bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying the Molecular Mechanisms by which Disruptor of Telomere silencing 1‐Like (DOT1L), a Histone 3, Lysine 79 (H3K79) Methyltransferase, Regulates Mammalian Hematopoiesis
Authors: Malcom Carrie , Piasecka‐Srader Joanna , Alvarez Nehemiah , Slawson Chad , Fields Patrick
Published: November 2023

Disruptor of Telomere silencing 1‐Like (DOT1L), is a histone 3, lysine 79 (H3K79) methyltransferase. The enzyme has been implicated in multiple processes, including activation of transcription, regulation of the cell cycle, leukemogenesis, and mouse embryonic development. Previous studies in our lab found that Dot1L deficiency results in an erythropoietic defect, leading to lethal anemia at around mid‐gestation (Feng et al., 2010). The precise molecular mechanism(s) by which DOT1L regulates embryonic hematopoiesis has not yet been elucidated and is the overall objective of this study. Specifically, we sought to determine whether the methyltransferase activity of DOT1L is essential for hematopoiesis. DOT1L is a large protein (1540aa) and involved in several, diverse processes, but only its methyltransferase activity has been documented. Additional functional domains of the protein might be responsible for its diverse activities.

We developed a murine embryonic stem cell (mESC)‐based culture system in which to examine the role of DOT1L in hematopoiesis, in vitro. Using the Cas9/CRISPR system, we created two different mutations in Dot1L in cultured mESCs: a Dot1L knockout and a Dot1L point mutant. Knockout mESCs contain a large, out‐of‐frame deletion in both alleles of Dot1L, while the point mutant contains a single amino acid change in the methyltransferase domain of Dot1L, thereby eliminating its methyltransferase activity, but preserving the rest of the protein. We then induced these Dot1L knockout and point mutant mESC clones, along with wildtype mESCs, to undergo hematopoiesis in culture. Our data showed that this protocol leads to similar erythropoietic defects as those observed in vivo. Dot1L knockout and point mutant mESCs are capable of forming the same types of hematopoietic colonies as wildtype mESCs (both erythroid and myeloid), but there is a decrease in colony size and number compared to wildtype. These results suggest that the methyltransferase activity of DOT1L plays a predominant role in the activity of the protein as a whole, and is entirely responsible for its function in facilitating early, murine hematopoiesis.

Support or Funding Information

R01 DK091277, Fields (PI), 04/01/12‐03/31/17NIH/NIDDKRole of the Histone Methyltransferase DOT1L in Embryonic Erythropoiesis
Decline in Arylsulfatase B and Overexpression of Galactose 6‐Sulfatase Both Increase DNA Methyltransferase Activity and Wnt Signaling in Prostate Stem Cells
Authors: Bhattacharyya Sumit , Feferman Leo , Tobacman Joanne Kramer
Published: November 2023

The enzymes arylsulfatase B and galactose 6‐sulfatase remove sulfate groups from sulfated glycosaminoglycans. ARSB removes 4‐sulfate groups from chondroitin 4‐sulfate and dermatan sulfate, and GALNS removes 6‐sulfate groups from chondroitin 6‐sulfate and keratan sulfate. In neonatal rat prostate, arylsulfatase B was predominant in the stroma, and GALNS in the epithelium. Other findings have demonstrated transcriptional effects due to decline in ARSB and the associated increase in chondroitin 4‐sulfation, leading to reduced binding of galectin‐3 with chondroitin 4‐sulfate. Increased nuclear galectin‐3 interacted with transcription factors Sp1 to increase the transcription of Wnt9A. Other studies demonstrated increased Wnt signaling following decline in ARSB, with increased nuclear beta‐catenin, increased nuclear DNA binding of TCF/LEF and c‐myc and increased expression of c‐myc. In this study, effects on DKK3 expression and DNA methyltransferase were evaluated, to determine if reduced expression of DKK3, an inhibitor of Wnt signaling, contributed to the increase in Wnt signaling following ARSB silencing. Methods included: growing human prostate stem cells (ATCC), silencing DKK‐3 by siRNA, measuring DNMT activity, determining DKK3 expression, analysis of DKK3 promoter methylation, and analysis of effects on Wnt signaling. ARSB was silenced and overexpressed, and GALNS was silenced and overexpressed, as previously, and the effects ascertained by measurements of enzyme activity. The effects on DKK3 expression, DNMT activity and expression, and Wnt signaling were detemined. Findings included: increased DNMT activity following ARSB silencing and GALNS overexpression, decline in DKK3 promoter activation and DKK3 expression following ARSB silencing and GALNS overexpression. Since studies have shown ARSB predominance in stroma and GALNS in epithelium, the similar effects of GALNS overexpression and ARSB silencing suggest a profound interaction related to sulfatase activity and chondroitin sulfation. Both ARSB silencing and GALNS expression lead to relative increase of chondroitin 4‐sulfation vs. chondroitin 6‐sulfation. These findings suggest that developmental regulation related to wnt signaling may be mediated by changes in chondroitin sulfation and sulfatase activity.
Kinetic studies of histone acetylation effect in nucleosome disassembly mediated by histone chaperone Nap1
Authors: Lee Jae Hyoun , Lee Tae‐Hee
Published: November 2023

