bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional Regulation of Cell Cycle Progression in T‐Cell Leukemia
Authors: Payne Jonathon Lee , Soliman Mario , Dovat Elanora , Kapadia Malika , Song Chunhua , Dovat Sinisa
Published: November 2023

The IKZF1 gene encodes the Ikaros protein that acts as a tumor suppressor in acute lymphoblastic leukemia (ALL). The role of Ikaros in T‐cell acute lymphoblastic leukemia (T‐ALL) has been documented by the presence of Ikaros deletions in a significant subset of human T‐ALL. Ikaros functions as a transcriptional regulator of a large set of its target genes and a tumor suppressor in B‐cell acute lymphoblastic leukemia. However, the molecular mechanisms of the tumor suppressor activity of Ikaros in T‐ALL is unclear. The use of quantitative chromatin immunoprecipitation (qChIP) showed that Ikaros binds to the promoter regions of the CDK2 and CDK6 cell cycle genes in primary T‐ALL cells in vivo. Ikaros gain‐of‐function experiments performed using retroviral transduction of T‐ALL cells showed that Ikaros overexpression in T‐ALL results in the reduced expression of CDK2 and CDK6, as evidenced by quantitative RT‐PCR (qRT‐PCR) and western blot. The knock‐down of Ikaros with shRNA in T‐ALL resulted in increased transcription of CDK2 and CDK6 as measured using qRT‐PCR. These data suggest that Ikaros can regulate cell cycle progression in T‐ALL by direct transcriptional repression of the CDK2 and CDK6 genes. Next, we studied the mechanisms that regulate Ikaros' ability to repress CDK2 and CDK6 in T‐ALL. Ikaros' function as a transcriptional repressor is regulated by its direct phosphorylation by Casein Kinase II (CK2). CK2 is an oncogenic kinase that is overexpressed in hematopoietic malignancies. We tested the effect of CK2 inhibition on Ikaros' ability to regulate transcription of CDK2 and CDK6 in human T‐ALL. Molecular inhibition with shRNA, as well as pharmacological inhibition of CK2 with a specific inhibitor, CX‐4945, resulted in reduced transcription of CDK2 and CDK6. This was associated with increased DNA‐binding of Ikaros to the promoters of CDK2 and CDK6 as indicated by qChIP. These data suggest that CK2 impairs Ikaros' ability to transcriptionally repress CDK2 and CDK6 and to regulate cell cycle progression in T‐ALL. Inhibition of CK2 restores Ikaros' function as a transcriptional repressor of CDK2 and CDK6 and as a regulator of cell cycle progression in T‐ALL. In conclusion, our results demonstrate that the Ikaros tumor suppressor can control cell cycle progression in T‐ALL via transcriptional regulation of CDK2 and CDK6 expression. Overexpression of CK2 impairs Ikaros' ability to repress CDK2 and CDK6 expression, which contributes to deregulation of cell cycle control in T‐ALL. Results reveal the therapeutic mechanism of targeted inhibition of CK2 as treatment for T‐ALL.
Transcriptome‐wide mapping of the miR‐122 targetome revealed its mechanistic role in the maintenance of liver homeostasis
Authors: Barajas Juan Martin , Luna Joseph , Teng Kun‐Yu , Darnell Robert , Ghoshal Kalpana
Published: November 2023

