dedicated to studying disordered macromolecules in solution using Small‐Angle X‐ray Scattering (SAXS). In SAXS, the solution scattering profile contains three dimensional information of macromolecules at nanometer scale in solution. The combination of a highly brilliant X‐ray source and advanced micro‐focusing optics at BioCAT enables significant insights into the structure and dynamic of bio‐macromolecules from both static and time‐resolved SAXS experiment.

The quality and interpretability of SAXS data is largely contingent on sample homogeneity. Size‐exclusion chromatography (SEC)‐SAXS, which was pioneered at BioCAT has therefore emerged as a powerful mode of data acquisition as it ensures optimal sample quality by purification immediately before exposure to x‐rays. In addition, we have also incorporated multi‐angle light scattering (MALS), dynamic light scattering (DLS), and differential refractive index (dRI) detectors in line with the SAXS camera thus enabling acquisition of a full complement of biophysical data typically required to structurally characterize biological macromolecules simultaneously with SAXS data.

The new frontier in the field is to study real‐time structural changes in biological systems as a response to very specific environmental stimuli such as a change in denaturant concentration, ionic strength, pH or ligand binding. Forefront time resolved SAXS techniques using chaotic mixing continuous flow, continuous laminar flow and stopped flow mixing are now available to a wide variety of bio‐medical projects at BioCAT primarily on account of being optimized over the past few years both for sample consumption and mixing efficiency.

Support or Funding Information

9 P41 GM103622
Exploring the Structural Impact of Unnatural Amino Acids on Protein Structure
Authors: Savidge Nicole S , Maurici Nicole , Brewer Scott H , Phillips‐Piro Christine M
Published: November 2023

Unnatural amino acids (UAAs) have the potential to serve as effective, site‐specific reporters of local protein structure and dynamics. However, in order to act as an effective probe, the UAA needs to be minimally perturbative to the native protein structure. One potential IR probe of local environment is 4‐nitro‐L‐phenylalanine (pNO2F). pNO2F has been genetically incorporated into the model protein superfolder green fluorescent protein (sfGFP) at a solvated and partially buried site individually in order to determine the structural impact of the presence of the UAAs on the native protein structure. The IR analysis of these protein constructs in addition to the protein crystal structures will be presented.

Support or Funding Information

• The F&M Hackman Scholars Program and Schappell Research Fund

• NIH R15 (2R15GM09330) to S.H.B/E.E.F.
Prediction of the Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) Protein Structure using bioinformatics tools
Authors: Lowerre Katie , Hemme Christopher , Espinosa Avelina
Published: November 2023

Entamoeba histolytica is an obligate anaerobic parasite that lacks energy producing mitochondria. Entamoeba histolyica's alcohol dehydrogenase 2 (EhADH2) is a bifunctional enzyme with aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activities that are essential for the organism's growth and survival. Alcohol dehydrogenase‐E enzymes (ADHE) are important targets for antimicrobial agents and ADHE enzymes are present in a variety of different organisms. Phylogenetic tools have suggested that EhADH2 is similar to other protist and bacterial bifunctional enzymes. This study will focus on protein analyses that include maximum likelihood predictions and pairwise deletions and comparisons. Non‐synonymous mutations and site information for the evolution of EhADH2 can be accessed through resources such as PAML. Additional methods using a ZDOCK, Discovery Studios, and the modelling program Chimera will be used to determine a 3D structure for EhADH2 (both N and C terminal domains) by comparing the protein to similar oxidoreductase patterns of an initial dimer formation defined for the Zymomonas mobilis ADH enzyme. Identification of inhibitor sites with enzyme inhibitor databases is helpful for EhADH2 and ADHE homologues by using effective laboratory‐tested pyrazoline derivatives capable of inhibiting enzyme activities and parasite growth.
Significance of charged residues in the catalytic sites of Escherichia coli ATP synthase
Authors: Ahmad Zulfiqar
Published: November 2023

Background

F1Fo ATP synthase, the smallest biological nanomotor, is the fundamental means of cellular energy production in almost all living systems. Oxidative or photophosphorylation in membranes of bacteria, mitochondria, and chloroplasts generates ATP from ADP and Pi (inorganic phosphate). The molecular mechanism of ATP synthase and generation of ATP depends on the designated catalytic site residues. We have been interested in identifying the specific Pi binding residues in the cyclical process of binding change mechanism. So far we have investigated eleven residues, βArg‐246, βAsn‐243, αArg‐376, βLys‐155, βArg‐182, αPhe‐291, αSer‐347, αThr‐349, αIle‐346, αIle‐348, and αGly‐351 based their proximity to the phosphate analogs AlF3 or SO42− in x‐ray structures of catalytic sites. We suspect that many other catalytic site residues including highly conserved αVISIT‐DG sequence residue αAsp‐350 plays important functional role in the catalytic sites.