We studied kinetics of nucleosome disassembly mediated by histone chaperone Nap1. We found that DNA and dimer motion is not synced during the disassembly of a nucleosome. Histone acetylation seems to not alter the pathway of disassembly, however, the acetylation affects the kinetic rates of each step of the disassembly, based on which we explained the biophysical meanings of the histone acetylation.
Histones are Targets for Modification by Glucose‐Derived Methylglyoxal
Authors: Galligan James J , Mitchener Michelle M , Wauchope Orrette R , Wang Tina , Rose Kristie L , Spiegel David A , Marnett Lawrence J
Published: November 2023

Diabetic nephropathy (DN) is the major cause of morbidity and mortality in diabetic patients. The sustained hyperglycemia associated with diabetes results in substantial oxidative stress and inflammation, leading to the generation of the reactive a‐oxoaldehyde, methylglyoxal (MGO). MGO has been shown to play an integral role in DN pathogenesis and its effects are often seen in DN patients, despite glycemic control, via a phenomenon called metabolic memory. Metabolic memory is hypothesized to result from alterations in histone posttranslational modifications. Here, we describe histones as targets for modification by MGO in a cell culture model of hyperglycemia. Utilizing novel LC‐MS/MS methodologies developed in our laboratory, histones were purified from cultured HEK293 cells treated with low (5mM) or high (25mM) glucose. Following exhaustive proteolytic digestion, absolute concentrations of histone posttranslational modifications were quantified by LC‐MS/MS. These analyses revealed both Lys and Arg‐derived MGO adducts, with the Arg hydroimidazalone (MG‐H) adducts being the most prevalent. To validate that these adducts were indeed derived from cellular MGO, glyoxalase 1 was knocked down, resulting in substantial increases in both cellular MGO and MG‐H adducts. Sites of MGO modification were identified via high‐resolution mass spectrometry, which revealed Lys and Arg adducts on all four‐core histones. We hypothesize that MGO adduction of histones offers a novel link between inflammation, hyperglycemia, and DN pathogenesis. The effects of these modifications on cellular homeostasis are the subject of current investigation.
Quantitative Histone H3 Modification Characterization of Differentiating Hematopoetic Murine Erythroleukemia (MEL) Cells Using Parallel Reaction Monitoring Mass Spectrometry
Authors: Hess Sonja , Moradian Annie , Raedle Matthias , Sweredoski Michael
Published: November 2023

Murine Erythroleukemia cells differentiate into hematopoetic cells that produce either embryonic or adult hemoglobin, dependent on the chosen inducer (histone deacetylase inhibitor butyric acid vs. DMSO). The differentiation has, at least in part, been associated with changes in histone modifications that are not readily discernible by classic biochemical analyses such as antibodies or classical bottom‐up mass spectrometry.

To establish a quantitative method for histone modifications, we developed a middle‐down proteomics workflow with electron transfer dissociation using a targeted acquisition mode, parallel reaction monitoring (PRM), on an Orbitrap Fusion.

We then monitored the twenty most abundant combinations of methylation and acetylation of the N‐terminal tail of histone H3. The N‐terminal H3 tail was separated primarily by its acetylation states using weak cation exchange hydrophilic interaction chromatography. In addition, some separation can also be achieved by its methylation states.

After extensive data evaluation, a minimum set of over 250 H3 proteoforms was identified. More importantly, quantitative acetylation and methylation maps were created. MEL cells treated with HDAC inhibitor butyric acid showed a bimodal distribution with one cluster showing significantly more acetylations and methylations than untreated samples and samples treated with DMSO. A second cluster emerged from the butryic acid treated samples showing significantly less acetylations and methylations than untreated and DMSO‐treated samples.

Furthermore, we developed a fragment ion map to visualize specific differences between treated and untreated samples.

Taken together the data presented here show that middle‐down proteomics with electron transfer dissociation using PRM is a novel, attractive mass spectrometry method for the effective analysis and quantification of histone tails.

Support or Funding Information

This project was supported by the Gordon and Betty Moore Foundation through grant GBM275, the Beckman Institute, HHMI (Fusion) and UO1 grant 4DNucleome.
Analysis of Long Noncoding RNA Chromatin Interactions by Chromatin Isolation by RNA Purification (ChIRP)
Authors: Asbrock Nick
Published: November 2023

Gene regulation plays a critical role in complex cellular processes such as development, differentiation, and cellular response to environmental changes. While the regulation of gene expression by transcription factors and epigenetic influences has been well studied over time, pervasive genomic transcription and the role of non‐coding RNAs in this process is a rapidly evolving field that remains to be thoroughly explored.

Chromatin Isolation by RNA Purification (ChIRP) is a method that allows discovery of the sites of interaction of chromatin‐associated RNAs (e.g. lncRNAs) with genomic DNA sequences by using probe‐based hybridization to target RNA molecules in chromatin. DNA can be isolated from recovered chromatin and analyzed by quantitative PCR or next generation sequencing (ChIRP‐seq). Alternatively, RNA may also be isolated to detect other RNA molecules that may be associated with the RNA of interest.

To enable the exploration of these RNA interactions in chromatin regulation, we have optimized the methods and developed ChIRP reagents. Using these reagents ChIRP experiments can be performed with reliable recovery of chromatin using lncRNA or other chromatin associated RNA as targets. Additionally, negative and positive control probe sets, and detection primers were developed to ensure first time success. We have performed ChIRP experiments with a HeLa cell lysate and capture oligos for the NEAT1 lncRNA. Isolated DNA was subjected to NGS library construction and sequenced on an Illumina HiSeq™ instrument. Sequences were aligned to the reference genome (hg19) and peaks were called. We successfully identified several NEAT1 binding sites in the genomic DNA sequence.

