bluuebunny/crossref_metadata_2025 huggingface dataset:
O‐GlcNAc Transferase and O‐GlcNAcase Interact with Mi2β at the Aγ‐Globin Promoter
Authors: Zhang Zhen , Costa Flávia C. , Tan Ee Phie , Bushue Nathan , Costello Catherine E. , McComb Mark E. , Whelan Stephen A. , Peterson Kenneth R. , Slawson Chad
Published: November 2023

Sickle Cell Disease (SCD), caused by a point mutation in the adult β‐globin gene, leads to chronic damage to multiple organs and stroke, as well as cardiovascular abnormalities and dysfunction. However, SCD patients are phenotypically normal if they carry compensatory mutations that result in continued expression of fetal γ‐globin. Thus, a logical clinical goal for treatment of SCD is to up‐regulate fetal γ‐globin synthesis. One mode of γ‐globin silencing occurs at the GATA binding sites located at −566 or −567 relative to the Aγ‐globin or Gγ‐globin transcription start site, respectively, and is mediated through the DNA binding moiety GATA‐1 (GATA Binding Protein 1) and its recruitment of co‐repressor partners, FOG‐1 (Friend of GATA‐1) and Mi2β (CHD4, Chromodomain Helicase DNA Binding Protein 4). Post‐translational modification of transcription factors can regulate their activity. One such modification is O‐GlcNAcylation, which is the attachment of a single N‐acetyl‐glucosamine moiety to serine or threonine residues on nuclear, cytoplasmic and mitochondrial proteins. O‐GlcNAc is added to proteins by O‐GlcNAc transferase (OGT) and removed by O‐GlcNAcase (OGA). Here, using ChIP (Chromatin Immunoprecipitation) assays, we demonstrate that both OGT and OGA interact with the Aγ‐globin promoter when Aγ‐globin is repressed in wild‐type murine CID (Chemical Inducer of Dimerization)‐dependent human β‐YAC (β‐globin locus yeast artificial chromosome) bone marrow cells (BMCs). In addition, Mi2β and OGT are recruited to the −566 Aγ‐globin GATA silencer site in mouse embryonic day E18 fetal liver when Aγ‐globin is repressed. Furthermore, we demonstrate that OGT and OGA interact with Mi2β and FOG‐1, and Mi2β and FOG‐1 are O‐GlcNAcylated using co‐immunoprecipitation experiments. In order to confirm Mi2β O‐GlcNAcylation, we mapped one O‐GlcNAc site at S85 by mass spectrometry. Our data suggested that OGT and OGA can regulate GATA‐1/FOG‐1/Mi2β repressor complex activity at the Aγ‐globin promoter.

Support or Funding Information

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (NIGMS) of the National Institutes for Health under grant P20 GM12345, National Institute of Diabetes and Digestive and Kidney Diseases R01 DK100595 to C. Slawson and K. Peterson, and by National Heart, Lung, and Blood Institute contract HHSN268201000031C and NIGMS grant P41 GM104603 to C. E. Costello.
The dynamics of TAL effector – DNA interaction reveals optimal number of targeted nucleotides for maximum binding specificity
Authors: Rinaldi Fabio Cupri , Wilkins Katherine Elizabeth , Bogdanove Adam Joseph
Published: November 2023

Transcriptional activator like effectors (TALEs) of plant pathogenic bacteria in the genus Xanthomonas have received special attention from the research community in recent years for their customizability as DNA targeting proteins. TALEs have evolved to bind DNA sequences in the promoters of host genes and upregulate expression. The DNA binding domain of TALEs is unique, comprising a series of modular repeats typically 34aa long that are essentially distinct only at positions 12 and 13, referred to as the RVD (repeat variable di‐residue). Individual base‐specific contacts by residue 13 across the repeats determine overall target specificity, with residue 12 playing a stabilizing role. Binding affinity and specificity of individual RVD‐nucleotide associations are increasingly well studied. However, there are still key questions concerning how TALEs can be designed to target DNA sequences with overall high binding affinity and specificity and how TALE target and off‐target sequences can be better predicted. Especially, the relationship between number of repeats and binding affinity has not yet been addressed quantitatively. For instance, in nature the average size of the TALE DNA binding domain is 17.5 repeats. Intuitively, one might assume that longer TALEs will have increased binding affinity and will be less likely to hit off‐target sites. We set out to test this assumption using biochemical methods to systematically study the relationship between the number of repeats of a set of designed TALEs and their respective binding affinities for the target DNA. We found that the binding affinity of TALEs in relation to their length saturates at approximately the average size of TALEs observed in nature. This suggests that increasing the number of repeats does not necessarily increase binding specificity and in fact has the opposite effect. Mathematically modeling the relationship supported this conclusion. Using the model improved the accuracy of TAL effector target prediction, providing empirical evidence in support of the model. This better understanding of the dynamics of TALE‐DNA interactions will benefit not only applications in biotechnology but also molecular characterization of the host‐pathogen interaction in the many important plant diseases caused by Xanthomonas spp.

