bluuebunny/crossref_metadata_2025 huggingface dataset:
O‐GlcNAc Regulates Erythroid Genes Controlled by GATA‐1
Authors: ZHANG ZHEN , Graw Stefan , Tan Ee Phie , Koestler Devin C. , Peterson Kenneth R. , Slawson Chad
Published: November 2023

Transcription factor GATA‐1 is essential for erythropoiesis and many GATA‐1 controlled genes are up‐regulated or down‐regulated as differentiation occurs. The mechanisms underlying how GATA‐1 target genes are regulated is unclear. One possible mechanism is through the post‐translational modification O‐GlcNAc. O‐linked N‐Acetylglucosamine (O‐GlcNAc) is added and removed from serine and threonine residues by the O‐GlcNAc processing enzymes, O‐GlcNAc‐transferase (OGT) and O‐GlcNAcase (OGA), respectively. In this study, we utilized a well‐established cell model of erythropoiesis, G1E‐ER4 cells, a murine GATA‐1 null erythroblast line that undergoes erythroid differentiation when GATA‐1 activity is restored by the addition of β‐estradiol. Interestingly, overall O‐GlcNAc levels dramatically decreased after GATA‐1 activation in G1E‐ER4 cells, with a slight increase of OGA and a decrease of OGT protein levels. We performed transcriptome analysis of G1E‐ER4 cells treated with β‐estradiol and/or Thiamet‐G (TMG, an OGA inhibitor). Approximately 6,800 transcripts expression patterns were changed following GATA‐1 activation. When the cells were treated with β‐estradiol and TMG, 1,100 genes changed expression patterns compared to cells treated with β‐estradiol only. The transcriptome profile changes induced by TMG likely alter GATA‐1 regulated pathways during erythropoiesis. Our data suggests that O‐GlcNAcylation plays a role in regulating erythroid gene expression controlled by GATA‐1.

Support or Funding Information

This work was supported by NIDDK Grant R01DK100595 (to C. S. and K. R. P.) and Biomedical Research Training Program from The University of Kansas Medical Center.
Molecular Characterization of HlyU, a Global Regulator of Vibrio vulnificus Virulence Genes
Authors: Choi Sang Ho , Lee Zee‐Won , Bang Ye‐Ji , Jang Kyung Ku
Published: November 2023

An opportunistic pathogen Vibrio vulnificus produces a large pore‐forming toxin, RtxA, which triggers necrotic and apoptotic cell death. Previous studies reported that a transcriptional regulator HlyU upregulates rtxA expression by direct binding to the promoter region of the rtxHCA operon. To examine the function of HlyU, the hlyU mutant was constructed and its virulence was evaluated. Compared with the wild type, the hlyU mutant exhibited low cytotoxicity toward mucin‐secreting HT‐29 MTX cells and reduced mortality in mice, suggesting that HlyU is essential for the virulence of V. vulnfiicus. To further identify the genes regulated by HlyU, transcriptomic profiles of the wild type and the hlyU mutant were analyzed by RNA‐seq. In addition to rtxA, genes encoding virulence factors such as hemolysin and phospholipase were down‐regulated in the hlyU mutant, implying that HlyU contributes to the V. vulnificus pathogenesis by regulating various virulence genes. To elucidate the mechanism of HlyU in rtxA regulation, the levels of rtxA and hlyU transcripts in V. vulnificus cells grown in different conditions were determined. Induction of the rtxA expression occurred in the wild‐type cells either exposed to the INT‐407 human epithelial cells or grown under anaerobic conditions, but not in the hlyU mutant, indicating that HlyU mediated the induction. Since the levels of hlyU transcript in the wild‐type cells were not significantly changed in those conditions, the rtxA activation might be attributed to altered activity rather than amounts of HlyU. To better understand this activity alteration of HlyU, C30, C96, and M87, which were predicted as essential amino acids from structural analysis, were mutated by site‐directed mutagenesis. Among the mutants, V. vulnificus expressing HlyU‐C30S showed a decreased rtxA level and cytotoxicity toward the HT‐29 MTX cells than the wild type, suggesting that C30 is a critical residue of HlyU for rtxA activation. Taken together, HlyU might act as a regulator for the various virulence genes and induce rtxA under host environment by altering its activity.
Regulation of Cellular Proliferation in B‐cell Acute Lymphoblastic Leukemia by Ikaros
Authors: Dovat Elanora , Payne Jonathon L , Song Chunhua , Desai Dhimant
Published: November 2023