In summary the methods developed allow unbiased discovery of RNA‐associated genomic DNA sequences, RNA sequences, and potentially proteins.
Alternatively spliced novel transcripts of mouse Nur77 gene identified using bioinformatics and molecular biology techniques
Authors: Abul Kalam Sayeed ur Rehman , Tabish Mohammad , Jairajpuri Mohamad Aman
Published: November 2023

Pre‐mRNA alternative splicing generates multiple mRNAs that code for different protein isoforms having similar, different or opposing functions. It is a highly regulated process that controls different pathways inside the cell. Identification of alternatively spliced isoforms is required in order to establish the functions of a gene. In this study, with the help of bioinformatics tools and molecular biology techniques, we have identified novel alternatively spliced transcripts of Nur77 gene in mouse. Nur77 is an intracellular transcription factor involved in cell cycle mediation, inflammation and apoptosis. Translocation of Nur77 protein from nucleus to mitochondria induces apoptosis. We have identified two new exons upstream of reported first exon located in the 5′ UTR of the gene. These exons give rise to two different transcript variants by alternative splicing. Different pos‐translational modifications of the N‐termini of conceptually translated transcripts were studied using ExPASy tools. Translation efficiency and predicted half life of protein isoforms were analysed by terminator analysis tool. The diversity at N‐termini of newly predicted proteins explains the diverse function of Nur 77 gene in mouse.
Identification and Characterization of a Novel Alternatively Spliced Isoform of IRF‐1 Gene in Mouse
Authors: Sarwar Tarique , Tabish Mohammad
Published: November 2023

Interferon regulatory factor 1 (IRF‐1) is one of nine members of the IRF family of transcription factors. It functions as a transcriptional activator or repressor of a variety of target genes by binding to an interferon stimulated response element (ISRE) in their promoters. IRF‐1 has been shown to play roles in the immune response, regulating apoptosis, tumour suppression and DNA damage. Alternative splicing of a single primary transcript of genes yields different mature mRNAs that can produce protein isoforms having diverse and even opposing functions. In the present study, using a combination of different bioinformatics tools, we have identified a novel transcript variant of IRF‐1 in mouse having a alternatively spliced new first exon. This new exon splices with existing exon 2 after replacing exon 1 of the repoted IRF‐1 generating a novel transcript. Using primers designed specific for the newly predicted first exon, the existence of the novel transcript variant was confirmed by RT‐PCR and semi‐nested PCR. This novel transcript was found to be differentially expressed in different tissues. PCR product corresponding to new splice variant was sequenced and was compared with the reported transcript. Using real time PCR, relative expression level of newly identified transcript variant with respect to the reported transcript was also studied. The novel transcript variant was found to have different transcription factor binding sites with a distinct promoter. Post translational modification analysis of conceptually translated novel transcript was also performed. The new protein variant display heterogeneous properties in having different phosphorylation and acetylation sites. Thus, alternative splicing of IRF‐1 producing a novel transcript increases the diversity of the gene.
Design of a Pre‐Trans‐Splicing Molecule to Generate a Soluble Extracellular Peptide Decoy to Block Activation of the Epidermal Growth Factor Receptor Pathway in Human Glioblastoma Cells
Authors: Falotico Sarah C
Published: November 2023

Although glioblastoma multiforme (GBM) is the most common brain malignancy, survival is limited to about 14 months. Current treatments are limited by the blood‐brain barrier. Tyrosine kinase receptors (TKRs) such as epidermal growth factor receptor (EGFR) are often overexpressed in GBM. The pre‐mRNA EGFR transcript consists of 28 exons and intervening sequences; the extracellular domain spans exons 1–16, the transmembrane domain corresponds to exon 17, followed by the intracellular domain at the 3′ end of transcript. In our novel approach, we have created an exogenous pre‐trans splicing molecule (PTM) with a poly‐A signal that is designed to splice to the upstream, extracellular region of the EGFR transcript. The early termination of the EGFR transcript leads to short soluble EGFR decoys, unbound from the cell membrane. More specifically, we have engineered a PTM designed with a binary antisense binding element directed against the EGFR transcript, a U7 snRNA spliceosome localization signal with a strong ‘kiss’ domain for stabilization, an intronic spacer region, a strongly recognized 3′ splice site, and a 3′ poly‐A signal. Potential targets within the EGFR transcript were ranked based on 5′ and 3′ splice site strength, presence of splicing elements and optimal intronic length. The junction of intron 15 and exon 16 was ultimately chosen for its relatively strong 5′ splice site, weak 3′ splice site, and long intronic length which provides a kinetic advantage for the trans‐splicing event. The intronic spacer region was designed devoid of splicing elements to provide a spacer between the antisense and 3′ splice site. The 3′ splice site consists of a branch point, the AG dinucleotide exclusion zone, a polypyrimidine tract, and a modified 3′ splice site with the crucial AG dinucleotide for activation. At the 3′ end the Simian Vacuolating Late 40 poly‐A signal was chosen based on its efficiency in vivo. The PTM was cloned into a plasmid adeno‐associated virus (pAAV) expression vector headed by the CMV promoter. The PTM therapy vector is being tested in human glioblastoma cell lines to determine trans‐splicing efficacy and the alteration in EGFR transcript and protein levels.
The role of histone variant H2A.Z in co‐transcriptional splicing in Saccharomyces cerevisiae
Authors: Neves Lauren Taylor , Douglass Stephen , Galivanche Anoop , Johnson Tracy
Published: November 2023