Support or Funding Information

Bayer CropScience (BCS)National Science Foundation (NSF)National Institutes of Health (NIH)
Mechanistic Study of ERCC6/CSB Enzymatic Regulation, and Its Roles in Transcription Elongation, and Chromatin Remodeling
Authors: Wang Dong
Published: November 2023

Cockyane Syndrome Group B protein (CSB/ERCC6), a highly conserved protein from yeast to human, belongs a member of a subfamily of SWI2/SNF2‐related chromatin remodelers. CSB is proposed to be among the first proteins to be recruited to the DNA lesion‐arrested RNA polymerase II (Pol II) and initiates a special DNA repair pathway, termed transcription‐coupled repair. In addition, CSB also plays crucial roles in regulating transcription elongation and maintenance of chromatin integrity. Human CSB mutations are associated with Cockayne syndrome, a severe autosomal‐recessive disorder clinically characterized by photosensitivity, physical and mental retardation, and premature aging. However, several important mechanistic questions remain elusive: 1) how the CSB enzymatic activities are regulated for its diverse biological functions; 2) how CSB interacts with Pol II transcriptional machinery; 3) What is the molecular mechanism of CSB chromatin remodeling.

Recently, we identified a novel conserved protein motif, termed the “leucine latch”, which plays a key role in autoregulation of CSB enzymatic activities. The presence of the leucine latch motif in organisms ranging from yeast to humans suggests a conserved mechanism for the autoregulation of CSB/ERCC6 in spite of the otherwise highly divergent nature of the N‐ and C‐terminal regions. This work provides important novel mechanistic insights into the autoregulation of CSB/ERCC6 proteins. We will also present our unpublished results toward the mechanistic understanding of roles of CSB in transcription elongation, and chromatin remodeling.

Support or Funding Information

NIH GM102362
Chromatin Remodeler EP400 Deposits H3.3 into Promoters and Enhancers During Gene Activation
Authors: Pradhan Suman Kalyan , Su Trent , Yen Linda , Jacquet Karine , Cote Jacques , Kurdistani Siavash , Carey Michael
Published: November 2023

Increasing evidence indicates that gene activation in metazoans is accompanied by deposition of the histone variants H2AZ and H3.3 into promoters and enhancers. The abundance of double variant nucleosomes correlates well with the levels of gene expression. However, knowledge is limited for how variant chromatin is assembled in regulatory regions and, more importantly, whether it plays a direct role in gene activation. To unravel the mechanism, we have recreated activator‐ and HAT‐stimulated transcription from H3.3/H2AZ variant chromatin in HeLa nuclear extracts. We further employed Immobilized template assays to identify variant chromatin specific chromatin proteins. Analysis of preinitiation complex (PIC) revealed that the chromatin remodeling factor EP400 was specifically enriched on variant versus canonical chromatin. In a cell based assay, EP400 was recruited to an integrated inducible promoter in U2OS cells in response to gene activation. Moreover, siRNA knockdown of EP400 led to diminished transcription and reduced assembly of H3.3‐containing chromatin, while having little or no effect on binding of the Mediator co‐activator. Remarkably, this observation was paralleled genome‐wide at promoters and enhancers, where knockdown of EP400 significantly affected H3.3 deposition, but not Mediator recruitment. We investigated the mechanism by in vitro histone exchange assay. EP400 is known to exchange H2AZ for H2A but in biochemical histone exchange assays, it also inserted H3.3 for H3.1 in a nucleosome dependent manner. Interestingly, a side‐by‐side comparison of histone exchange assay indicates that reverse exchange, i.e. exchange of canonical histone into variant chromatin is much less efficient. However, H3.3 exchange into variant chromatin is highly efficient. We reason that this dynamic exchange of H3.3 with H3.3 may play a role in enhaced transcription. What confers this specificity remains an open question. To this end we are currently exploring the contribution of Brd8, a bromodomain containing protein that resides in the same complex and happens to interact directly with EP400. Indeed, we have preliminary evidence from modified histone peptide array experiments that suggest that Brd8‐EP400 duo is significantly enriched in H3K18ac and H3K27ac histone marks. This raises further likelihood of collaboration between EP300 and EP400 in the context of gene activation. Our data support a model in which EP400 links variant chromatin assembly directly to gene activation but downstream of PIC assembly in a pathway parallel to pre‐initiation complex assembly but necessary for transcription on chromatin.

Support or Funding Information

L.Y. is supported by the Ruth L. Kirschstein National Research Service Award GM007185
Long non‐coding RNA GAS5 suppresses TGFβ‐Smad signaling via intrinsic rSBE elements
Authors: Tang Rui
Published: November 2023

Transforming Growth Factor β (TGF‐β) signaling plays an important role in various physiological and pathological progresses. TGF‐β transmits its signals through Smad proteins. Long noncoding RNAs (lncRNAs) are noncoding RNA molecules longer than 200 nucleotides. LncRNAs regulate gene expression via various mechanisms from chromatin modulation to protein degradation. However, lncRNA function in TGF‐β/Smad signaling is still unknown. In this study, we demonstrate for the first time that lncRNA Growth Arrest Specific 5 (GAS5) binds to Smad proteins. GAS5 contains multiple RNA‐based Smad binding elements (rSBEs) that competitively bind Smad proteins and prevents Smads binding to SBE in TGF‐β target gene promoter and thus negatively regulates TGF‐β/Smad signaling. Moreover, artificial RNAs or other lncRNA containing rSBE elements effectively suppress TGF‐β signaling, suggesting that rSBE suppression of TGF‐β/Smad signaling is a common mechanism for the function of multiple lncRNAs. Functionally, GAS5 inhibits TGF‐β‐induced fibroblast activation in vitro and suppresses skin and kidney fibrosis in vivo in mouse models. Taken together, we have uncovered a novel mechanism for the regulation of TGF‐β signaling by lncRNA, which may be useful for developing therapeutic to treat TGF‐β/Smad‐related diseases.
Circadian lnc to EDN1‐AS: A Novel, Long, Non‐Coding RNA Antisense to the EDN1 Gene
Authors: Solocinski Kristen , Barilovits Sarah J. , Welch Amanda K. , Wingo Charles S. , Cain Brian D. , Gumz Michelle L.
Published: November 2023