B‐cell acute lymphoblastic leukemia (B‐ALL) is the most common malignancy of childhood. IKZF1 encodes the Ikaros protein that acts as a tumor suppressor in B‐ALL. In high‐risk B‐ALL that is characterized by poor prognosis, the activity of Ikaros as a tumor suppressor is impaired. The goal of our research is to define the mechanisms through which Ikaros regulates cellular proliferation in high‐risk B‐ALL. In gain‐of‐function experiments, we overexpressed Ikaros in B‐ALL cells via retroviral transduction. Ikaros overexpression results in transcriptional repression of a large set of genes that promote cell cycle progression and several genes that promote the Phosphoinositide 3‐kinase (PI3K) pathway. This was associated with increased Ikaros binding at promoters of these genes. Ikaros loss‐of‐function experiments in which Ikaros in B‐ALL was downregulated by transfection with a specific Ikaros shRNA, resulted in increased transcription of Ikaros target genes. These results suggest that Ikaros can regulate the cell cycle and PI3K pathway in B‐ALL by functioning as a transcriptional repressor of the genes that promote cell cycle progression and the PI3K pathway. Since both cell cycle control and the PI3K pathway are dysregulated in high‐risk B‐ALL, these data provide a mechanism for how impaired Ikaros function results in the development of high‐risk B‐ALL. To determine the mechanism trough which Ikaros represses transcription of genes that regulate cellular proliferation in B‐ALL, we performed fine mapping of Ikaros binding and identified the epigenetic signature of the promoters of Ikaros target genes in B‐ALL cells using serial quantitative chromatin immunoprecipitation (qChIP). Results showed that Ikaros binding to the promoters of its target genes is associated with the formation of repressive chromatin that is characterized by the presence of specific histone modifications – H3K27me3 or H3K9me3. These data suggest that Ikaros represses the transcription of genes that regulate cellular proliferation via chromatin remodeling. In summary, the presented data demonstrate that Ikaros controls cellular proliferation in B‐ALL by direct transcriptional repression of genes that promote cell cycle progression and the PI3K pathway via epigenetic mechanisms. The loss of Ikaros function results in the deregulation of cell cycle control and the PI3K pathway and the development of high‐risk B‐ALL. Results provide the groundwork for further studies of the epigenetic regulation of gene expression in B‐ALL.

Support or Funding Information

Supported by St. Baldrick's Foundation Summer Fellowship (E.D.)
MSK‐1 mediated Histone H3 phosphorylation is critical for ethanol‐induced inhibition of IL‐2 gene transcription in CD4+ T lymphocytes
Authors: Ghare Smita , Joshi‐Barve Swati , McClain Craig , Barve Shirish
Published: November 2023

Immune suppression due to decreased CD4+ T cell function is a significant pathogenic component of multiple diseases including alcoholism, HIV infection and aging; however, the underlying mechanisms are not yet fully understood. We have previously shown that immunosuppressive effects of alcohol involve a decrease in the expression of interleukin‐2 (IL‐2), which critically regulates T cell proliferation, clonal expansion and homeostasis. In the present study we investigated the role of ethanol‐induced promoter histone modifications in the context of dysregulated IL‐2 gene expression. Chromatin immunoprecipitation (ChIP) analysis revealed that ethanol significantly prevented TCR inducible transcriptionally permissive histone H3 modifications at IL‐2 promoter in CD4+ T lymphocytes. Specifically, ethanol treatment inhibited phosphorylation of H3 serine 10 (H3S10ph) which then coordinately down regulated histone H3 lysine 4 tri‐methylation (H3K4Me3) and H3 lysine 9 acetylation (H3K9Ac). Notably, data showed that ethanol mediated transcriptional repression at IL‐2 promoter involves inhibition of Mitogen and Stress activated protein kinase (MSK‐1). Treatment of CD4+ T cells with MSK‐1 inhibitor further confirm the role of MSK‐1 mediated phosphorylation in regulating IL‐2 gene expression. Importantly, ethanol inhibited MSK‐1 activation via PP1 activation and could be reversed by okadaic acid‐phosphatase inhibitor treatment of CD4+ T lymphocytes. Overall, these observations suggest that, ethanol‐induced MSK‐1 inhibition and resultant decrease in promoter histone phosphorylation constitute a significant pathogenic mechanism underpinning the loss of IL‐2 expression and subsequent CD4+ T cell function associated with alcohol‐induced immune suppression.

Support or Funding Information

This work was supported by NIH grants.
Upregulation of CYP17A1 by Sp1‐mediated DNA Demethylation Confers Temozolomide Resistance through DHEA‐Mediated Protection in Glioma
Authors: Hsu Tsung‐I
Published: November 2023

Steroidogenesis‐mediated production of neurosteroids is important for brain homeostasis. Cytochrome P450 17A1 (CYP17A1), which converts pregnenolone to dehydroepiandrosterone (DHEA) in endocrine organs and the brain, is required for prostate cancer progression and acquired chemotherapeutic resistance. However, whether CYP17A1‐mediated DHEA synthesis is involved in brain tumor malignancy, especially in glioma, the most prevalent brain tumor, is unknown. To investigate the role of CYP17A1 in glioma, we determined that CYP17A1 expression is significantly increased in gliomas, which secrete more DHEA than normal astrocytes. We found that as gliomas became more malignant, both CYP17A1 and DHEA were significantly upregulated in temozolomide (TMZ)‐resistant cells and highly invasive cells. In particular, the increase of CYP17A1 was caused by Sp1‐mediated DNA demethylation, whereby Sp1 competed with DNMT3a for binding to the CYP17A1 promoter in TMZ‐resistant glioma cells. CYP17A1 was required for the development of glioma‐cell invasiveness and resistance to TMZ‐induced cytotoxicity. Additionally, DHEA markedly attenuated TMZ‐induced DNA damage and apoptosis. Together, our results suggest that components of the Sp1‐CYP17A1‐DHEA axis, which promotes the development of TMZ resistance, may serve as potential biomarkers and therapeutic targets in recurrent glioma.