In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre‐messenger RNA (pre‐mRNA). Although RNA‐synthesis and RNA‐processing have typically been studied as biochemically distinct reactions, recent studies have shown these two processes to be spatio‐temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited in to the chromatin by the SWR1‐complex and is typically found near the 5′ ends of protein‐coding genes. H2A.Z has been implicated in transcription elongation and polymerase processivity. Here we show that H2A.Z, encoded by HTZ1 in yeast, is important for efficient splicing of intron‐containing genes, and splicing in cells lacking H2A.Z is impaired under suboptimal conditions, such as mutant splicing complexes or suboptimal splice sites. HTZ1, is particularly dependent on an intact U2 snRNP, as it shows extensive genetic interactions with components of the U2 snRNP and U2 snRNP associated proteins. Consistent with this, H2A.Z is required for the splicing of introns containing a mutated splice site. RNA‐seq reveals that intron‐containing genes with non‐consensus branchpoint sequences are particularly sensitive to loss of H2A.Z. We find that deletion of HTZ1 results in persistent association of the U2 snRNP with nascent RNA, suggesting altered spliceosomal rearrangements in the absence of H2A.Z. Together, these results demonstrate that histone variant H2A.Z is required for efficient splicing of a subset of intron‐containing genes and indicate a role for H2A.Z in co‐transcriptional spliceosome assembly.

Support or Funding Information

Ruth L. Kirschstein National Research Service Award GM007185.
Discovery of mammalian regulators of ribosome biogenesis
Authors: Farley Katherine , McCann Kathleen , Merkel Janie , Surovtseva Yulia , Baserga Susan J.
Published: November 2023

It is well known that in cancer an increased size and number of nucleoli correlates with increased malignant potential. Nucleoli make ribosomes, the essential protein synthesizers of the cell. Previously we have shown (PLoS Genetics 2012) that disruption of nucleolar function by depletion of the ribosome biogenesis factors UTP4 or NOL11 caused a change in the number of nucleoli per cell from 2–3 to only 1. Taking advantage of this relationship between number and function, we exploited the changes in nucleolar number to discover new regulators of nucleolar function in human cells via a high‐content, high‐throughput, genome‐wide siRNA screen. This screen successfully identified approximately 200 proteins whose depletion caused a change in the number of nucleoli per cell from 2–3 to only 1. The function of a subset of these hits in ribosome biogenesis has been validated by oligonucleotide deconvolution. Of these high‐confidence, validated hits, 22 were chosen for further study, including both nucleolar and non‐nucleolar proteins. While the nucleolar proteins are directly involved in ribosome biogenesis, the non‐nucleolar proteins likely reflect new pathways of nucleolar regulation in mammalian cells. Out of the 22 tested hits, we have identified new roles for 20 hits in regulating either the RNA polymerase I transcription of the ribosomal DNA (8/22), the processing of the pre‐ribosomal RNA (19/22), or in the maintenance of the ribosomal DNA chromatin state (2/22). These hits increase the complexity of ribosome biogenesis, extending it beyond the nucleolus to extra‐nucleolar protein regulators of nucleolar function.

Support or Funding Information

NIGMS
Chemical Ligation Synthesis of Isotope‐Labeling Compatible Selenoprotein S
Authors: Subramoney Jay , Zhang Zhengqi , Cheng Rujin , Rozovsky Sharon
Published: November 2023

Selenoproteins contain the 21st amino acid selenocysteine (Sec) and play vital roles in human health, such as antioxidant defense. Although selenoproteins are expressed naturally in the human body, procuring sufficient quantities for biochemical and biophysical characterization by heterologous expression is challenging. Low yield in genetic incorporation is due to the Sec codon being identical to the UGA stop codon in mRNA. An alternative approach for their preparation is chemical ligation, a semi‐synthetic method that relies upon an amide bond formation to generate peptides and proteins from their complementary fragments. Here, we describe a new method for chemical ligation of selenoprotein S (SelS) which is compatible with simple and low cost isotopic labeling and does not rely on complete chemical synthesis of a selenoprotein. SelS is a member of the endoplasmic reticulum associated protein degradation (ERAD) pathway, a protein machinery devoted to relocating misfolded proteins from the ER to a cytoplasmic protein degradation complex. The active site lies close to the C‐terminal where a sole Sec is paired with a Cysteine 13 residues away. Previously we have characterized the activity of SelS prepared by heterologous expression, however, structural characterization with the Sec in place was prohibited by the low yield. In our approach a Sec‐containing fragment is expressed in E. coli and used as a source of the selenopeptide during the chemical ligation. This has the advantage of a high yield, with simple and efficient ligation while being compatible with isotopic‐labeling of selenoproteins. Useful applications of this method include incorporation of Selenium‐77 and Carbon‐13 for structural and biochemical characterization by NMR spectroscopy.

Support or Funding Information

This work was funded by the National Institute of General Medical Science
The PINK‐Parkin Circuit in Mitochondrial Quality Control
Authors: Liu Xuedong , Zhang Conggang , Peng Yinghua , Bunker Eric , Lee Schuyler
Published: November 2023

Mutations in E3 ubiquitin ligase Parkin and Ser/Thr protein kinase Pink1 cause autosomally inherited parkinsonism. Genetic studies have shown that Pink1 and Parkin act in the same pathway that regulates mitochondria dynamics, motility, and quality control. Activation of this pathway can be triggered by a variety of cellular stressor signals that cause mitochondrial damage.