Endothelin‐1 (ET‐1) is a peptide signaling molecule that acts as a potent vasoconstrictor in the vasculature whereas in the kidney it has natriuretic (sodium‐excreting) effects. ET‐1 is a critical regulator of blood pressure and dysregulation of ET‐1 signaling can result in several pathologies including hypertension and chronic kidney disease. It is widely accepted that the main mechanism for regulation of ET‐1 expression is through transcriptional control of the gene that encodes ET‐1, Edn1. However, we have discovered a long, non‐coding RNA (lncRNA) antisense to the Edn1 gene, called EDN1‐AS, which represents a putative mechanism for regulation of ET‐1 expression. Strand‐specific RT‐PCR has demonstrated that EDN1‐AS is present in multiple human cell lines as well as authentic human kidney RNA. We have also detected EDN1‐AS in mouse kidney and aorta. Preliminary data indicate possible sex and localization differences in EDN1‐AS expression. Expression also seems to be regulated by the circadian clock as EDN1‐AS expression oscillates in human kidney proximal tubule (HK2) cells synchronized with dexamathasone. EDN1‐AS expression is conserved in both humans and mice and appears to be regulated in multiple ways. Therefore, we hypothesize that EDN1‐AS represents a novel regulatory mechanism of ET‐1 expression and may present a novel therapeutic target for diseases caused by aberrant ET‐1 expression.

Support or Funding Information

This work was supported by NIH DK085193 and DK098460, the ASN Foundation for Kidney Research to MLG, 2T32HL083810 to KS, 2T32HL083810 to AKW, and NIH DK082680 to BDC and CSW.
Synthesis and Characterization of Ribonucleic Acid Aptamers Targeted at Aspergillus Cell Surface Carbohydrates
Authors: Sheridan Megan R. , Engelke David R. , Rohlman Christopher E.
Published: November 2023

Aspergillosis is a disease characterized by the degradation of the respiratory system due to Aspergillus fumigatus fungal hyphae. The fungal infection is slow to establish and current methods provide only minimal rates of detection and high mortality rates in immuno‐suppressed patients. RNA aptamers have the potential to bind selectively to cell surface carbohydrates of the fungus, and aid in the detection of aspergillosis, where fluorescent labelling could be used in the identification of infection. In this study RNA aptamers targeted at the Aspergillus cell surface carbohydrate beta‐D‐glucan were selected from a 40 nucleotide randomized sequence population of 1020 to 1024 variants. RNA aptamer pools were synthesized using PCR amplification to generate the initial DNA template pool and through subsequent rounds of selection. RNA aptamers were generated via in vitro transcription of the template pool utilizing modified NTPs. Successful aptamers were selected through nine successive rounds of binding to beta‐D‐glucan, reverse transcription, and cDNA template pool amplification. We will describe the characterization of the sequence diversity of the aptamer pool as well as its binding characteristics.
Editing of Kv1.1 RNA Transcripts is Altered by Human Mutations Respons‐ible for Episodic Ataxia Type‐1
Authors: Kiddie Elizabeth Ferrick , Patterson Kathleen M. , Reenan Robert A. , Rosenthal Joshua J. C. , Emeson Ronald B.
Published: November 2023

Transcripts encoding the Kv1.1‐subtype of voltage‐gated potassium channel un‐der‐go an adenosine‐to‐inosine (A‐to‐I) RNA editing event resulting in the con‐ver‐sion of a genomically‐encoded isoleucine (AUU) to a valine codon (IUU) at amino acid position 400 (I400V). Previous studies have shown that this single amino acid change alters the recovery rate following channel inactivation and has been pro‐posed to mod‐u‐late the kinetics of neuronal repolarization due to its location within the predicted ion pore of the channel. Numerous nonsense and mis‐sense muta‐tions in the Kv1.1 gene (kcna1) have been associated with the hu‐man po‐tas‐sium channelopathy, Episodic Ataxia type‐1 (EA1), which is char‐ac‐ter‐ized by epi‐sodes of stress‐induced ataxia and myokymia, and can be associated with epi‐leptic seizures. Three human EA1 mu‐ta‐tions (V404I, I407M, and V408A) are within close prox‐imity to the I400V editing site, and are contained within the RNA duplex structure required for A‐to‐I con‐ver‐sion. We have employed both in vitro and in vivo systems to determine the impact that these EA1 mutations might have on Kv1.1 RNA editing, as well as determined the electro‐phys‐iological changes in chan‐nel function derived from tran‐scripts containing EA1 mutations with either an edited or non‐edited status. In vitro tran‐scribed RNA substrates containing EA1 mutations were incubated with nuclear extracts isolated from cells overexpres‐sing the ADAR2 editing enzyme. Each of the EA1 mu‐ta‐tions dif‐fer‐en‐tial‐ly de‐creased A‐to‐I conversion of Kv1.1 RNAs in relation to their over‐all prox‐im‐ity to the editing site. Furthermore, a mouse model of EA1, encoding the human V408A mutation at the endogenous kcna1 locus, also re‐vealed a significant decrease in Kv1.1 editing in all tissues examined. Analysis of EA1 mutant chan‐nels using a Xenopus oocyte expression system, in a non‐edited (I400) or edited (V400) background, revealed significant changes in the elec‐‐tro‐physiological char‐ac‐teristics of the channel. These observations suggest that altered Kv1.1 function in EA1 could result from both the missense mutation and alterations in editing ef‐fici‐ency. These results also are con‐sis‐tent with a gen‐et‐ically‐modified mouse mod‐el of Kv1.1 editing, where mice sole‐ly expressing the non‐edited Kv1.1 iso‐form exhibit stress‐in‐duced ataxia and an increase in induced‐sei‐zure suscepti‐bil‐i‐ty.