Support or Funding Information

This project was supported by grants from MOST (MOST 103‐2320‐B‐038‐046‐MY3, 105‐2911‐I‐038‐503 and 105‐2320‐B‐038‐062) provided by Ministry of Science and Technology in Taiwan.
The Sp1 in Astrocyte plays an important role in Neurogenesis
Authors: Hung Jan‐Jong , Chang Wen‐Chang
Published: November 2023

Sp1 as a transcriptional factor regulates many genes related to various physiological conditions such as cellular survival, death, and development, and it also involves in many diseases such as cancer. In the past, we found that Sp1 could be up regulation through H2O2‐mediated IRES‐dependent translational pathway to protect neuron from ischemic damage, indicating that Sp1 might be crucial for the physiology or pathology of brain. Our recent data indicate that higher level of Sp1 is expressed in astrocyte compared to neuronal cells, implying that Sp1 might be critical for the function of astrocyte. To elucidate the function of Sp1 in astrocyte, we constructed a conditional functional knockout of Sp1 specific in astrocyte. Our recent results reveal that loss of Sp1 in astrocyte affect the morphology and function of astrocyte, leading neuron loss and synapse formation defect, subsequently affect the abilities of learning and excise. To address the molecular mechanism(s) show that many genes in astrocyte regulated by Sp1 control the levels of glutamate and other growth factors, which are important for neuron function and survival. In conclusion, Sp1 in astrocyte regulates many genes related to neurogenesis.
Transcriptional Regulation by Mediator Kinases during Starvation or Proliferation
Authors: Rubin Jonathan D. , Dowell Robin D. , Taatjes Dylan J.
Published: November 2023

The objective of this research is to better understand the role of Mediator kinase activity in transcriptional regulation. Mediator is a multi‐subunit protein complex necessary for regulated transcription of all RNA polymerase II (RNAPII) transcripts. Mediator kinases (CDK8 and CDK19) form 4 subunit modules which reversibly associate with Mediator affecting transcription in poorly understood ways. Overexpression of CDK8 has been shown to correlate with tumor aggressiveness and mortality in colorectal cancer patients and the kinase activity of CDK8 and CDK19 promotes proliferation of acute myeloid leukemia in cells and in vivo. The kinase activity associated with these enzymes and how this relates to transcriptional regulation is not well understood because specific inhibitors of Mediator kinase activity have only recently been discovered.

The study of genome‐wide phenomena has been aided greatly by the development of next generation sequencing. A recent sequencing technique, global run‐on with sequencing (GRO‐Seq), measures genome‐wide nascent transcription and allows for the unprecedented characterization of general transcription machinery. Serum response facilitates the study of transcriptional regulation by coordinating cells and increasing transcription at known genes involved in serum response. We performed GRO‐Seq on cells during serum response with and without the specific Mediator kinase inhibitor Cortistatin A (CA).

Preliminary results show that qualitatively, genes involved in serum response have increased GRO‐Seq read counts at initiation regions following Mediator kinase inhibition. RNAPII pausing as determined by a pausing index is significantly increased following CA treatment for a large number of genes. Gene ontology (GO) enrichment analysis revealed that these paused genes are associated with metabolic processes. Additionally, this dataset was analyzed using a newly developed technique which detects eRNAs from GRO‐Seq datasets and measures transcription factor (TF) motif enrichment at eRNA epicenters. We found that MNT was significantly more enriched at eRNAs following Mediator kinase inhibition with CA. MNT is a known antagonist of MYC, a major downstream effector of MAPK signaling, and is a key regulator of the MYC transcriptional network in vivo. These results suggest that Mediator kinase activity is involved in activating metabolic pathways during serum response potentially through the regulation of transcriptional pausing or elongation and TF activation.

Support or Funding Information

NIH SCR Training Grant ‐ T32 GM08759
Using artificial transcription factors to induce differentiation into cardiomyocytes
Authors: Heiderscheit Evan Anthony , Eguchi Asuka , Wleklinski Matthew J , Spurgat Mackenzie C , Ansari Aseem Z
Published: November 2023

Differentiation of pluripotent cells has become an effective way to study diseases in vitro as well as to engineer tissues for regenerative medicine. However, the discovery of natural transcription factors that specifies cells to desired fates is challenging, expensive, and labor‐intensive. Instead of taking a trial and error approach toward identifying key regulatory genes, artificial transcription factors (ATFs), capable of regulating genes independently of the endogenously expressed proteins, can provide a way to study the transcriptional networks of specific cell states in an unbiased manner. By using a genome‐scale library of ATFs, each comprised of a zinc finger DNA‐binding domain, thousands of genes can be tested in parallel. We applied this forward genetic approach in the differentiation of human pluripotent stem cells into cardiomyocytes. Cardiomyocyte differentiation relies on the temporal regulation of Wnt signaling via small molecule inhibitors. First, a Wnt signaling agonist (CHIR99021) is used to specify pluripotent cells into a mesodermal lineage, followed by treatment with an antagonist (IWP2) to induce cardiomyocyte differentiation. While this method of generating cardiomyocytes is efficient, the off‐target effects of the inhibitors used in this protocol remain unknown, and therefore, the gene networks activated by the inhibitors are not completely understood. By replacing the function of these inhibitors with ATFs, the specific genes involved in differentiation can be identified. The combinations of ATFs identified in the screen were capable of replacing IWP2, an inhibitor that prevents proper Wnt secretion. After confirming that these ATFs are capable of replacing the function of IWP2, cognate site identification was used to determine the optimal binding motifs preferred by the identified ATFs. These binding motifs were then mapped to the genome to identify genes with an ATF binding site within 1 kb of the transcriptional start site. The genes identified in this screen provide further insights into the transcriptional networks controlling the differentiation of human stem cells into cardiomyocytes, and the tools used in this project can be implemented in other biological contexts where cell fate‐defining regulatory networks have been more elusive.
Haematological and miRNAs (let‐7g, miR‐21, miR‐141) expression modulation profile in serum samples of Human prostate cancer
Authors: JAMES AYORINDE BABATUNDE , FADAKA ADEWALE OLUWASEUN , MAGBAGBEOLA OLUBUNMI ABIOLA , OTURU ABAYOMI , KOLAWOLE OLUFEMI OMOWUNMI , OGUNJIMI ADEBISI , OSHODI TEMITOPE , HABEEBU MUHAMMED , ONAWOGA FATIMA OMODEINDE , AJOGBEJE EBENEZER OLALEYE
Published: November 2023

MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. This study investigated the haematological and miRNA expression modulation profile in serum samples of human prostate cancer.

Twenty‐two study participants with age range between 59 and 82yeaars were included in the study after informed consent. Blood samples of the participant were obtained and both haematological parameters and miRNA analysis (miR‐21, miR‐141 and let‐7g) were performed using Bc‐3200 auto‐haemalogical analyzer and RT‐PCR.

The result shows that there was no correlation between the haematological profile and the modulation of miR‐21, miR‐141 and let‐7g. There was no significant difference in the haematological profile between the healthy control and prostate cancer (PCa) patients. However, miR‐21, miR‐141 and let‐7g (p<0.0001) expressions were significantly up regulated although, miR‐21 expression was more pronounced in the serum of the study participants relative to healthy control group.

Analysis of miR‐21, miR‐141 and let‐7g might have clinical utility as a supplement to prostate specific antigen screening.
Cyclosporin A Alters Expression of Renal MicroRNAs: New Insights Into Calcineurin Inhibitor Nephrotoxicity
Authors: Bai Yun , King Clayton , Francis Cynthia , Gooch Jennifer
Published: November 2023

Background

Calcineurin inhibitors are powerful immunosuppressants that revolutionized organ transplantation. However, non‐immune effects of the calcineurin inhibitor, cyclosporin A (CsA), have significantly hindered their use. Specifically, nephrotoxicity, which is associated with tubulointerstitial fibrosis, inflammation, and podocyte damage, affects up to half of all transplant patients. Calcineurin is involved in many aspects of kidney development and function. Therefore, mechanisms of CsA‐induced nephrotoxicity are complex and not yet fully understood. MicroRNAs (miRs) are short non‐coding RNAs that regulate protein‐coding RNA expression through post‐translational repression of target messenger RNAs (mRNAs). MicroRNA dysregulation is known to be involved in kidney diseases including fibrosis.

Objective

This study investigates the microRNA expression changes induced by cyclosporin A.

Methods and Results

We compared the renal microRNA expression profiles between mice that received CsA (20mg/kg) or vehicle daily for 6 weeks. The results demonstrated that CsA induced significant changes in renal microRNA expression. We used combined criteria of False Discovery Rate (≤0.1), fold change (≥2) and median signal strength (≥50) and identified 76 expression level significantly changed microRNAs. This approach identified microRNAs previously linked to renal fibrosis including let‐7d, miR21, miR29, miR30, miR130, miR192, and miR200 as well as microRNAs that have not been reported to be related to nephrotoxicity or immunosuppression. Pathway analysis of microRNA/mRNA changes highlighted the Wnt, TGF‐β, mTOR, and VEGF pathways. The mRNA expression profiles were compared in the same samples. The change of mRNA and microRNA profiles showed close correlations. To validate that the observed microRNA and mRNA expression level changes in mice kidney tissue were directly related to CsA treatment, the expression of three microRNAs (miR21, miR186, and miR709) and three mRNAs (bmpr1a, smurf1 and smad 7) were compared in the HEK293 cell line. A similar trend of expression level change was induced by CsA treatment in all selected microRNAs and mRNAs in the in vitro cell model. These data provide a roadmap for future work to study the role of the known and novel candidate microRNAs in the mechanism of nephrotoxicity and future therapeutic potential.

Conclusions

Our study profiled expression of microRNA and mRNA genes in mouse kidney after kidney fibrosis has been induced (the major sign of kidney toxicity) and compared the pairwise expression profiles. The results indicate that CsA induces an extensive change in both microRNA and mRNA gene expressions compared to control mice. In addition, the microRNA and mRNA profiling results show a close correlation.

Support or Funding Information

NIH1R15M113120‐01
Non‐coding RNA Editing Involved in Adipose Dysfunction during Aging
Authors: Seidler Abbagael , Marcelo Aileen , Page Jamika , Santanam Nalini
Published: November 2023