Methods

Mutagenic analysis of αAsp‐350 along with other catalytic site residues was performed to generate single and double mutants. Growth patterns were observed on limiting glucose and succinate plates. MgPi protection assay against NBD‐Cl induced inhibition was performed to reveal the direct or indirect role of residues in Pi binding.

Results

Lately, we found that αThr‐347 of the VISIT‐DG sequence is the sixth important residue involved in phosphate binding other than βLys‐155, βArg‐182, αArg‐376, βArg‐246, and αSer‐347 while αIle‐346 and αIle‐348 are required for the maintenance of catalytic site along with βAsn‐243, αPhe‐291, and αGly‐351. Currently, we are exploring the role of negatively charged αAsp‐350 in the Pi binding sub‐domain of the catalytic sites of ATP synthase.

Support or Funding Information

This work was supported by the National Institutes of Health Grant GM085771 and A.T. Still University‐Kirksville College of Osteopathic Medicine Biomedical Science Graduate Program Funding #850‐611.
The Ribosome‐Associated Complex Suppresses [PSI+] Prion Formation in Yeast
Authors: Kelly Christina , Tessitore Thomas , Taddeo Jessica , Cameron Dale M
Published: November 2023

Prions are self‐propagating infectious protein conformations. In mammals prions underlie the transmissible spongiform encephalopathies, a set of invariably fatal neurodegenerative disorders associated with misfolding of the mammalian prion protein, PrP. In recent years increasing numbers of fungal proteins capable of infectiously propagating altered conformations have been discovered. To gain insight into the mechanisms by which cells regulate prion formation, propagation and toxicity, we previously examined the role of the ribosome‐associated complex (RAC) chaperones in the formation and toxicity of the [PSI+] prion in yeast. The [PSI+] prion results from a self‐propagating amyloid form of the essential translation termination factor Sup35, and is manifested phenotypically as elevated levels of nonsense suppression. The RAC consists of the Hsp70 chaperone Ssz1 and the Hsp40 chaperone Zuo1, which anchors the complex onto ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. Previously we showed that yeast cells lacking Zuo1, and thus lacking RAC function on ribosomes, exhibit higher frequencies of spontaneous and induced [PSI+] formation. Cells expressing variants of Zuo1 that are unable to associate with ribosomes, or that are unable to stimulate Ssb ATPase activity, exhibit similarly high levels of prion formation. These findings are consistent with a role for the RAC in chaperoning nascent Sup35 to prevent misfolding of the N‐terminal prion domain as it emerges from the ribosome. Zuo1 can additionally function as transcriptional co‐activator for the pleiotropic drug resistance pathway, which enhances resistance to various drugs and environmental toxins. Its role in this pathway requires unfolding of the C‐terminal domain of Zuo1, which prevents its association with ribosomes. Since a variety of environmental stress conditions are known to induce prion formation, RAC dissociation from ribosomes under these conditions may facilitate prion formation. Our current work aims to understand the mechanism by which Zuo1 dissociation from ribosomes is triggered in response to environmental stress.

Support or Funding Information

This work was supported by an award from the National Institutes of Health National Institute of General Medical Sciences to DMC.
The Role of ER‐resident Lectin Chaperone UGT1 in MHC Class I Peptide Loading
Authors: Arshad Najla , Cresswell Peter
Published: November 2023

The phenomenon of antigen processing and presentation aids the immune system in distinguishing between normal and diseased cells. Antigen presentation by MHC class I heavy chain‐β2m heterodimers (MHC‐I) to CD8+ T cells requires association of MHC‐I with peptides, a process that occurs in in the ER. Binding is facilitated by the Peptide Loading Complex (PLC), which consists of the TAP transporter, the ‘peptide editor’ tapasin, which is disulfide linked to the thiol‐oxidoreductase ERp57, and a lectin chaperone, calreticulin (CRT). Heterodimers without peptides are inherently unstable and are not expressed on the cell surface. Even when MHC‐I associates with a peptide, only those molecules loaded with a peptide deemed “optimal” by quality control mechanisms in the ER proceed to the cell surface.