Support or Funding Information

Joel G. Hardman Chair in Pharmacology (RBE)Multidisciplinary Training in Molecular Endocrinology (METP) NIH/NIDDK – T32 DK007563 (EFK)Ruth L. Kirschstein National Research Service Award NINDS F31 NS087911 (EFK)
RNA Editing‐Mediated Modulation of Calcium‐Dependent Activator Protein for Secretion 1 (CAPS1) Function
Authors: Emeson Ronald B , Ulbricht Randi J , Lazarenko Roman M , DelBove Claire E , Zhang Qi
Published: November 2023

CAPS1 is a priming factor, involved in the regulated release of hormones, peptides and neurotransmitters from large dense‐core secretory granules and synaptic vesicles. Tran‐scripts encoding CAPS1 are modified by an adenosine‐to‐inosine (A‐to‐I) RNA editing event in which a genomically‐encoded glutamate (GAG) is converted to a glycine (GIG) codon. This non‐synonymous amino acid change is located within the essential carboxyl‐terminal domain of the protein which mediates the CAPS1 interaction with peptide and hormone‐containing vesicles, suggesting that RNA editing modulates the contribution of CAPS1 to evoke vesicular release. Editing of CAPS1 was found to be conserved in vertebrate species from humans to zebrafish, indicating its importance in regulated secretion, an essential cellular function in the nervous and endocrine systems. Quantitative analysis of editing for CAPS1 transcripts in dissected mouse brain regions and peripheral endocrine organs revealed that editing was highest in the cortex of the brain (~24%) and lowest in the testes (~2%). Furthermore, the sequences necessary for editing were found to be contained within a predicted duplex region of the pre‐mRNA formed by two halves of a 32 base‐pair inverted repeat surrounding the editing site (exon 27) and within intron 27. Electrophysiological analyses of primary neuronal cultures from postnatal rat hippocampus revealed that neuronal synapses were enhanced by overexpression of the non‐edited CAPS1 isoform. Surprisingly, FM 1–43 dye loading and unloading studies suggested that synaptic vesicle release and retrieval were less complete with the non‐edited CAPS1 isoform. Tracking the fate of synaptic vesicle protein recycling during exocytosis and endocytosis, using a red fluorescence pH‐sensor (pHTomato) fused to the synpatophysin luminal domain, revealed that the non‐edited CAPS1 isoform promoted fast, but incomplete, fusion (i.e. kiss‐and‐run) that is often exerted by docking vesicles. These data suggests that CAPS1 editing may serve as a molecular mechanism to fine tune synaptic connectivity through less appreciated vesicle docking and recycling kinetics at pre‐synaptic terminals. These studies provide insights into the functional importance of CAPS1 editing in the regulation of neuronal network connectivity, as well as suggest a broader role for CAPS1 RNA editing in vertebrate physiology.

Support or Funding Information

Joel G. Hardman Chair in Pharmacology (RBE) and NIH OD008761 (QZ)
Phosphorylation‐independent recruitment of 14‐3‐3 to tristetraprolin partially impairs deadenylation by blocking Ccr4, but not Pan2, association
Authors: Nunez Castrejon Ana M. , Tun Maria , Nava Raul , Kraemer Laura , Clement Sandra L.
Published: November 2023

The regulation of mRNA stability by RNA binding proteins plays a key role in the modulation of gene expression. The RNA‐binding protein tristetraprolin (TTP) binds to AU‐rich elements (AREs) in the 3′ untranslated regions (UTRs) of transiently expressed mRNAs encoding cytokines, growth factors, and proto‐oncogenes and stimulates their decay by recruiting a number of mRNA decay enzymes including the Ccr4 and Pan2 deadenylase complexes. Phosphorylation of serines 52 and 178 of TTP by the p38‐activated MAP kinase MK2 impairs TTP's ability to stimulate mRNA decay by preventing the recruitment of these deadenylases. Phosphorylation of TTP also creates a binding site for the protein 14‐3‐3, although it is unclear whether 14‐3‐3 association is required to impair TTP function. In order to determine whether 14‐3‐3 association is sufficient to prevent deadenylase recruitment in the absence of phosphorylation, we replaced serines 52 and 178 in TTP via site‐directed mutagenesis with either a peptide sequence known to bind 14‐3‐3 independently of phosphorylation or with an altered version of this peptide sequence that does not. The ability of Myc‐tagged wild‐type and mutant forms of TTP to bind FLAG‐tagged Ccr4, Caf1, or Pan2 deadenylases was tested in anti‐FLAG co‐immunoprecipitation assays from transiently transfected Human Embryonic Kidney (HEK) 293T cell lysates. Mutant forms of TTP that bound 14‐3‐3 independently of phosphorylation were unable to interact with the either the Ccr4 or Caf1 proteins but retained their ability to associate with Pan2 whereas forms of TTP that failed to bind 14‐3‐3 bound all three proteins. These results suggest that 14‐3‐3 association with TTP independently of phosphorylation does not fully recapitulate the effect of phosphorylation.