Liver cancer has the second highest mortality rate among all malignancies, which implies a lack of molecular understanding of disease development and progression necessary to combat this deadly disease. miR‐122 is a conserved liver‐specific miRNA, that maintains metabolic homeostasis, suppresses tumor development, and promotes HCV replication. Loss of miR‐122 is associated with loss of hepatic phenotype, gain of malignancy‐associated features, and poor patient prognosis in hepatocellular carcinoma (HCC) patients. Recent in vivo work in our miR‐122 knockout (KO) mice have established miR‐122 as a bona fide tumor suppressor. These data suggest it is critical to identify the miR‐122 targetome in the liver to better understand its function. Previously, in silico prediction was the go‐to‐method for identification of miRNA targets. However, these methods are dependent on known or evolutionarily conserved functionality of the miRNA‐Ago complex and without reference to cellular context, can result in a high number of false positives and false negatives. To overcome this, the laboratory of Dr. Robert Darnell introduced a novel unbiased high‐throughput method known as Ago‐dHiTS‐CLIP (Ago‐CLIP) which has been applied in wild‐type (WT) and miRNA KO platforms to systematically and biochemically identify transcriptome‐wide miRNA‐mediated Ago maps. To this end, in collaboration with Dr. Darnell's laboratory, we performed Ago‐CLIP analysis in livers of 6 week‐old WT and KO mice as well as in benign human liver expressing relatively high levels of miR‐122 and matching HCC tumor exhibiting down‐regulation of miR‐122. Ago‐CLIP identified ~800 miR‐122 targets in mice and ~1400 in humans with only 309 targets conserved in both. Surprisingly, a majority of miR‐122 sites are in coding exons (30%) and 3′‐UTRs (30%) along with sites in the 5′UTR, introns, etc. RNA‐seq analysis revealed specific upregulation of these 3′UTR and CDS targets in miR‐122 KO livers, suggesting these are functional miR‐122 targets. Furthermore, Liver and Hepatocellular Carcinoma (LIHC) data from The Cancer Genome Atlas (TCGA) revealed alterations in 26 conserved miR‐122 target genes are predictive of overall patient prognosis. BCL9, an exclusive CDS target and a critical component of Wnt/β‐catenin signaling, was found to be significantly predictive of patient survival and may play a key role in liver tumor progression. In summary, our analysis identified key targets involved in liver homeostasis and revealed a miR‐122 target‐signature predictive in human HCC.

Support or Funding Information

This work was supported by NIH grants RO1CA193244 and R01CA19324406S1 (K. Ghoshal).
miRNA‐mediated crosstalk between Wnt3a and TGFβ3 in osteoblast differentiation
Authors: Fushimi Shigeko , Nohno Tsutomu , Nishimatsu Shin‐ichiro , Katase Naoki , Terada Kumiko , Katsuyama Midori , Demura Masashi , Saijoh Kiyofumi , Nagatsuka Hitoshi , Katsuyama Hironobu
Published: November 2023

miRNAs play roles in regulating gene expression at the post‐transcriptional level through fine‐tuning of the protein‐encoding gene expression, and are involved mainly in regulating important biological processes in different cell and tissue types, including osteoblast differentiation. Canonical Wnt signaling is also known to play pleotropic roles in regulating cell differentiation during mammalian osteogenesis, although involvement of miRNAs is not established for Wnt signaling in osteoblast differentiation. We examined here the role of candidate miRNAs expressed differentially after Wnt3a expression during osteoblast differentiation.

Overexpression of Wnt3a resulted in increased transcription of the alkaline phosphatase gene, but in decreased transcription of the Runx2, collagen 1a and osteocalcin genes in osteoblastic MC3T3‐E1 cells. Analysis of miRNA expression profile showed that 14 miRNAs were up‐regulated and 21 miRNAs were down‐regulated after Wnt3a overexpression in MC3T3‐E1 cells. TGFβ3 is considered to be a candidate target gene that was down‐regulated by one of miRNAs with database search. Transfection of the miRNA mimic into MC3T3‐E1 cells significantly inhibited TGFβ3 mRNA level and then resultant protein expression, whereas overexpression of Wnt3a increased TGFβ3 mRNA level and protein expression.

These results suggest that the miRNA is involved in osteoblast differentiation as a critical regulatory factor, mediating Wnt3a signaling pathway and TGFβ3 signaling pathway.