Adipose tissue is one of the key endocrine organs that maintain lipid and glucose homeostasis. Adipose dysfunction increases upon aging, and results in dramatic alterations in fat mass, distribution and function. This leads to age associated metabolic dysfunction and increased risk to cardiometabolic diseases. Continuous replenishment of mature adipocytes by differentiation of pre‐adipocytes is a must for normal adipose function. We recently showed decrease in adipogenesis during aging was due to dysfunction in microRNA's (miRNA) involved in adipogenesis (miR‐143). This dysfunction may be attributed to changes in enzymes involved in biogenesis of miRNA (DROSHA, DICER) or enzymes involved in modifying its function (ADAR). We determined the differences in the expression levels of these enzymes in Fisher 344 x Brown Norway hybrid rats (FBN) at ages 8 mo and 25 mo old (n=3). There were sex specific differences in the levels of both biogenesis and editing genes in peri‐gonadal adipose tissue from FBN rats with aging. The DROSHA and DICER levels were higher but editing enzymes (ADARs) lower in female rats and the reverse in male rats with aging. Sanger sequencing of miR‐143 (a key miRNA in adipogenesis) showed edited sequence in old rats compared to younger rats. Sex‐specific post‐transcriptional regulation of miRNA(s) involved in adipogenesis during aging in whole adipose tissue will also be explored in pre‐adipocytes and mature adipocytes from aging rats and correlated to metabolic changes leading to insulin resistance.

Support or Funding Information

Supported by NIH‐NIA‐R15 AG051062 (NS); Marshall University Biomedical Sciences Graduate Program, the Marshall University Foundation Summer Research Internship for Minority Students Fund, and by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence
“Listening in” ‐ The cross‐talk between mother and infant through exosomal microRNAs in breast milk
Authors: Kraft Jamie Dale , Altosaar Illimar
Published: November 2023

Breast milk is an intricate source of key nutritional complexes required to nourish an infant as well as facilitate the development of the infant's immune system. During the postnatal period, the gastrointestinal tract (GIT) matures following enteral feeds and colonization with commensal bacteria. The development of the GIT could be delayed or altered in a premature infant, thus predisposing the infant to inflammatory diseases such as necrotizing enterocolitis (NEC). NEC is the leading cause of death among premature infants. The pathophysiology is currently unknown, however, prematurity, formula feeding and an abnormal intestinal microbiota are thought to cause an inflammatory response in the intestine which can lead to NEC and sepsis. Breast milk is the gold standard of infant nutrition and is largely thought to help prevent NEC.

The components of breast milk responsible for the development of the GIT have yet to be identified, although, novel research has discovered the presence of microRNA (miRNA) in breast milk exosomes. Due to stable structure, exosomes are protected against degradation, suggesting breast milk exosomal miRNAs continue to be biologically active following ingestion by the infant. miRNAs have previously been described as functioning in cellular communication, implying a method of “listening in” to the cross‐talk between mother and infant via breastfeeding.

To characterize miRNAs and identify the role they play in the development of the GIT, exosomes from breast milk must be isolated in a reproducible method to extract miRNAs. Isolating and storing procedures of unprocessed breast milk are underexposed. In recent studies, it has been revealed that storing unprocessed breast milk at −80°C or −4°C lead to cell death within the milk, causing contamination of the native breast milk exosomes. The profiling of miRNA fluctuates depending on the milk fraction analyzed, preparation of milk and exosome fraction as well as the miRNA extraction protocol. Extra care should be taken when preparing samples and following extraction procedures in this area of research. During this early stage of exploring the ways in which the mother communicates with her newborn, it is necessary to collaborate with other research groups to standardize procedures and to synchronize approaches.
Positive Charge in the n‐region of the Signal Peptide Contributes to Efficient Post‐translational Translocation of Small Secretory Proteins
Authors: Liu Ming , Sun Jinhong , Xiong Yi , Cui Jingqiu , Guo Huan
Published: November 2023

A majority of secretory proteins in mammalian cells undergo signal recognition particle (SRP) dependent co‐translationally translocation across the endoplasmic reticulum (ER). However, due to minimal time for SRP recognition, many small secretory proteins can be post‐translationally tanslocated into the ER. Several chaperones and cellular machinery have been identified to be involved in post‐translational translocation of these small secretory proteins. However, the intrinsic signals of small secretory proteins that contribute to efficient post‐translational translocation remain unknown. Here, we analyzed signal sequences of eukaryotic secretory proteins and found that the percentage of the proteins with positive charge in the n‐region of their signal peptide (SP) increases substantially as the polypeptides become shorter. Eliminating the n‐region positive charge selectively impaired translocation of small secretory proteins. This translocation impairment appeared to be mainly caused by the defect of post‐translational translocation. Slowing down protein translational rate of small secretory proteins could alleviate their translocation defects caused by loss of SP N‐terminal positive charge. Together, these data revealed a previously unrecognized role of n‐region positive charge in ensuring efficiency of post‐translational translocation of small secretory proteins.

Support or Funding Information

This work was supported by NIH RO1‐DK088856, and by the research grants from The National Natural Science Foundation of China 81070629, 81370895, 81570699.
In vivo and in vitro studies of RRF (ribosome recycling factor) revealed that its major function is to release mRNA from the post‐termination complex and not splitting of the ribosomal subunits
Authors: Quaglia Fabio , Kaji Hideko , Kaji Akira , Inokuchi Yoshio
Published: November 2023

In prokaryotes two adjacent ORF are often linked with an overlapping combination of termination and initiation codons with a total of 4 or 5 nucleotides, for example UAAUG or AUGA. In these junctions, ribosomes at the stop codon are released by RRF and some of them re‐bind to the nearby AUG and start translating the downstream ORF. In the absence of RRF, the ribosome at the stop codon remains on the mRNA and would read it in frame with the termination triplet. We studied the role of RRF in these junctions in vitro and in vivo. For in vivo studies we used an E. coli strain with a temperature sensitive mutation of RRF so that we were able to inactivate the function of RRF at the non‐permissive temperature (39°C). We show that for correct reading of the downstream ORF, AUG is essential. The shorter the upstream ORF the lower the downstream reading. Introduction of a complementary sequence to the 3′‐terminal region of 16S rRNA into the mRNA increased downstream reading from AUGA. Shortening of the upstream ORF to 4 codons completely abolished the downstream reading of UAAUG. This suggests that if Shine‐Dalgarno (SD) sequence is near the termination codon, the RRF‐released ribosome is attracted by the SD sequence to the extent that it loses the downstream movement after it is released.