Support or Funding Information

This project was supported by a California State University Program for Education and Research in Biotechnology (CSUPERB) New Investigator Grant to SLC. ANC received a stipend from the Cal Poly College of Science and Mathematics 2015 Summer Research Program. MT received a stipend and research funds from the Allan Hancock College/Cal Poly SLO Bridges to the Baccalaureate program funded by the National Institutes of Health.
Effects of Aging and Nitrogen Deprivation on Nonfunctional rRNA Decay in Saccharomyces cerevisiae
Authors: Zekan David , LaRiviere Frederick J
Published: November 2023

Nonfunctional Ribosomal RNA Decay (NRD) is a quality control pathway in Saccharomyces cerevisiae capable of degrading functionally defective mature rRNAs. Two types of NRD are known: 18S NRD which removes rRNAs with mutations in the small subunit decoding site and 25S NRD which removes rRNAs with mutations in the large subunit peptidyl transferase center. 18S NRD is translation dependent and occurs after ribosomal subunit joining on the mRNA. 25S NRD is mechanistically distinct; 25S rRNA degradation is translation independent and occurs prior to ribosomal subunit joining. We are interested in understanding this pathway because rRNAs are usually very abundant, generally comprising greater than 80% of the total cellular RNA, and very stable, with half‐lives on the order of several days. Therefore, natural NRD substrates in the form of damaged rRNAs may increase as cells age. Moreover, defective rRNAs may accumulate in NRD‐deficient cells. General ribosome turnover also increases under certain cellular stress conditions. For example, nitrogen deprivation results in degradation of both small and large ribosomal subunits by ribophagy. However, the effect of nitrogen starvation on NRD is unknown. Therefore, we are interested in analyzing the effects of both chronological aging and nitrogen deprivation on 18S and 25S NRD. To do this, we are measuring growth phenotypes and rRNA expression levels in yeast grown in normal or nutrient‐deprived media for various lengths of time. These experiments use either wild‐type or NRD‐deficient yeast strains transformed with rRNA expression plasmids that encode functional and/or nonfunctional versions of the 18S and 25S rRNAs. The plasmid‐derived rRNAs also contain short, unique sequence tags that allow for specific detection of these rRNAs in the presence of total cellular rRNA. Data collection and analysis are currently ongoing. We hope that these studies might provide insight into the accumulation of defective rRNAs over time in chronologically aged cells and the effects of nitrogen deprivation on this novel rRNA decay pathway.

Support or Funding Information

This work is supported by the Dr. G. Ashley Allen Student Research Grant at Washington and Lee University.
Dephosphorylation of the RNA‐ Binding Protein Tristetraprolin by PP2A in LPS‐Stimulated Macrophages
Authors: Smolgovsky Sasha A. , Caldwell Michael E. , Parsons Ben M. , Cahill Megan E. , Clement Sandra L.
Published: November 2023

Reversible post‐translational processes can regulate gene expression by rapidly changing levels of messenger RNA (mRNA), and consequently, levels of the proteins they encode. For example, the RNA‐binding protein tristetraprolin (TTP) stimulates mRNA decay by binding to adenylate‐‐uridylate‐‐rich elements (AREs) in the 3′ untranslated region of target mRNAs and recruiting mRNA decay enzymes. Various external stimuli can induce phosphorylation of TTP by the p38 MAPK‐‐activated kinase MK2. Phosphorylation of TTP inhibits the recruitment of mRNA decay factors and results in the transient stabilization of target transcripts. Dephosphorylation of TTP allows TTP to resume mRNA decay, thereby preventing the constant production of these target proteins. Previous studies have implicated the heterotrimeric phosphatase PP2A in the dephosphorylation and subsequent reactivation of TTP, but it is unknown how and when this phosphatase is targeted to TTP. In order to better characterize the TTP dephosphorylation process, we used SDS‐PAGE and western blotting to observe the relative abundance and phosphorylation of TTP in cell lysates from RAW 264.7 murine macrophages stimulated with lipopolysaccharide (LPS). TTP levels were highest approximately 12 hours after LPS induction, with dephosphorylation commencing roughly 16 hours after LPS induction. We also observed the relative abundance of components of the PP2A enzyme including the catalytic (C) and three candidate regulatory subunits (B, B′ and B″) of the heterotrimer. We found that PP2A C, B and B″ protein levels remained relatively constant throughout the time course. In contrast, levels of PP2A B′ (B56) were not detectable until 12 and 14 hours after LPS stimulation and declined shortly thereafter. The simultaneous increase of PP2A B′ abundance and TTP dephosphorylation suggests that this regulatory subunit may play a role in targeting PP2A to TTP. We are currently performing co‐immunoprecipitation assays between TTP and PP2A subunits in order to determine whether the interaction between these proteins is regulated.
Characterization of the ubiquitin binding properties of the deadenylase Pan2
Authors: Swinney Zachary , Teruya Jonathan , Vengoechea Nicole , Clement Sandra L.
Published: November 2023