Here, we present evidence that PINK1‐Parkin has both cytoprotective and pro‐apoptotic functions. PINK1‐Parkin operates as a molecular switch to dictate cell fate decisions in response to different cellular stressors. Cells exposed to severe and irreparable mitochondrial damage agents such as valinomycin can undergo PINK1‐Parkin‐dependent apoptosis. The proapoptotic response elicited by valinomycin is associated with the degradation of Mcl‐1. PINK1 directly phosphorylates Parkin at Ser65 of its Ubl domain and triggers activation of its E3 ligase activity through an autocatalytic mechanism which amplifies its E3 ligase activity towards Mcl‐1. Autocatalytic activation of Parkin bolsters it accumulation on mitochondria and apoptotic response to valinomycin. Our results suggest that PINK1‐Parkin constitutes a damage‐gated molecular switch that governs cellular context‐specific cell fate decisions in response to variable stress stimuli. We developed an analog sensitive PINK1 allele and demonstrated that PINK1 is required for triggering Parkin translocation to mitochondria, but is dispensable for sustaining Parkin accumulation and consequent mitophagy.

Support or Funding Information

This work was supported by grants from a Butcher Award from the University of Colorado and National Institutes of Health CA107098 and GM113141 to Xuedong Liu. The ImageXpress MicroXL was supported by a NCRR grant S10 RR026680 from NIH.
Optimizing the Enzymatic Activity and Storage Method of the Metacaspase SUMO‐Scp3
Authors: Proper Danielle E , Fox Kristin M
Published: November 2023

Metacaspases are heterodimeric proteases found in plants, fungi, and protozoa. More specifically, metacaspases are known as cysteine proteases due to a cysteine/histidine catalytic dyad present in their active site [1]. The nucleophilic cysteine thiol group present in metacaspases catalyzes the cleavage of proteins after arginine or lysine residues. This mechanism is essential in their functions to induce programmed cell death and the clean up of protein aggregates [2]. Currently, the best‐known substrate for this enzymatic reaction is Z‐GRR‐AMC [3]. The objective of the present study was to optimize the enzymatic activity and storage method for one of the five metacaspases in the fungus Schizophyllum commune, Scp3. The protein is only expressed in E. coli when it is fused to the small ubiquitin modifying protein, resulting in SUMO‐Scp3. By altering the components of the metacaspase assay buffer, it was determined that the assay buffer resulting in the highest activity of SUMO‐Scp3 contained 5.0mM CaCl2, 50mM Tris at pH 7.5, 100mM NaCl, 5M DTT, and 500μM Z‐GRR‐AMC. Experiments revealed that in order for SUMO‐Scp3 to maintain its activity, it needs to be stored in 20% glycerol at −80°C. Glycerol acts as a stabilizer and helps prevent damage to SUMO‐Scp3 during freezing and thawing. Currently, we are working on scaling up the production of active SUMO‐Scp3 in order to run experiments to determine the Km and Vmax of SUMO‐Scp3. Preliminary experiments have determined the Km for SUMO‐Scp3 to be 2.2 μ;M. By obtaining these kinetic constants, we hope to be able to compare them with other metacaspases.
Adaptor Guided Proteolysis Initiates on the Ribosome
Authors: Puri Neha , Woo Perry , Mehta Preeti , Choksi Arnav , Karzai Wali
Published: November 2023

Objective

To characterize the capture of tmRNA‐tagged proteins by the SspB adaptor on the ribosomes.

Methods

We utilized in vitro enzyme kinetics and in vivo ribosome enrichment assays to study SspB‐ribosome interactions and the consequential degradation of tagged protein. We isolate total ribosomes and determine the relative amounts of SspB associated with the ribosomes by specifically enriching for ribosomes translating the reporter mRNA of choice.

Results

Adaptor proteins extend the substrate range and specificity of AAA+ proteases. The SspB adaptor protein enhances the efficiency of ClpXP mediated proteolysis of tmRNA‐tagged proteins. Our investigations into whether ribosomes serve as a hub for initiating directed proteolysis reveal that SspB captures tag‐bearing proteins on tmRNA‐recued ribosomes in a trans‐translation dependent manner, in a process reliant on distinct recognition signals within the tmRNA degron. Defined alterations in the SspB and ClpX recognition elements of the tmRNA peptide sequence, or the peptide‐binding groove of SspB, have adverse effects on the efficiency of SspB ribosome recruitment and degradation of marked proteins.

Conclusions

Our studies suggest that SspB scans translating ribosomes to capture tmRNA tagged proteins and guide their delivery to ClpXP for directed proteolysis. Our inquiries have also unveiled the capacity of SspB to capture additional nascent polypeptides that harbor genetically encoded yet degenerate forms of the tmRNA degron and modulate their intracellular abundance through selective proteolysis.

Support or Funding Information

NIH
Expression and Purification of Active S. commune Metacaspase Proteins
Authors: Jones Brandon M , Fox Kristin M
Published: November 2023

Our objective is to produce purified active metacaspase proteins native to the fungus Schizophyllum commune using an E. coli expression system, compare their structural and functional properties, and understand the role of the N‐terminal prodomain in metacaspase function. Metacaspases are caspase homologs that participate in the clearance of protein aggregates, ER stress, and programmed cell death. Five S. commune genes encoding metacaspase proteins have been identified, Scp1–5. With the exception of Scp3 which lacks a prodomain, all other identified S. commune metacaspases contain an N‐terminal prodomain. While several of the metacaspase proteins have been expressed in E. coli, currently only one of these proteins, Scp3, displays activity upon overexpression. Production of active protein was accomplished by fusing Scp3 to the protein small ubiquitin modifying protein (SUMO), which has been shown to increase solubility of foreign proteins in E. coli.

