bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cldn18−/− mice reveal novel role for YAP signaling in regulation of distal lung epithelial stem/progenitor cell homeostasis
Authors: Borok Zea , Zhou Beiyun , Flodby Per , Castillo Dan R , Luo Jiao , Yu Fa‐Xing , McConnell Alicia , Varghese Bino A , Li Guanglei , Conti Peter S , Liu Yixin , Sunohara Mitsuhiro , Liebler Janice M , Yang Chenchen , Marconett Crystal N , Laird‐Offringa Ite A , Minoo Parviz , Guan Kun‐Liang , Stripp Barry R , Crandall Edward D 
Published: November 2023

Claudins are integral tight junction (TJ) proteins that contribute to cell polarity and regulate paracellular permeability to ions and solutes. Claudin 18 (CLDN18) is one of the most highly expressed claudin family members in lung alveolar epithelium. To investigate the role of CLDN18 in alveolar homeostasis, we recently generated Cldn18−/− mice that demonstrate increased lung solute permeability and alveolar fluid clearance (AFC) compared to wild type (WT) controls. Lungs of Cldn18−/− mice are markedly enlarged due to increased abundance and proliferation of alveolar epithelial type II (AT2) cells, known progenitors of distal lung epithelium, with resultant parenchymal expansion. Cldn18−/− AT2 cells grown with MLg fibroblasts in 3‐dimensional (3D) culture show increased colony forming efficiency (CFE), suggesting increased progenitor capacity. Given its known role in regulating stem/progenitor cell proliferation and organ size, we investigated a potential role for Yes‐associated protein (YAP) signaling in mediating the proliferative phenotype of Cldn18−/− lung progenitors. Progenitor cell activation was accompanied by activation of YAP, as evidenced by increased nuclear YAP, increased expression of YAP target genes in lungs of Cldn18−/− mice, and increased YAP and decreased phospho‐YAP (p‐YAP) by western analysis in isolated Cldn18−/− AT2 cells and by immunofluorescence in Cldn18−/− AT2 cells in 3D culture. Treatment with the YAP inhibitor verteporfin (VP, 100 mg/kg) reduced AT2 cell proliferation in vivo (EdU+NKX2.1+/total cells: 1.36 ± 0.10% vehicle vs 0.93 ± 0.06% VP, p<0.05) and decreased lung size (lung dry weight/body weight ratios (mg/g): 3.56 ± 0.08 vehicle vs 2.93 ± 0.13 VP, p<0.05) in Cldn18−/− mice. Inhibition of YAP with VP (0.75 μM) or shRNA decreased colony size (~50% vs control) and number (30–50% vs control) and reduced proliferation (Ki67+ cells at 10.7 ± 0.4% VP vs 29.2 ± 3.7% vehicle) of Cldn18−/− AT2 cells in 3D culture, while overexpression of CLDN18 decreased YAP nuclear localization, cell proliferation, CFE and YAP activity. These results reveal a novel role for YAP signaling in regulation of distal lung epithelial progenitor cell homeostasis and identify a role for TJ proteins, in particular CLDN18, in regulating YAP activity and organ size. Overall, they suggest a mechanism whereby growth‐promoting signals are transduced from TJ to the nucleus that has important implications for modulating stem/progenitor cell function and regeneration following injury.Support or Funding InformationNational Institutes of Health, Hastings and Whittier Foundations
YAP1 stimulates angiogenesis and lung regeneration through the Angiopoietin‐Tie2 pathway
Authors: Mammoto Akiko , Mammoto Tadanori 
Published: November 2023

Angiogenesis, the formation of new blood capillaries, plays key roles in organ development and regeneration. Inhibition of angiogenesis through the blockade of angiogenic signaling pathways impairs adult lung regeneration. Previously we have reported that Wnt co‐receptor, low‐density lipoprotein receptor‐related protein 5 controls lung vascular and alveolar regeneration. The Hippo signaling transducer, Yes‐associated protein (YAP1), cooperates with Wnt‐b‐catenin signaling and controls organ size and regeneration. Here, we have demonstrated that YAP1 knockdown decreases angiogenic factor receptor Tie2 expression and inhibits endothelial cell sprouting and lung epithelial morphogenesis in vitro. YAP1 knockdown also attenuates angiogenesis and alveolar epithelial cell recruitment into the hydrogels implanted on the mouse lungs and inhibits compensatory lung growth after unilateral pneumonectomy in adult mice. These findings suggest that YAP1 plays an important role in lung vascular and alveolar regeneration and modulation of YAP1 expression in alveolar capillary endothelial cells may be a novel strategy for lung regeneration.Support or Funding InformationBoston Children's Hospital Career Development Awards
Messenger RNA Stability Drives the Expression of Antibiotic Resistance Methyltransferase Independently of Ribosome Stalling
Authors: Los Katia , Yap M.‐N. Frances 
Published: November 2023