Messenger RNA (mRNA) stability plays an important role in regulating gene expression in eukaryotic cells. While a number of endo‐ and exo‐nucleolytic enzymes act on mRNA, removal of the poly(A) tail, or deadenylation, is often the first and rate‐limiting step in mRNA decay. This process is initiated by the Poly(A) Nuclease (Pan2) complex and completed by the Ccr4/Caf1/Not complex(1). In addition to its poly(A) specific exonuclease domain, the Pan2 protein also contains a conserved domain belonging to a class of deubiquitinating enzymes (DUBs). Amino acid sequence alignments of Pan2 with other DUBs show that two of the three essential amino acids required for catalysis are not conserved, making it unlikely that Pan2 retains the ability to remove ubiquitin. However, the strong conservation of residues known to play a role in ubiquitin binding suggests that Pan2 might have retained its ability to bind ubiquitin. In order to test whether Pan2 binds ubiquitin, we performed a ubiquitin binding assay by incubating ubiquitin‐agarose resin with FLAG‐tagged Pan2 expressed in transiently transfected Human Embryonic Kidney (HEK) 293T cells and observed that Pan2 binds to ubiquitin‐agarose, but not to agarose lacking ubiquitin. The ability of Pan2 to bind ubiquitin agarose was abolished by pre‐incubation of cell lysates with excess amounts of free ubiquitin. In order to identify ubiquitinated Pan2 binding partners, anti‐FLAG co‐immunoprecipitations are currently being performed in lysates from cells co‐expressing FLAG‐tagged Pan2 and Myc‐ubiquitin‐tagged proteins. The observation that Pan2 binds ubiquitin raises the intriguing possibility that mRNA deadenylation by Pan2 may be regulated by ubiquitin‐dependent protein interactions.

Support or Funding Information

This work was supported in part by a grant to S.L.C. from the Cal Poly Extramural Funding Initiative Program.
Exploring GTP Control of Bacterial Respiratory System Specific Chaperone Interaction with its Substrate Protein's Twin‐Arginine Signal Peptide
Authors: Cherak Stephana Julia , Turner Raymond J
Published: November 2023

Many bacterial respiratory redox enzymes depend on the twin‐arginine translocase (Tat) system for translocation and/or membrane insertion. Tat substrates contain an N‐terminal twin‐arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone ‐ redox enzyme maturation protein (REMP) – to help mediate final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin‐arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase (DmsABC). DmsD adopts monomer, dimer, and unique folding forms that appear under pH control. All alternate DmsD forms retain the ability to bind DmsAL. As well, it is now suggested that DmsD cooperatively binds guanosine‐5′‐triphosphate (GTP), which elicits a dissociative effect on the DmsD to DmsAL interaction. Thus, the primary aim of this study was to analyze DmsD thermal stability when subjected to both GTP association and variance in pH. We have utilized two in vitro protein‐protein interaction techniques of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF). DSF experiments, yielding melting temperatures as a determinant of the associated thermal stability, showed an increase in DmsD stabilization upon DmsAL binding. Between pH values of 5.0 and 6.0, DmsD demonstrated the least amount of DmsAL release. This pH range also corresponds to a transition in DmsD folding forms. When DmsD was first subjected to GTP binding, subsequent variance in pH decreased melting cooperativity by the appearance of a pretransition peak, but did not affect melting temperature. However, if alteration in pH occurred prior to GTP binding, the pretransition peak was attenuated, and an incremental increase in melting temperature was observed. These findings illustrate that a greater affinity for DmsAL promotes an increase in DmsD thermal stability. As well, pH shift results in formation of a more uniform DmsD population, with subsequent GTP binding permitting an increase in thermal stability. These results support the idea that H+ and GTP play roles as molecular regulators in the biochemical pathway of DmsAB protein maturation – Tat system targeting mechanism.

Support or Funding Information

Research was supported by a Canadian Institutes of Health Research operating grant (MOP‐49422) to RJT.
Differential action of purifying selection explains the evolution of the major chaperone sub‐network in humans
Authors: Hess Kyle , Ellis Jacqueline , Nguyen Peter , Nikolaidis Nikolas
Published: November 2023

The cellular stress response (CSR) is one of the most conserved mechanisms present in all species as it allows organisms to adapt to their ever‐changing environments and survive. The most important orchestrator of the CSR is a group of proteins called molecular chaperones, which regulate protein homeostasis and cell survival. Although the molecular chaperone networks are critical for cell health and disease in humans, the impact of the evolutionary process on their function remains largely unknown. In this study, we explored the microevolution process of an important component of the human chaperone network, composed by Hsp70s, Hsp40s, and BAGs, by analyzing single nucleotide polymorphisms (SNPs). The results can be summarized as follows: (1) in 92% of the genes the number of non‐synonymous SNPs (nsSNPs) is higher than the number of synonymous SNPs (sSNPs). (2) Sixty five percent of the genes had a significantly higher SNP density than the surrounding genes. (3) More than 80% of the genes had significantly higher SNP density within their exonic regions as compared to both intronic and un‐translated regions. (4) The majority of genes contained more sSNPs than nsSNPs within known functional regions (domains), while the number of sSNPs is similar between domains and non‐domain protein regions. (5) On average 50% of the genes contained a nsSNP on an amino acid position of known function. (6) Calculations of synonymous and non‐synonymous distances revealed the action of purifying selection, the intensity of which varied dramatically both between and within the gene families. To test some of these predictions we generated recombinant protein variants corresponding to the wild‐type and mutated HSPA1A, the major human heat‐inducible gene. We then used Isothermal titration calorimetry (ITC) and determined that most mutations only marginally altered the binding affinity to both nucleotide (ATP and ADP) and protein substrates. However, one mutation revealed alterations in the way the molecules interact and the reaction products are formed. Collectively, these results suggest that strong purifying selection due to functional constraints shaped the evolution of the major chaperone sub‐network in humans. The nsSNPs, which are either predicted or showed to alter the function of a particular protein, may either represent adaptations to particular environmental pressures, disease states, or are mutations that have not been eliminated yet from the population.
Naturally occurring mutations alter the function of HSPA1A, a key regulator of the cellular stress response in humans
Authors: Oliverio Ryan Michael , Hess Kyle , Nikolaidis Nikolas
Published: November 2023