For in vitro studies we used the PURE system so that we were able to omit RRF in the reaction mixture. We have confirmed, in vitro, the essence of the in vivo observation described above using a mRNA having the following sequence: “GGGAAUUCAAAAAUUUAAACAGGUAUACAUACU AUG UUU ACG AUU ACU ACG AUC UUC UUU ACG AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG AUU ACU ACG AUC UUC UUU ACG UAAUG CGU CUG CAG GCA UGC AAG CUA A24A” (Bold character is the junction sequence broken underline is the Shine‐Dalgarno sequence). In the presence of RRF, the downstream reading starts from AUG causing the incorporation of [14C]‐Leucine (CUG and CUA). On the other hand in the absence of RRF, the first triplet read was UGC of UAAUGCGUC and [14C]‐Valine was incorporated due to the codon GUC. Upstream reading was detected by [3H]‐phenylalanine incorporation (due to UUUs and UUCs). With this assay we also showed that Fusidic Acid was able to inhibit RRF at lower concentration than that necessary to inhibit translocation (monitored by the incorporation of [3H]‐phenylalanine), causing the inhibition of the incorporation of [14C]‐Leucine. Moreover, using ribosome with tethered subunits (1), we were able to show that the splitting of the ribosomal subunits was not necessary in the recycling reaction, demonstrating that recycling of the ribosome take place also in the absence of the splitting of the ribosomal subunits.
Deconstructing the Peptide Specificity of TCR Recognition
Authors: Riley Timothy P , Mendoza Juan , Hellman Lance , Garcia KC , Baker Brian
Published: November 2023

T cell receptors (TCRs) have emerged as a new class of therapeutics, most prominently for the treatment of cancer where they are key components of new cellular therapies as well as soluble biologics. Although in many ways similar to antibodies, TCRs differ in the complexity of the receptor‐ligand interface: whereas antibodies can be elicited to almost any antigens, TCRs are restricted to linear peptides presented by class I or class II MHC proteins. Additionally, TCRs do not undergo affinity maturation, and bind with weak‐to‐moderate affinities and reduced specificity.

The off target and organ‐specific toxicities observed in cancer immunotherapy present an obstacle to T cell based therapeutics. A recent clinical trial underscored the need for improved methods to define TCR specificity after melanoma patients treated with TCR engineered T cells suffered from fatal cardiovascular toxicity arising from the unpredicted recognition of a muscle specific peptide. To address this drawback to T cell based immunotherapies, we developed a novel protein engineering approach to define the peptide specificity of a given TCR. Here, a large scale peptide library underwent directed evolution in a yeast display system, where interactions with the melanoma reactive DMF5 TCR acted as the guiding selection pressure. After this technique identified a panel of putative cross reactive peptides, sequence analysis and computational modeling followed by kinetic binding experiments and X‐ray crystallography determined the DMF5 TCR recognizes two distinct classes of peptides through chemically distinct mechanisms. This information led to the rational, structure based design of additional cross reactive peptides and introduces a unique approach to screen the human proteome for TCR targets to avoid unanticipated autoimmune reactions in response to therapeutic TCRs.

Support or Funding Information

This project was supported, in part, with support from the Indiana Clinical and Translational Sciences Institute funded, in part by Grant Number UL1TR001108 from the National Institutes of Health, National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award.
Determining histone deacetylase 8 substrates using non‐natural amino acids
Authors: Lopez Jeffrey , Haynes Sarah , Majmudar Jaimeen , Martin Brent , Fierke Carol
Published: November 2023

The histone deacetylase family is comprised of 18 enzymes that catalyze the removal of an acetyl group from the ɛ‐position of a lysine residue. Lysine deacetylation is a post translational modification that affects over 6000 different macromolecules and has been implicated in various types of cancers and neurodegenerative diseases. To elucidate and identify HDAC substrates, we have incorporated the photo reactive, non‐natural amino acid – p‐benzonyl‐4‐phenylalanine (pBpa) into HDAC8, a structurally well characterized isozyme of this family with a largely unknown substrate pool. Using both photo crosslinking and proteomics will allow us to identify both substrates and binding partners of HDAC8 and enhance the understanding of how this enzyme is regulated in vivo. In addition, this will validate the approach as a feasible system to probe other HDAC isozymes and their specificities.