Support or Funding Information

Authors declare that we have no conflicts of interests.
Role of Long Non‐Coding RNA Alive in Response to Angiotensin II in Vascular Smooth Muscle Cells
Authors: Amaram Vishnu , Das Sadhan , Leung Amy , Reddy Amar , Lanting Linda , Natarajan Rama
Published: November 2023

Angiotensin II (Ang II) is a key pro‐inflammatory and growth‐promoting factor involved in the development of cardiovascular diseases (CVDs) like hypertension and atherosclerosis by promoting vascular smooth muscle cell (VSMC) dysfunction. Long noncoding RNAs (lncRNAs) are important regulators of gene expression and are associated with disease processes. Using high throughput RNA‐sequencing, we recently showed that several novel lncRNAs are differentially regulated by Ang II in VSMC. Here we investigated the role of one of these novel lncRNAs in Ang II mediated VSMC dysfunction. We found that an Ang II regulated lncRNAs in VSMC is transcribed from H3K27ac marked active enhancer regions and perhaps functions as an enhancer RNAs (eRNAs). This lncRNA, which we named “Ang II induced long noncoding RNA in vascular cells with enhancer function” (Alive), was upregulated by Ang II in rat and human VSMCs in a time dependent manner. Alive expression was also induced by platelet derived growth factor (PDGF) in rat VSMC. The expression of the mouse ortholog of Alive was increased in vivo in aortas of Ang II infused mice. Reporter luciferase assays showed that the putative Alive enhancer region indeed functions as a transcriptional enhancer. RNA‐FISH and subcellular fractionation by RT‐qPCR experiments showed that Alive is predominantly localized in the nucleus. Alive transcript is located 117 kb upstream of an Ang II responsive protein coding neighboring gene Aggrecan (Acan) that codes for an extracellular matrix protein and is associated with CVDs. Functional analysis revealed that knockdown of Alive with siRNAs or GapmeRs in rat VSMC decreased the expression of not only Alive, but also its neighboring gene Acan. Furthermore, knockdown of Alive resulted in the upregulation of inflammatory genes such as IL6 and Ptgs2. Currently, CRISPR‐dCas9 mediated activation of Alive expression is being tested to determine the effects of gain‐of‐function of Alive in VSMCs. These results suggest that Alive functions as a novel anti‐inflammatory lncRNA and its expression is increased in response to Ang II to protect against the Ang II associated VSMC dysfunction involved in CVDs.

Support or Funding Information

NIH, NHLBI.
Regena/NOT2 is Essential for Gene Silencing by MicroRNAs
Authors: Zbornik Tika , Andersen Katherine , Bowden Sean , Reinke Catherine
Published: November 2023

A forward genetic screen in Drosophila melanogaster has yielded genetic evidence demonstrating a requirement for the CCR4/NOT complex subunit Regena/NOT2 in gene silencing. A single mutation in Regena/NOT2 generated by ethyl methanesulfonate mutagenesis leads to increased expression of a GFP reporter that is fused to the 3′UTR of the Drosophila Bearded gene and sensitive to microRNA‐mediated gene silencing. MicroRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). MicroRNA‐mediated silencing is accomplished by miRISC through translation block, target mRNA degradation, or a combination of these mechanisms. To determine whether the observed requirement of Regena/NOT2 for gene silencing is dependent upon the activity of microRNAs, the effect of a mutation in Regena/NOT2 on a GFP‐based reporter with disrupted microRNA‐binding sites and insensitive to microRNA‐mediated gene silencing was assayed. No change in reporter expression was observed in the presence of a Regena/NOT2 mutation when the reporter lacked microRNA‐binding sites, supporting a microRNA‐dependent role for Regena/NOT2 in gene silencing. We are further exploring the specific role of Regena/NOT2 in gene silencing by determining whether this CCR4‐NOT complex component is required for translational block, transcript degradation, or both, and characterizing a putative role for Regena/NOT2 in microRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the forward genetic screen aim to identify additional essential miRISC components, to elucidate the complex molecular mechanisms of microRNA‐mediated silencing.

Support or Funding Information

This work was supported by a grant from the M.J. Murdock Charitable Trust
A Bifunctional Fusion Enzyme with DNA polymerase and dUTPase Activities
Authors: Dash Asim Kanti , Bhamidipati Sravya , Rashid Mohammad Bazlur
Published: November 2023

Construction of a fusion enzyme by recombinant DNA technology could be useful in obtaining more efficient bifunctional enzymes. However, two open reading frames in tandem often interfere with each other's functions. We have previously demonstrated that when a relatively smaller dUTPase enzyme is tagged on either side of a thermostable DNA polymerase it did not affect polymerization function. Here, we will further reveal that both the enzymatic domains of these fusion enzymes also do not muddle each other's functions indeed. Furthermore, both of these affinity purified fusion proteins appeared to be ‘undisruptable dimers’ in denaturing protein gels and as confirmed by MALDI‐MS/MS analyses.