Currently, through PCR amplification and Gibson assembly, plasmid vectors encoding SUMO‐Scp1 and SUMO‐Scp5 fusion proteins without their respective prodomains (SUMO‐Scp1nopro and SUMO‐Scp5nopro) as well as SUMO‐Scp5 with its prodomain (SUMO‐Scp5pro) have been produced. E. coli have been transformed using these plasmids and after growth at 27°C, SUMO‐Scp1nopro, SUMO‐Scp5nopro, and SUMO‐Scp5pro have been produced based on SDS‐PAGE analysis. SUMO‐Scp1nopro has been partially purified using a nickel affinity column. This is the first time an S. commune metacaspase has been produced with an intact prodomain in soluble form.
Assessing Substrate Engagement by Single Molecules of the AAA+ Protease ClpXP
Authors: Saunders Reuben A. , Stinson Benjamin M. , Sauer Robert T.
Published: November 2023

ClpXP, a AAA+ protease, binds ssrA‐tagged protein substrates, unfolds them, and translocates the polypeptide into a sequestered peptidase chamber for proteolysis. Although the sites of ssrA interaction in the ClpXP pore have been characterized, the dynamic steps by which initial binding leads to unfolding and degradation are poorly understood. Specifically, it is unclear whether initial binding is reversible or commits a substrate to unfolding and degradation. The kinetics of substrate degradation measured directly in population studies are also substantially slower than the kinetics expected from the single‐molecule rates of unfolding and translocation, raising the possibility that degradation initiation is a slow step in the ClpXP activity cycle.

To clarify the kinetic mechanism of substrate engagement, we established a single‐molecule assay based on fluorescence quenching to assess substrate binding and release by ClpXP directly. A set of substrate proteins with a wide range of unfolding stabilities was labeled with a fluorescence quencher. We then bound fluorescently labeled ClpXP to a glass surface and used total internal reflection fluorescence microscopy to monitor single‐molecule fluorescence in the presence of different concentrations of substrate. The single‐molecule assay was validated by comparison with solution‐binding experiments.

We observed many instances in which ClpXP binds and releases ssrA‐tagged substrates without degrading them. Commitment is a comparatively rare event and is slow or rate‐limiting in the kinetic process of degradation. Release without unfolding occurs more frequently for stably folded substrates. However, we also observe lengthy bound dwell times for stably folded substrates, which we identify as extended unfolding attemp

Support or Funding Information

This research was supported by the US National institutes of Health grant GM‐101988 (R.T.S.). R.A.S. was supported by the MIT Undergraduate Research Opportunities Program.
Understanding Dysregulated Collagen‐I Proteostasis Linked to Osteogenesis Imperfecta
Authors: Doan Ngoc‐Duc , DiChiara Andrew S. , Wong Madeline Y. , Taylor Rebecca J. , Shoulders Matthew D.
Published: November 2023

Osteogenesis imperfecta (OI) is an inherited disease most commonly caused by autosomal dominant mutations in collagen‐I genes. Unfortunately, there is currently no cure for OI. Treatment options are symptomatic and limited to the administration of anti‐resorptive bone drugs that do not address the underlying causes of OI and have substantial side effects. Disrupted collagen‐I proteostasis caused by the production of mutant, misfolding collagen‐I strands is a key pathological mechanism underpinning OI. Biochemical strategies targeted at improving intracellular collagen‐I folding and quality control mechanisms could help to resolve OI‐associated disruptions in collagen‐I proteostasis, but first require a detailed mechanistic understanding of the fundamental processes of wild‐type and mutant collagen‐I maturation. We created a new cellular platform for biochemical studies of collagen folding and misfolding. Osteosarcoma Saos2 cells, which produce and secrete endogenous collagen type‐I, were used as the cell line of choice for the construction of an OI model for proteomics studies. Single colony Saos2T‐REx cells were produced by transduction with a lentiviral vector encoding the tetracycline repressor under a CMV promoter. HA‐Tagged wild‐type Col1A1 or Col1A2 or mutant Col1A1 genes under a tetracycline‐inducible promoter were then introduced into the Saos2T‐REx cells using lentiviruses. The collagen mutations introduced were Col1A1‐Gly247Ser, Col1A1‐Gly352Ser, Col1A1‐Gly883Ser and Col1A1‐Cys1299Trp. The tetracycline‐inducible expression of HA‐tagged collagen‐I proteins in these Saos2T‐REx cells was confirmed by Western blotting using antibodies against both the HA‐tag and collagen‐I. Importantly, HA‐tagged collagen was found to be inserted into triple‐helical collagen‐I molecules. In addition, the unfolded protein response was not activated by introduction of HA‐tagged collagens in Saos2T‐REx cells. We next employed a covalent crosslinking enhanced, mass spectrometry‐based proteomics strategy to quantitatively establish the differential interactomes of wild‐type and OI‐causing collagen‐I variants. Our proteomic data identified >75 different novel proteins that might play a role in collagen‐I proteostasis, in addition to all currently known collagen‐I interactors including Hsp47, BIP, PDI, CRTAP, and more. Our mechanistic findings in follow‐up studies are revealing new players in cellular collagen‐I folding and quality control that may play an important role in OI pathology.
Mutational Editing of Strained New Protein Folds with Deletion Mutations
Authors: Pattengale Sarah R. , Kumirov Vlad K. , Szyszka Taylor N. , Cordes Matthew H.J.
Published: November 2023