An essential question in molecular evolution is how mutations enable organisms to adapt and survive their environments. At the cellular level, this same question relates to the ability of cells to adapt and survive stressful conditions, such as disease. Cellular adaptation to stress is regulated and maintained by the cellular stress response system. Molecular chaperones, and, in particular the 70‐kDa heat shock proteins (Hsp70s) are key orchestrators of the stress response system, and alterations in their function have direct physiological consequences. Therefore, it is of vital importance to determine how natural mutations alter the function of these proteins and how such changes affect cellular and organismal adaptation. To answer this question we tested whether natural single nucleotide polymorphisms (SNPs) found on HSPA1A, the major stress inducible Hsp70 gene in humans, alter protein function. Specifically, the wild‐type human HSPA1A sequence was subcloned into mammalian expression vectors, and the mutated gene variants were generated using site‐directed mutagenesis. We first determined whether any of these mutations affected the intracellular localization of HSPA1A within mammalian cells. These experiments were performed by tagging HSPA1A to GFP, using fluorescent dyes to stain the nucleus, mitochondria, and lysosomes, to view where the proteins localize via confocal microscopy. These assays revealed that the mutants and the WT protein had similar subcellular localization. Next, we determined whether the mutations affected the ability of HSPA1A to prevent cell death by inhibiting the formation of protein aggregates caused by poly‐glutamine carrying huntingtin proteins. This assay determined that two of the mutations caused increased cell death as compared to the WT, suggesting that some mutations alter the chaperone function of HSPA1A. Furthermore, live‐dead assays showed that human cells carrying some of these mutations had significantly less resistance to heat stress than cells expressing the WT protein. Given that these natural variants are either population‐specific or clinical we suspect that the observed functional differences alter the ability of cells, and the individuals carrying them, to cope with stress and adapt to environmental perturbations.
Prion ‐ like protein polymerisation underlies signal transduction in innate immunity: the emergence of a universal mechanism ?
Authors: O'Carroll Ailis , Ve Thomas , Lau Derrick , Giles Nichole , Moustaqil Mehdi , Bhumkar Akshay , Hunter Dominic , Kobe Bostjan , Boecking Till , Sierecki Emma , Gambin Yann
Published: November 2023

The innate immune system uses a multitude of receptors such as Toll‐like Receptors and Nod‐like Receptors to sense pathogens and danger signals. Upon their activation, a signalling cascade is initiated culminating in the transcription and translation of cytokines and other proteins vital for host protection. Recent studies have discovered that an array of these intracellular signalling proteins assemble into oligomeric signalling platforms, consequently amplifying the transmission of the signal from the stimulated receptor to the activation of relevant transcription machinery.

Unexpectedly, it was also discovered that some of these proteins present in innate immune signalling such as the inflammasome adaptor protein ASC and the mitochondrial membrane‐associated protein MAVS, can form fibrils. These fibrils can elongate in a prion‐like manner to create super‐sized signalling platforms.

In this study, we screened over 100 innate immune intracellular signalling proteins using single molecule fluorescence. We defined the oligomerisation and aggregation propensity of these proteins and track the specific signature of polymerisation. We identified multiple candidates forming super‐sized protein aggregates; through protein seeding experiments, we were able to conclude that ~20 of these proteins could self‐propagate aggregation as found in prion‐like mechanisms.

These innate immune cytosolic proteins are not isolated to one signalling pathway but transverse most, if not all, signalling pathways of innate immune defence, inflammation and cell death. Intriguingly, some of these proteins could act to regulate and even inhibit the signalling cascade through their prion‐like protein propagation.

Together these findings reveal a universal mechanism where the creation of polymer‐like platforms increases the number of binding sites and dramatically amplifies the signal. Our observations also suggest a complex interplay among the major signalling cascades of the innate immune system, in which prion‐like polymerization underlies the backbone of intracellular communication.
A HECT domain ubiquitin ligase mediates the degradation of unassembled nuclear proteins in mammalian cells
Authors: Xu Yue , Ye Yihong
Published: November 2023

Protein quality control mechanisms are essential for cells to eliminate misfolded or unassembled polypeptides and therefore to maintain proteomic integrity, but little is known about how unassembled nuclear proteins are degraded in mammalian cells. Here we have established model substrates to study this process. Using these substrates, we demonstrate that the degradation of unassembled cytosolic and nuclear proteins both require the ubiquitin selective chaperone p97 and its cofactor Npl4. However, unlike unassembled cytosolic proteins whose degradation involves the UBR family ubiquitin ligases, unassembled nuclear factors depend on a distinct ubiquitin ligase. By screening candidate ligases using small interference RNAs, we establish the HECT domain ligase HUWE1 as a ubiquitin ligase required for efficient turnover of unassembled nuclear proteins. Interestingly, HUWE1 is predominantly localized in the cytoplasm, suggesting the possibility of translocation of nuclear factors to the cytosol for ubiquitination by HUWE. Using a SILAC‐based proteomic method, we have identified endogenous HUWE1 substrates, and consistent with the proposed role in nuclear protein quality control, many of the identified proteins are component of nuclear complexes involved in nuclear acid metablism. Our study revealed a new protein quality control process that degrades unassembled nuclear proteins in mammalian cells.
ER protein quality control and lipid droplets: Unexpected functional connections
Authors: Olzmann James A
Published: November 2023