Support or Funding Information

Faculty Research Support Fund from the Office of Sponsored Programs at the University of Houston‐Clear Lake, Texas.
Extra‐ribosomal function of bacterial ribosomes: Modulation of enzyme activities
Authors: Shekhtman Alexander , DeMott Chris , Majumder Subhabrata , Reverdatto Sergey
Published: November 2023

The intracellular concentration of bacterial ribosome is linearly proportional to the growth rate and can occupy up to 20% of the cell volume or about 50 mg/mL. This high concentration facilitates weak binding between cytosolic proteins and ribosomes and can affect protein biochemistry. By using a combination of in‐cell and in vitro biophysical techniques, we show that adenylate kinase (ADK) and dihydrofolate reductase (DHFR) bind to ribosomes via specific interaction surfaces, and the respective co‐factors ATP and NADPH also bind to ribosomes both specifically and non‐specifically. As a consequence, ribosomes modulate the enzymatic activity of ADK and DHFR by interfering with substrate availability. Ribosome binding also impedes protein diffusion, an important determinant for reaction rates. Using fluorescence recovery after bleaching (FRAP), we show that green fluorescent protein (GFP) diffusion is significantly slower in the presence of ribosomes in vitro, comparable to that observed intracellularly. These results strongly suggest that ribosomes play a previously under‐appreciated, non‐translational role in regulating the cellular activity of enzymes.

Support or Funding Information

This work was supported by National Institutes of Health Grant 5R01GM085006 to A.S.
Assembly and disassembly of the hibernating bacterial 100S ribosome
Authors: Basu Arnab
Published: November 2023

Translation is an important biological process critical for all life. Suppression of ribosome biogenesis and translation have been linked to bacterial persistence and slow growth inside the animal hosts. In Gram‐positive human pathogen Staphylococcus aureus, a ribosome‐binding protein called hibernation‐promoting factor (HPF) dimerizes the 70S ribosome to form the translationally inactive 100S complex. The formation of 100S ribosome in S. aureus is distinct than that of the majority of the gammaproteobacteria. In S. aureus the formation of 100S ribosome dimer begins at log phase and continues throughout the stationary phase, whereas the 100S dimer formation is specifically induced during the stationary phase in gammaproteobacteria (such as E. coli). Importantly, E coli requires a helper protein called ribosome modulation factor (RMF) in addition to HPF to promote 70S dimerization. The biological significance of the bacterial 100S ribosome has been elusive for six decades. We found that the loss of hpf reduces S. aureus survival and causes rapid degradation of ribosomes during stationary phase. Disrupting the 100S formation only derepresses the translation of a subset of genes. The reason for the selectivity of HPF‐dependent translation attenuation is unclear. Our recent work suggests a model by which the 100S ribosome serves as a reservoir for long‐term S. aureus viability because the 100S ribosomes may be more resistant to ribonuclease degradation. We propose that during stationary phase, the 100S ribosomes in the wild‐type are dissembled by an unidentified dissociation factor into subunits and may be recycled for a new round of translation. In contrast, the hpf mutant exhausts all ribosome pools upon entry to the late stationary phase, leading to cell death. Using biochemical and genetic approaches, we identified a bona fide dissociation factor responsible for the disassembly of 100S ribosome. Because the dissociation factor antagonizes the stability of 100S ribosome, we examined the temporal expression of HPF and the dissociation factor to elucidate how they impact the homeostasis of 100S ribosomes. We also performed chemical crosslinking to map the position of HPF on the ribosome that may account for its translational inhibition function. These results provide a plausible explanation for the constitutive production of 100S ribosome that is intimately related to S. aureus pathophysiology.