There are 1,200 known protein folds, some of which evolved from other folds. The consequences and causes of evolutionary fold switching are not well known. The fold switch that occurs in the Cro protein family is an example of new protein fold evolution. There are two major fold groups of Cro proteins: Cro proteins that have an ancestral all‐a fold and those that have a descendant a+b fold. Early forms of the a+b Cro fold, represented by prophage Pfl6 Cro protein, exhibited conformational strain in the turn connecting the first two helices. Two groups of a+b Cro proteins that emerged later show deletions in this turn, either in G14 or T15, with apparent relief of conformational strain. To determine whether a G14 deletion could account for the strain relief, we introduced a G14 deletion into the strained Pfl6 Cro using site‐directed mutagenesis. Using circular dichroism (CD), nuclear magnetic resonance (NMR), and fluorescence anisotropy (FA), we characterized the stability, structure, and DNA binding affinity, respectively, of this Pfl6 variant. The G14 deletion slightly increased the protein's thermal stability, partially relieved the strain in the helix 1–2 turn, and did not significantly change the DNA binding affinity compared to wild‐type Pfl6. These findings suggest that new protein folds may emerge through transitional forms that are edited through deletion and/or insertion mutations to remove awkward features.

Support or Funding Information

This research is funded by an NIH grant (R01 GM066806).
Structural analysis of CCT‐bound folding Intermediates for mTORC1 subunits Raptor and mLST8
Authors: Ludlam William Grant , Aoba Takuma , Cuéllar Jorge , Makaju Aman , Franklin Sarah , Valpuesta José M , Willardson Barry M
Published: November 2023

The mTOR signalling complex 1 (mTORC 1) is a master regulator of cell growth and a high‐value drug target. While mTORC1 function has been studied extensively, little is known about how its subunits are folded and assembled together. Using a combination of cryo‐electron microscopy (cryo‐EM) and chemical crosslinking coupled with tandem mass‐spectrometry (XL‐MS), we investigated the mechanism by which the chaperonin CCT and its co‐chaperone, PhLP1, participate in the folding of mTORC1 subunits Raptor and mLST8. 2D cryo‐EM reconstructions of the raptor‐CCT complex show a mass completely filling the CCT folding cavity and extending out from it. This evidence supports the claim that CCT folds large proteins, such as Raptor, that do not fit inside its cavity by accommodating individual domains inside the cavity while the rest of the protein remains outside the cavity. 2D cryo‐EM reconstructions of the PhLP1‐mLST8‐CCT complex show that mLST8 sits inside the cavity while PhLP1 spans the cavity. XL‐MS data for the mLST8‐CCT complex indicate that mLST8 is oriented on one side of the cavity in contact the CCTα and CCTγ subunits. These results suggest different mechanisms for mLST8 and Raptor folding by CCT, one involving PhLP1 and the other involving a single domain Raptor.

Support or Funding Information

Grants from the National Institute of Health: EY012287, GM07855
mRNA Quality Control Is Activated Due To Chromium (VI) Mediated Oxidative Damage
Authors: Garcia‐Doherty Naiara , Larson Abbi L , Six Marshall , Segal Scott P
Published: November 2023

Chromium VI (Cr(VI)) is a genotoxic stressor that is known to cause 8‐oxoG bases to form in DNA. However, little is known regarding whether Cr(VI) has an effect directly at the level of mRNA. Prior data showed that treatment with Cr(VI) results in the selective assembly of P‐bodies, but not stress granules. This selective P‐body assembly requires translational repression to occur. This leads to a model in which mRNA either transcribed from damaged genes, or mRNA damaged directly by the Cr(VI) is sent to P‐bodies for degradation. To test between these two possibilities, strains in which transcription has been repressed by actinomycin D were subsequently treated with Cr(VI). Upon the dual treatment, P‐body assembly was unaffected, suggesting that Cr(VI) may have the ability to damage mRNA, and it is these mRNA that are, in turn, be localized to P‐bodies.

Several mRNA quality control pathways exist in eukaryotes that potentially shuttle mRNA through P‐bodies for subsequent degradation. Of significance, are the Nonsense Mediated Decay (NMD) pathway, which degrades mRNA with premature termination codons, and the No‐Go pathway, which degrades mRNA in which ribosomes have stalled during translation. To test which pathway(s) may be activated in response to Cr(VI), deletion mutants were used to reduce or eliminate the activity of specific pathways. Upon reduction of NMD activity, no effect on P‐body assembly was seen in response to Cr(VI). However, reducing No‐Go decay, resulted in a significant reduction in P‐body assembly. Based on this data, it is likely that the increase in P‐body assembly through Cr(VI) can directly damage mRNA. This could potentially lead to stalled ribosomes during translation and subsequent activation of the No‐Go decay pathway.
Determining Cellular and Viral Interaction Partners of the Kaposi's Sarcoma‐associated Herpesvirus Protein ORF55
Authors: Randolph Keyera , Rivas Hembly , Gaglia Marta Maria
Published: November 2023