Endoplasmic reticulum‐associated degradation (ERAD) mediates the recognition and destruction of proteins from the early secretory pathway. Substrates of the ERAD pathway include terminally misfolded proteins, unoligomerized subunits of multimeric complexes, and ER‐resident enzymes. Determining how substrates are recognized and targeted for degradation is paramount to combating the over 70 diseases associated with disrupted ER protein folding (e.g. Cystic fibrosis). Integrating proteomic and functional genomic platforms we recently constructed the first ERAD interaction network in mammalian cells. This systems‐level analysis led to our discovery that the p97/VCP‐recruitment factor UBXD8 relocalizes from the ER to lipid droplets (LDs) in response to excess fatty acids. UBXD8 recruitment of p97/VCP to the LD surface stabilized LDs by inhibiting the rate‐limiting enzyme in lipolysis, ATGL. In the current study we employed the acyl‐CoA synthetase inhibitor triacsin c to explore a potential role for LDs or lipid biosynthetic pathways in ERAD. Our analyses indicate that treatment with triacsin c impaired ERAD substrate glycan trimming, dislocation, and degradation. However, disruption of LD biogenesis by inhibiting the conversion of diacylglycerol to triacylglycerol had no effect on ERAD kinetics, arguing against a direct role for LDs in ERAD. Acyl‐CoA synthetases are also required for fatty acid modifications such as palmitoylation. Exploiting a click chemistry approach we identified multiple ERAD components that are palmitoylated, and treatment with the palmitoylation inhibitor 2‐bromopalmitate impaired ERAD substrate degradation. Our data support a model in which a subset of ERAD components traffic to LDs and participate in a LD regulatory complex. Although we find that LDs are dispensable for ERAD, our data reveal an unexpected role for fatty acid modifications in the regulation of ERAD machinery.

Support or Funding Information

This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R00DK095921) and the National Institute of General Medical Sciences (R01GM112948).
Test‐driven development improves GRNsight: a web application for visualizing models of gene regulatory networks
Authors: Varshneya Anindita , Anguiano Nicole A. , Samdarshi Mihir , Dionisio John David N. , Fitzpatrick Ben G. , Dahlquist Kam D.
Published: November 2023

GRNsight is a web application and service for visualizing models of gene regulatory networks (GRNs). A gene regulatory network consists of genes, transcription factors, and the regulatory connections between them which govern the level of expression of mRNA and protein from genes. GRNmap, a MATLAB program that performs parameter estimation and forward simulation of a differential equations model of a GRN, can mathematically model the dynamics of GRNs. GRNsight automatically lays out the network graph based on GRNmap output spreadsheets. GRNsight uses pointed and blunt arrowheads, and colors the edges and adjusts their thicknesses based on the sign (activation or repression) and magnitude of the GRNmap weight parameter. Visualizations can be modified through manual node dragging and sliders that adjust the force graph parameters. We have now implemented an exhaustive unit testing framework using Mocha and the Chai assertion library to perform test‐driven development where unit tests are written before new functionality is coded. This framework consists of over 135 automated unit tests that examine about 450 test files to ensure that the program is running as expected. Error and warning messages have a three‐part framework that informs the user what happened, the source of the problem, and possible solutions. For example, GRNsight returns an error when the spreadsheet is formatted incorrectly or the maximum number of nodes or edges is exceeded. The completion of the testing framework marks the close of development for version 1 (the current release stands at version 1.12). In version 2.0 of GRNsight, a new feature will be implemented that colors the nodes (genes) based on the expression data. The user will have the choice to display the experimental data or the data produced by the forward simulation feature in GRNmap. GRNsight is available at http://dondi.github.io/GRNsight/.

Support or Funding Information

This work was partially supported by NSF award 0921038 (B.G.F., K.D.D.), a Kadner‐Pitts Research Grant (K.D.D.), the Loyola Marymount University Summer Undergraduate Research Program (A.V.) and the Loyola Marymount University Rains Research Assistant Program (N.A.A.).
Information extraction from native mass spectra
Authors: Guan Shenheng
Published: November 2023

Native MS spectra of large protein assemblies are typically characterized by low signal‐to‐noise ratios and broad peak shapes. Accurate peak detection is challenging due to additional issues such as high degrees of peak overlap and highly interleaved charge envelopes. A comprehensive algorithm has been developed to address peak identification, overlapping deconvolution, and charge state assignment in native mass spectra. Our open source software provides a novel and practical tool for those problems and is capable of extracting signals of large protein assemblies from heterogeneous, complex, and noisy native mass spectra.

Nonspecifical binding of ligands to large protein complexes is commonly observed in native MS spectra. Here we also present a deconvolution model to discriminate specific bindings of a ligand to a multi‐protein complex target from the nonspecific interactions. Using the deconvolution algorithms with two level modeling steps, specific binding constant(s) can be effectively separated from the nonspecific ones. The nonspecific binding can be modeled by a simple power inverse function. An additional advantage of the method is that the target concentration can be more accurately obtained.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (1S10OD016229, 8P41GM103481, GM49985, GM36659, AI‐21144, NS059690, GM109896, AG021601, AG002132, and AG010770).