Proteins involved in this quality control include the ER‐resident lectin chaperones calnexin and CRT that interact with MHC‐I via its conserved N‐linked glycan. Once the peptide‐associated MHC‐I leaves the PLC, it can be a substrate for the enzyme UDP‐glucose:glycoprotein glucosyltransferase 1 (UGT1), which selectively reglucosylates the N‐linked glycan of the MHC‐I heavy chain. The glucosylated MHC‐I can then re‐associate with CRT in the PLC, allowing for additional rounds of peptide binding. This process is reminiscent of the ubiquitous calnexin‐calreticulin cycle and has been adapted by the cell for optimal antigen presentation by MHC‐I. However, in this case, MHC‐I is a folded and functional molecule.

This study aims to understand the mechanism by which UGT1 recognizes a peptide/MHC‐I complex. Biochemical assays using purified UGT1 and the human MHC‐I allele HLA‐A2 unexpectedly showed that there is no strong correlation between the affinity of the associated peptide and reglucosylation of the HLA‐A2 glycan. Recognition may be based on conformational changes in MHC‐I induced by the peptide. Consistent with this, published hydrogen/deuterium exchange studies show that such changes occur near the glycosylation site, and are independent of the affinity of the peptide for MHC‐I.

Support or Funding Information

Cancer Research Institute Irvington Fellowship and The National Institutes of Health:R01AI09720605
Functional characterization of natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible 70kDa heat shock gene in humans
Authors: Oliverio Ryan , Nikolaidis Nikolas
Published: November 2023

The cellular stress response system is responsible for maintaining cellular homeostasis during periods of stress caused by environmental insults including pathogens and disease. Molecular chaperones, and, in particular the 70‐kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system. Therefore, we hypothesized that natural occurring variations in the sequence of Hsp70s will affect cellular and subsequently species adaptation. To investigate the relationship between adaptation, disease susceptibility, and Hsp70s' variation we tested whether natural single nucleotide polymorphisms found on HSPA1A, the major stress inducible Hsp70 gene in humans, alter its function. Specifically, the wild‐type human HSPA1A sequence was subcloned into mammalian expression vectors, and the mutated gene variants were generated using site‐directed mutagenesis. We then determined whether any of these mutations affected the intracellular localization of HSPA1A. These experiments were performed by using GFP‐tagged HSPA1A and fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, and visualized using confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Furthermore, we determined whether the mutations affected the ability of HSPA1A to prevent cell death by inhibiting the formation of protein aggregates caused by poly‐glutamine carrying huntingtin proteins. This assay determined that one of the mutations caused increased cell death as compared to the WT, suggesting that it may alter HSPA1A's ability to prevent protein aggregation. We also determined whether these mutations affect the ability of HSPA1A to refold heat‐denatured luciferase. These assays revealed that only one mutation resulted in significantly lower levels of refolded enzyme, suggesting a putative loss‐of‐chaperone function. Lastly, live‐dead assays revealed that human cell lines carrying two of these mutations had significantly less resistance to heat and ethanol stress than cells expressing the WT protein. Given that these natural variants are found at a very low frequency in humans we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress.

Support or Funding Information

This project was supported by funds from NIH and CSUF to NN
Dual function of the trigger factor chaperone in nascent protein folding
Authors: Kaiser Christian , Maciuba Kevin , Liu Kaixian
Published: November 2023

Multi‐domain proteins often require help from molecular chaperones to fold productively, even before the ribosome has finished their synthesis. The mechanisms underlying chaperone function remain poorly understood. Using optical tweezers to study the folding of elongation factor G (EF‐G), a model multi‐domain protein, we find that the N‐terminal G‐domain in nascent EF‐G polypeptides folds robustly. The following domain II, in contrast, fails to fold efficiently. Strikingly, interactions with the unfolded domain II convert the natively folded G domain to a non‐native state. This non‐native state readily unfolds, and the two unfolded domains subsequently form misfolded states, preventing productive folding. Both the conversion of natively folded domains and non‐productive interactions among unfolded domains are efficiently prevented by the nascent chain‐binding chaperone trigger factor. Thus, our single‐molecule measurements of multi‐domain protein folding reveal an unexpected role for the chaperone: It protects already folded domains against denaturation resulting from interactions with parts of the nascent polypeptide that are not folded yet. Previous studies had implicated trigger factor in guiding the folding of individual domains, and interactions among domains had been neglected. Avoiding these early folding defects is crucial, since they can propagate and result in misfolding of the entire protein.