Support or Funding Information

5‐R01‐GM‐040602 C.A.F.; F31‐GM‐116619 J.E.L.
Multiple Approach to Determine Protein‐Protein Binding Affinity of Calcineurin Homologous Protein Isoforms 1 and 2 and the Sodium Hydrogen Exchanger Isoform 1
Authors: Marshall Chelsea N , Wallert Mark Anthony , Provost Joseph John
Published: November 2023

Protein‐protein interactions direct many important cellular functions. The ability to characterize and quantitate these interactions is importance when studying the mechanisms of biological processes. We are interested examining protein protein interactions experiments between Calcineurin Homologous Protein Isoforms 1 and 2 (CHP1&2) with the Sodium Hydrogen Exchanger 1 (NHE1). NHE1 is a ubiquitous transmembrane protein shown to be of importance in lung cancer proliferation and metastasis. CHP1 and CHP2 are calcium binding proteins that interact with proteins on the plasma membrane and nuclear proteins/transcription factors. Both isoforms share a 61% amino acid sequence homology, both bind to nearly the same residues of NHE1 and have been suggested to bind NHE1 with different levels of affinity. Thus to understand the nature of CHP1/2 – NHE1 interactions we wish to develop a quantitative interaction assay. However, CHP proteins possess several solvent exposed hydrophobic residues making expression, stable solubility and precipitation problematic. To investigate the interaction between these proteins, we have generated several fusion proteins: GST‐NHE1‐CBD (CHP binding domain), 6X His CHP1, 6XHis CHP2 and RFP/GFP His tagged CHP1 and CHP2. We investigated protein‐protein interactions using fluorescent thermal shifts, bait‐pray pull down assays, circular dichroism thermal shifts and quartz crystal microbalance measurements to determine the application of each technique to determining binding affinities.
Characterization of ppGpp‐Binding Proteins in Escherichia coli
Authors: Nguyen Duc Tien , Jung Che Hun
Published: November 2023

Guanosine‐5′‐diphosphate 3′‐diphosphate (ppGpp) is a bacterial signal molecule that generates stringent responses under conditions of deprivation and amino acid deficiency. In recent years, substantial progress has been made in our understanding of the molecular mechanisms of ppGpp metabolism and ppGpp‐mediated regulation. Although many studies focus on studying direct and indirect targets of ppGpp regulation, the overall mechanism of ppGpp is still not clear. In this study, ppGpp was immobilized on NHS‐activated Sepharose. ppGpp binding proteins were isolated by affinity chromatography, and further separated by 2‐D gel electrophoresis. The resolved proteins were identified by MALDI‐TOF MS. The tryptic hydrolysates were also analysed by LC‐MS/MS. Among the identified proteins, some proteins of interest were cloned and over‐expressed. The cloned proteins (pepB, iadA, map, dld, ldh, gldA, lon, ycbZ, pyrD, lpdA) were tested for their binding to ppGpp by fluorescence spectroscopy and were indentified as ppGpp‐binding proteins with various Kds. Interestingly, we found ppGpp inhibit membrane D‐lactate dehydrogenase non‐competitively with Ki 48 mM and fermentative D‐lactate dehydrogenase competitively with Ki 120 mM. These results provide that this method will be very effective for screening proteins which new plausible ppGpp regulated proteins. And further studies on these proteins will expand the scope for the functions of ppGpp.

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BK21 PLUS (Brain Korea 21 Program for Leading Universities & Students), National Research Foundation (NRF) of Korea
Structural and Biochemical Characterization of a Periplasmic Lipoprotein with a Role in Adaptation of Salmonella to Magnesium Limitation
Authors: May John F , Rasmussen Damien M , Davie Timothy J , Vaneerd Cody , Groisman Eduardo A , Bhattacharyya Basudeb
Published: November 2023

Living cells require magnesium ions for many fundamental biochemical processes. Certain pathogenic bacteria can thrive in environments with limiting magnesium, and the ability to adapt to magnesium limitation can be critical for bacterial virulence. We have used genetic and biochemical approaches to gain insight into how Salmonella enterica, a major bacterial cause of food‐borne illness, adapts to low magnesium. Using a genetic screen in a Salmonella mutant defective for adaptation to low magnesium, we identified a periplasmic lipoprotein protein that promotes growth in low magnesium. This Salmonella protein, which has a monomeric molecular weight of 19.7 kDa, contains a periplasmic localization sequence followed by a single domain of unknown function that is conserved among enterobacteria. To elucidate its function, we have purified and crystallized this protein as well as obtained atomic‐resolution X‐ray diffraction data. Circular dichroism spectroscopy indicates that the protein consists predominantly of beta‐sheet secondary structure, and the diffraction data indicate there is likely more than one protein monomer in the asymmetric unit. Overall, our results demonstrate a physiological function in magnesium ion homeostasis for a previously uncharacterized periplasmic lipoprotein in Salmonella, and we have obtained the first atomic‐resolution structural data for a member of this family of proteins. These discoveries will provide insight into molecular mechanisms of bacterial adaptation to magnesium limitation.

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This research is supported by University of Wisconsin‐La Crosse Undergraduate Research and Creativity Grants (DR, TD), the McNair Scholars Program at UW‐La Crosse (DR), and a University of Wisconsin‐La Crosse Faculty Research Grant (JM). The authors would also like to thank Professor James Keck (University Wisconsin, Department of Biomolecular Chemistry) for graciously allowing access to the Advanced Photon Source. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE‐AC02‐06CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817). GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB‐12002) and the National Institute of General Medical Sciences (AGM‐12006).
Towards the structure of PE5‐PPE4‐EspG3 heterotrimer from Mycobacterium smegmatis to elucidate PE‐PPE dimer recognition by cognate EspG
Authors: Williamson Zachary , Ciocca William , Reed Robert , Korotkov Konstantin
Published: November 2023