Kaposi's sarcoma (KS) is a cancer caused by Kaposi's sarcoma‐associated herpesvirus (KSHV). KS mainly affects immunocompromised patients such as AIDS patients and transplant patients. There are few available treatments for KS and none of them directly targets KSHV infection. KSHV alters host gene expression, a process thought to promote viral replication. At present the viral factors involved in the regulation of host gene expression are unknown. Our lab screened for viral factors that are involved in host gene modulation using reporter assays and identified the KSHV protein ORF55 as a potential new gene regulator. We found that ORF55 causes reporter RNA and protein levels to decrease by two‐fold. We hypothesize that ORF55 plays a role in gene regulation through interactions with cellular proteins. We used the results of a published mass‐spectrometry screen of cellular proteins that interact with 90 different KSHV proteins to select candidate ORF55‐interacting proteins, and we focused on several proteins involved in signaling proteins and transcriptional regulation. We overexpressed ORF55 and tagged version of the candidate interacting cellular proteins and used co‐immunoprecipitation followed by western blotting to determine if these proteins are interacting with ORF55. Our results suggest that ORF55 does not interact with RACK1, 14‐3‐3 protein ɛ and the transcriptional regulator TRIM28. Further studies will be performed for the remaining cellular proteins of interest.
Aim32p is a Novel Member of the Erv1 Translocation Pathway within the Mitochondrial Intermembrane Space of Saccharomyces cerevisiae
Authors: Dabir Deepa Vinay , Nguyen Kevin , Koehler Carla
Published: November 2023

The mitochondrion is a key player in basic cellular homeostasis in eukaryotic cells, contributing to the synthesis of ATP, prevention of oxidative damage from reactive oxygen species and regulation of cell death. In yeast, most mitochondrial proteins are synthesized in the cytoplasm and imported post‐translationally into the organelle. One particularly interesting translocation pathway is the Erv1 oxidative folding pathway, a disulfide relay system comprising of Erv1 and Mia40 that couples the folding, oxidation and import of proteins into the mitochondrial intermembrane space (IMS). Besides import of proteins, Erv1 has diverse functions in the IMS; these include reoxidation of Mia40 upon release of the client protein, maturation of cytosolic iron‐sulfur cluster‐containing proteins and in the biogenesis of the TIM22 translocation pathway. Considering that Erv1 has several key functions in the IMS, we hypothesized that additional Erv1 partner proteins exist that function as electron acceptors and/or substrates. In this study, we elucidate the biological functions of Aim32p; a newly identified Erv1‐binding protein. Aim32p is an IMS‐ resident and under native conditions co‐exists in a complex with Erv1 and Osm1. Osm1 is a fumarate reducatase and has been recently postulated to function as an anaerobic electron acceptor of Erv1. Our structure predictions based on sequence analysis and homology modeling suggest that Aim32p is a putative thioredoxin‐ like ferredoxin protein. Although the function of most thioredoxin‐like ferredoxins remains unknown, the presence of a putative thioredoxin fold and a ferredoxin domain in Aim32p is highly suggestive of a role in redox sensing and electron transfer. Interestingly, loss of Aim32p also confers severe sensitivity to DNA damaging agents, MMS (Methyl methanesulfonate) and Hydroxyurea (HU). Thus, Aim32p may have a dual role in the mitochondrial IMS, participating in electron transfer reactions with Erv1 singularly or as a redox couple along with Osm1, and function in DNA damage stress response. Importantly, this study connects the redox‐regulated Erv1 translocation pathway to DNA damage stress response, and consequently its importance and versatility in several fundamental processes of the mitochondria.

Support or Funding Information

This work is supported by the Kadner Pitts grant.
Analysis of tectonin I and II and tectonin‐related proteins from Physarum polycephalum
Authors: Matsuda Takuya , Morita Rihito , Minami Yoshiko
Published: November 2023

Physarum polycephalum, a true slime mold, is a lower eukaryote with a unique life cycle that consists of plasmodium, spore, amoeba, and sclerotium stages. The plasmodia consist of a huge, multinuclear cell that grows without cytokinesis. When the plasmodia are cultured in liquid medium, the cells segregate into numerous small‐walled masses called microplasmodia. The current study focused on the role of tectonin, a lectin‐like protein of the microplasmodia. Microplasmodia express tectonin I and II isoforms. Tectonin II has a larger molecular weight than tectonin I, and the two‐third of amino acid sequence of tectonin II is homologous to the entire sequence of tectonin I. Both tectonin I and II sequences contain six WD repeat motifs that form a beta‐propeller structure, a motif that is generally found in diverse proteins related to signaling processes. Tectonin‐like domains have been found in various types of proteins in a wide range of organisms. Examples include human tectonin (TECPR) and horseshoe crab galactose‐binding protein (GBP). The function of these tectonin‐like proteins has been well studied, whereas that of Physarum tectonin is not well characterized.

We have previously reported that tectonin I is expressed during every stage of the Physarum cell cycle. However, haploid‐stage amoebas and spores do not express tectonin II. Therefore, it is believed that tectonin I and II may have distinct functions. Immunofluorescent intracellular localization has shown that tectonin I and II localize to plasmodia cell surface protrusions, suggesting that both proteins may function at the cell membrane and facilitate signal transduction, endocytosis, exocytosis, or other processes. To elucidate the function of tectonin, microplasmodia cell fractionation was performed. Although tectonin I and II were thought to be soluble proteins according to amino acid sequence analysis, both proteins separated into the membrane fraction. Solubilization experiments were performed using detergents, salts, ions, and various buffers to test protein release from the membrane surface. Tectonin I and II were released from the membrane when plasmodia were exposed to an alkaline buffer or to a buffer containing deoxycholate. Next, pull‐down assays were performed to analyze the presence of tectonin‐associated proteins using GST‐tectonin I and II followed by immunoprecipitation using anti‐tectonin I and II antibodies, respectively. Only a few proteins that putatively interacted with tectonin I and II were found. Finally, we confirmed that microplasmodia secrete tectonin‐like proteins in the culture medium during cultivation. We will present preliminary analyses of how tectonin interacts with tectonin‐related proteins.