Support or Funding Information

Edward Mallinckrodt Jr Foundation, and The Pew Charitable Trusts.
Carboxyl‐Terminus of TFG Regulates Directional Movement of COPII Transport Carriers
Authors: Block Samuel , Hanna Michael , Audhya Anjon
Published: November 2023

Roughly one fourth of all proteins encoded by the human genome are integral membrane proteins that participate in a large number of vital biochemical processes, such as ion transport and signal propagation. To achieve functionality, these proteins must travel through the secretory pathway, in which they are modified and trafficked to their specific organelles. The journey along the secretory pathway originates at subdomains on endoplasmic reticulum (ER) tubules, known as ER exit sites (ERES). Here, newly synthesized proteins embedded in the ER membrane, as well as soluble cargoes, are packaged into vesicles that bud off the ER in a process dependent on the COPII machinery. While the minimum components needed to generate a COPII vesicle have been identified, the mechanism that facilitates the coordinated anterograde transport of these carriers remains poorly understood. Our lab has previously identified TFG (Trk‐fused gene) as an essential component of the coordinated efflux of COPII vesicles from ERES. TFG contains an intrinsically disordered region at its carboxyl‐terminus capable of homo‐oligomerization in Vitro, and is essential for its co‐localization with COPII components in Vivo. Given these data, we hypothesized that the carboxyl‐terminus of TFG regulates the movement of COPII vesicles in a process dependent upon self‐oligomerization, as well as interactions with other components of COPII vesicles. To test this hypothesis, we used affinity chromatography with recombinant GST tagged TFG and a preparation of rat liver cytosol to isolate potential interacting partners, which can be further characterized by mass spectrometry.
Probing the Pal‐Peptidoglycan Interaction
Authors: Phadke Shivani , Stanton Sarah , Pierce Jeanetta , LaClair Ciara , Hall Cianna , Michel Lea
Published: November 2023

Peptidoglycan‐associated lipoprotein (Pal) is a dual‐oriented lipoprotein in Escherichia coli (E. coli) that interacts non‐covalently with peptidoglycan (cell wall) and other outer membrane and periplasmic proteins. By comparing Pal to its homologue in another Gram‐negative bacterium, our team was able to design point mutations at possible interaction sites between Pal and peptidoglycan. Recombinant Pal proteins (wild‐type and mutants) were expressed in E. coli and purified using His‐tag column chromatography. Multiple protocols were employed to purify peptidoglycan from Escherichia coli cells, with varying results. Using an enzyme‐linked immunosorbent assay (ELISA), we were able to assess the binding affinity of each Pal mutant to peptidoglycan, compared to wild‐type protein. Preliminary data suggest that several key residues help to modulate the Pal‐peptidoglycan interaction, and site‐directed mutants of Pal bind differentially to peptidoglycan compared to wild‐type Pal.

Support or Funding Information

The Camille and Henry Dreyfus Foundation: Special Grant Program in the Chemical Sciences (L. Michel).
Identifying serine proteases involved in HMPV F cleavage
Authors: Kinder Jonathan Tyler
Published: November 2023

Human metapneumovirus (HMPV) is a recently identified pneumovirus which causes severe acute respiratory tract infections in children, elderly and immunocompromised patients worldwide. Though this viral pathogen is ubiquitous and responsible for a large number of respiratory tract infection‐associated hospitalizations, there are no vaccines or antiviral therapies other than supportive therapy. HMPV encodes for three surface glycoproteins including the small hydrophobic protein, the attachment protein and the fusion protein (F). The main mediator of viral infectivity is the F protein, which attaches to host cell surface receptors and promotes fusion between the viral and cellular membranes to mediate successful entry. However, in order to mediate viral infection, the HMPV F protein must be proteolytically processed by host serine proteases into a metastable form. TMPRSS2 is a serine protease that has been shown to cleave HMPV F and allow for proper protein function and subsequent infection1. Other serine proteases that are hypothesized to be involved in HMPV F cleavage include HAT, Matritpase, KLK5 and KLK12, all of which have been shown to process influenza hemagglutinin. Using transiently expressed HMPV F in Vero cells, we have verified the requirement of a serine protease to cleave HMPV F. Data on analysis of the ability of both transmembrane and secreted serine proteases to process HMPV F into its fusogenically active form will be presented.