Support or Funding Information

Pew Scholars Program
Evolution and natural variation of HSPA1A, the major stress inducible gene, in humans
Authors: Nguyen Peter , Kdeiss Brianna , Ord Sara , Hess Kyle , Oliverio Ryan , Nikolaidis Nikolas
Published: November 2023

HSPA1A is the major stress inducible 70‐kDa Heat shock protein (Hsp70) in humans. This protein is a critical component of the cellular stress response and alterations in its function have been associated with several disease phenotypes, such as cancer, and neurodegenerative conditions. However, the extent of HSPA1A's natural variation within and between species remain largely unknown. Therefore, we investigated how this gene evolved in humans and other mammals. Our results reveal that in all Eutherian mammals HSPA1A is actually two almost identical proteins coded by two tandemly arranged genes. This extreme level of sequence conservation, both at the nucleotide and protein levels, can be explained by a gene conversion‐like mechanism that resulted in sequence homogenization. However, gene conversion alone cannot explain the observed evolutionary pattern of all the mammalian heat‐inducible genes, which contain several non‐amino acid altering (synonymous) mutations. Therefore, we conceptualized that both gene conversion and purifying selection act to conserve these Hsp70 genes. This notion is also supported by analyses of HSPA1A variation in humans, which revealed that all mutations have a very low frequency and that the majority of these mutations is synonymous. The presence of purifying selection was further strongly implied by determining whether natural variants affect the major chaperone functions of HSPA1A. Specifically, recombinant proteins corresponding to the wild‐type (WT) and five mutated variants were tested for their ability to hydrolyze ATP. These experiments revealed that the mutations did not significantly alter the rate of ATP hydrolysis. Then Isothermal Titration Calorimetry was employed to test whether the mutations alter binding of HSPA1A to ATP, ADP, and protein substrates. These experiments showed that only one mutation increased the entropy of the reactions. Lastly, the effects of these mutations on protein stability was determined by calculating the protein's melting temperature (Tm). This assay revealed that three mutations had significantly higher Tm as compared to the WT protein. These experiments suggest that although purifying selection acts to preserve the primary functions of HspA1A, other secondary functions of the molecule may be altered. Collectively, our results constitute an evolutionary paradigm in which gene conversion, purifying selection, and neofunctionalization act conversely to conserve a critical component of the heat shock response.

Support or Funding Information

This project was supported by funds from NIH and CSUF to NN
Identification of a pharmaceutical therapeutic for Nod2, a protein mutated in Crohn's Disease, through development of a screen using split GFP complementation
Authors: Wastyk Hannah Constance , Hou Ching Wen , Grimes Catherine
Published: November 2023

The human body houses over 10 trillion bacterial cells to make up our microbiome. These bacteria can be commensal or pathogenic, and it is the role of our innate immune system to delineate between the two. Innate immune receptors, such as Nod2, are responsible for binding to bacterial cell wall fragments. Nod2 binds to bacterial cell wall fragment, muramyl dipeptide (MDP), which leads to subsequent downstream activation of the NF‐κB pathway and a protective inflammatory response. Mutations in Nod2 are correlated with an increased susceptibility to Crohn's disease. Nod2 mutations that decrease its stability disrupt the downstream signaling response. Initial work identifies a chaperone protein, Hsp70, which binds to Nod2 and assists its stability and activity. The interaction of Nod2 and Hsp70 was characterized to suggest that Hsp70's ATPase activity is dispensable for Nod2 stabilization. A truncated 71 amino acid domain of Hsp70 was shown to bind and stabilize Nod2 (wild type and Crohn's variant). Here, we go a step further by expressing the minimized peptide chain in cells and measuring the relative signaling response via a NF‐kB luciferase assay. The 71 minimized peptide chain was also used as a pharmacological tool through the principle of split Green Fluorescent Protein (GFP) complementation for development of a screen for pharmaceutical therapeutics. The peptide and Nod2 were tagged and detected with engineered self‐assembling fragments of GFP. When the peptide and Nod2 are bound, fluorescence is detected. Each pharmacoperone was individually incubated with the fluorescent protein complex. Effective pharmacoperones should competitively bind to Nod2, and thus disrupt the binding of the peptide and lead to lower fluorescence. The most promising pharmacoperones from the screen will be further analyzed for Nod2 stability and signaling in cell based assays. Successful identification of a pharmacoperone that mimics the natural stabilization of Hsp70 can be applied to restore function of a number of disease‐relevant mutated proteins.

Support or Funding Information

The authors would like to thank the following sources of funding: the Chemistry‐Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through Nucleus.