Mycobacteria use a specialized Type VII Secretion (T7S) system in order to translocate protein substrates across their highly hydrophobic didermal cell envelope. In the pathogenic species the T7S system is crucial for the secretion of various virulence factors. There are five T7S loci in the mycobacterium genome, classified ESX‐1 through ESX‐5. ESX‐1, ESX‐3, and ESX‐5 have been previously shown to secrete substrates, while there is lacking evidence that ESX‐2 and ESX‐4 are functional systems. All three of the functional systems secrete substrates from the large PE and PPE protein families, named for their Pro‐Glu and Pro‐Pro‐Glu residues respectively. Nearly all PE proteins are found in a heterodimer with a specific PPE protein. A system‐specific chaperone, EspG, for the PE/PPE dimers is encoded by each functional ESX. Each EspG protein interacts with the PPE substrate of the dimer via a hh motif that is conserved within secreted PPE proteins. Yet, the PPE substrates are only recognized by their cognate EspG partners. The goal of this study is to solve the structure of the PE5/PPE4/EspG3 trimer from ESX‐3 and perform structural comparison to the homologous ESX‐5 trimer to uncover key residues that elicit the specificity of the EspG interaction.
Characterization of the Role for the N‐Terminal Domain on Sil1 Activities as a Nucleotide Exchange Factor and Reductase
Authors: Pareja Kristeen Alcaide , Sevier Carolyn
Published: November 2023

The accumulation of reactive oxygen species (ROS) can lead to oxidative stress and play a role in human diseases such as cancer and neurodegenerative diseases. We are interested in studying how cells use ROS as signaling molecules to maintain a balanced redox environment. Using yeast as our model system, we have previously shown that the Hsp70 in the endoplasmic reticulum (ER), BiP, acts as a direct sensor of peroxide, a type of ROS produced during protein folding. BiP has two major domains that regulate its function as a chaperone: an ATPase domain and a substrate‐binding domain. Under ER oxidative stress, yeast BiP's conserved cysteine gets modified/oxidized by peroxide. Modification of BiP's cysteine causes a conformational change, which enhances BiP's ability to bind to substrates, leading to protection of cells under ER oxidative stress. When cells go back to non‐stress conditions, reversibility of BiP's cysteine modification is important for normal cellular function and survival. We have evidence that the protein Sil1, a known nucleotide exchange factor (NEF) of BiP, can remove the modification on BiP's cysteine. We have shown that in addition to acting as a NEF, Sil1 has an unexpected reductase activity that depends on two cysteines, Cys‐52 and Cys‐57, located at the N terminus. While the N‐terminal domain is required for Sil1 activity as a reductase, Sil1 is competent as a NEF even in the absence of the N‐terminal domain. Our goal is to further elucidate the molecular details relating to how the N‐terminal region of Sil1 impacts both Sil1 NEF and reductase activity by utilizing a variety of biochemical and biophysical approaches. Our preliminary data suggest that NEF activity is influenced by the presence of the N terminus. Our current data also suggest the N‐terminal domain may adopt multiple conformations, allowing for regulation of Sil1 NEF activity and potentially its activity as a reductase. Of note, mutations in the Sil1 gene have been found in over half of patients with Marinesco‐Sjögren Syndrome (MSS), a rare autosomal disease characterized by early onset cataracts, cerebellar ataxia, and delayed mental development. One such mutations maps near the N‐terminal domain, and we expect that characterization of full‐length Sil1 may also lend further insight into how mutations in Sil1 can cause MSS.

Support or Funding Information

NSF GRFP and SUNY Diversity Fellowship
An Alternative Structural Model of Activation for the Anti‐Anti‐σ Factor PhyR and Interaction with the Anti‐σ Factor NepR
Authors: Luebke Justin L , Eaton Daniel S , Sachleben Joseph R , Crosson Sean
Published: November 2023

Protein phosphorylation is a universally conserved post‐translational modification. In bacteria, aspartyl phosphorylation generally controls transcriptional regulatory proteins, enabling cells to respond to chemical and physical changes in their local environment. In the large and diverse alphaproteobacteria class of bacteria, stress monitoring is primarily regulated by the general stress response (GSR). PhyR is the master regulator of the GSR and is a two‐domain anti‐anti‐σ factor containing a phosphorylatable response regulator receiver domain and a σ‐like output domain. PhyR titrates the anti‐σ factor NepR away from its cognate σ factor in a phosphorylation‐dependent manner to ultimately control global changes in gene expression. The binding site of NepR within PhyR is largely occluded at the domain interface. It has long been hypothesized that phosphorylation of PhyR results in undocking of the two domains to expose the high‐affinity NepR binding site; however, the foundations by which phosphorylation of the PhyR receiver domain controls structure and function remains largely undefined. Small‐angle X‐ray scattering (SAXS) and molecular dynamics (MD) simulations indicate phosphorylation is insufficient to drive PhyR opening. Recent biophysical and biomolecular NMR data indicate intrinsically disordered regions of NepR play a significant role in PhyR activation. These interactions drive an unstructured to structured transition of NepR and are required to facilitate both PhyR phosphorylation and PhyR‐NepR binding. This leads to a model where phosphorylation is necessary but insufficient to expose the high affinity NepR binding site of PhyR and interactions between PhyR and NepR prior to phosphorylation are a necessary component of the structure‐function relationship of these two core regulatory proteins.

Support or Funding Information

NIH 5R01AI1707159