Macrolides, lincosamides, and streptogramin B (MLSB) are structurally distinct antibiotics that are clinically successful in treatment of bacterial infections. Their efficacy has decreased due to the rapid emergence of resistant bacterial strains. Cross‐resistance to MLSB antibiotics often arises through the production of erythromycin resistance N‐6‐methyltransferase (Erm). The enzyme modifies the common MLSB binding site within the ribosomal exit tunnel and reduces the drug binding affinity to the ribosome. Previous model based on the ermCL‐ermC system suggests that the synthesis of Erm is induced through the antibiotic‐stalled ribosome that translates the co‐transcribed upstream leader sequence. Ribosome stalling induces a structural rearrangement of erm transcript and exposes the otherwise masked erm Shine‐Dalgarno (SD) sequence, subsequently leading to translational initiation. Curiously, many resistant clinical isolates carry mutated versions of leader sequence, including the ribosome‐stalling null mutants. The seemingly paradoxical role of leader peptide‐dependent ribosome stalling in MLSB resistance remains poorly understood.We used the ermBLEF‐ermB operon from the human pathogen Staphylococcus aureus as a model to determine how the expression of ermB, unlike other homologous erm genes, is inducible by a broader spectrum of MLSB antibiotics. Our data show that disrupting the translation of ErmBL abolishes ribosome stalling but results in high basal levels of ErmB that is well correlated with elevated ribosome methylation and antibiotic resistance, suggesting that a ribosome stalling‐independent determinant is required for ermB expression. Subsequently, we found that both MLSB antibiotic treatment and abrogating ErmBL translation upregulate the basal ermB mRNA levels and prolong the ermB mRNA half‐life. Furthermore, antibiotic exposure reduces mRNA cleavage at a site immediately downstream of the ermB SD sequence. The data confirmed that the expression of ermB could be upregulated by antibiotics via stabilization and accumulation of its mRNA. Finally, genome‐wide transcriptome analysis showed that the stabilizing effect of antibiotics on mRNA is not limited to ermBL‐ermB; antibiotics representing a ribosome stalling inducer and a non‐inducer co‐regulate similar sets of genes and increase the half‐lives of the majority of analyzed transcripts.Collectively, our data suggest a model by which the upregulation of ErmB is achieved through disrupted translation of the leader peptide, which normally acts as a break to fine‐tune the cellular ErmB levels. Tight regulation of ErmB is necessary because hypermethylation of ribosome may impose a fitness cost on the bacterium.Support or Funding InformationThis work is supported by the NIH, the Pew Charitable Trust (Pew Scholars in the Biomedical Sciences), the Edward Mallinckrodt Jr. Foundation, and SLU President's Research Fund.
Regulation of Protein Synthesis by Post‐translational Modification of Eukaryotic Translation Elongation Factor 1A
Authors: Sharma Pragati , Mateyak Maria K , He Donming , Perez Winder B , Kinzy Terri G 
Published: November 2023

Protein synthesis is an intricate multi‐step process accomplished through a series of concerted actions of several different factors. Post initiation, translation elongation continues in a cyclic manner with the delivery of cognate aa‐tRNA to ribosomal A site, peptide bond formation and translocation to P‐site. The broad mechanism of action of elongation factors is known, however the regulation of translation elongation remains poorly understood. To safeguard timely and accurate gene expression, the protein synthesis machinery must be strictly regulated. Indeed the eukaryotic Elongation Factor 1A (eEF1A) shows extensive post‐translational modifications, both phosphorylation and methylation. In Saccharomyces cerevisiae, global proteomic analyses have reported at least 15 residues in eEF1A that are phosphorylated under different growth conditions. We have created a set of corresponding alanine (non‐phosphorylatable) and aspartate (phosphomimetic) mutations in these residues to study their phenotypic as well as enzymatic effects. Our in vivo and in vitro analyses in budding yeast show that S53D, Y416D and T430D mutant alleles are unable to support cell growth as the only form of eEF1A, while S18D, T259D and Y416A show slow growth and an initiation defect manifested by Gcn2 activation. Further experiments to test the translation fidelity, in vitro enzymatic activity and their ability to interact with cofactors are ongoing. In addition to translation elongation, eEF1A has been implicated in a wide variety of cellular processes, for example cytoskeleton organization through actin bundling, protein quality control, viral invasion and replication, etc. This work is aimed at identifying the events and factors that modulate eEF1A activity ensuring careful control of gene expression and appropriate cell function.Support or Funding InformationNIH grant RO1 GM057483
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 InformationNIH ‐ 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 InformationHHMI 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 InformationThis 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 InformationProvost Research Support Award, The Chemistry Department Sellers Fund, Eastern Michigan University
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 InformationThis 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

BackgroundManganese 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).MethodsChromatography 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.ResultsUsing 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.ConclusionWe 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 InformationSupported by NIH grant 5 K01 AA022372‐04(Maria Lauda Tomasi)