Support or Funding Information

University of Kentucky department of cellular and molecular biochemistry
Identification of Different Binding Partners of the F‐BAR Proteins Cdc42 Interacting Protein 4 (CIP4) and Formin Binding Protein 17 (FBP17) in Cortical Neurons
Authors: McDermott Maeve E , Taylor Russell J. , Taylor Kendra L. , Dent Erik W.
Published: November 2023

During neuronal development, neurons perform many dynamic processes, including migration and differentiation, which require the coordination of movements between both the plasma membrane and the underlying cytoskeleton. The Cdc42 Interacting Protein 4 (CIP4) subfamily of F‐BAR proteins contains an N‐terminal F‐BAR domain that can interact with the plasma membrane and C‐terminal HR1 and SH3 domains that can interact with Rho GTPases and actin associated proteins, respectively. F‐BAR proteins, which function in detecting and inducing membrane curvature, can effectively link the plasma membrane and the cytoskeleton. Previous studies have shown that CIP4 and another family member, Formin Binding Protein 17 (FBP17), exhibit distinct functions in neurons. CIP4 tends to accumulate in the extending peripheral membrane, inhibiting neurite protrusion, while FBP17 tends to localize in membrane tubules, function in endocytosis, and assist in neurite/axon outgrowth. In this work, we used Western blot and immunoprecipitation assays to show that CIP4 and FBP17 interact with different subsets of proteins to produce their distinctive functions. We have shown previously that CIP4 colocalizes with actin‐associated proteins such as WASP Family Verprolin‐Homologous Protein 1 (WAVE1) and Disheveled Associated Activator of Morphogenesis 1 (DAAM 1). Here we will determine if WAVE1 and DAAM1 form a complex with CIP4 and if FBP17 interacts with a different set of proteins than CIP4 in cortical neurons, including N‐WASP (Neural Wiskott‐Aldrich syndrome protein) and dynamin. Results from these studies will help determine how two very similar proteins function in entirely different processes in developing neurons.
Affinity and Structural Characterization of Human AT Hook Motif Variants
Authors: Dobbins Kaitlyn , Buchmueller Karen L
Published: November 2023

High Mobility Group A (HMGA) proteins are a family of architectural transcription factors that control the expression of many genes. Overexpression of such proteins has been linked to metastasis in numerous cancers. HMGA proteins bind to the minor groove of DNA through two or three positively‐charged AT hook motifs, each containing the invariant sequence Arg‐Gly‐Arg‐Pro. We endeavor to investigate the effects of changing the amino acids flanking this invariant sequence using fluorimetry, circular dichroism, and filter binding. The peptides were designed to have varying flanking sequences based on amino acid occurrences in human AT hook proteins. Binding characteristics and the dynamic structure of the amino acid sequences will be elucidated by our analysis and will inform the development of HMGA‐targeting therapeutics.

Support or Funding Information

Beckman Scholars Program; Arnold and Mabel Beckman Foundation
Analysis of Complex Interactions Between the Essential Subunits, Pam16, Tim44, and Tim50, of the Hsp70‐based Mitochondrial Protein Import Machinery
Authors: Yan Nicholas Lok , Ting See‐Yeun , Schilke Brenda , Craig Elizabeth A
Published: November 2023

Because the vast majority of mitochondrial proteins are synthesized in the cytosol, efficient protein import is essential for mitochondrial function and cell viability. The mitochondrial protein import motor, which is tethered to the matrix face of the inner membrane, plays a critical role. This motor translocates preproteins from the cytosol, across both mitochondrial membranes, into the mitochondrial matrix.

This biological machine is driven by the molecular chaperone mtHsp70, which binds translocating preproteins entering the matrix in an ATP‐dependent manner. Both mtHsp70 and its co‐chaperone Pam18, which performs the critical function of stimulating Hsp70's ATPase activity, are tethered to the translocon by Tim44. While mtHsp70 directly interacts with Tim44, evidence suggests that Pam18 is tethered via Pam16, with which it forms a heterodimer. However, how Pam16 interacts with Tim44 is not well understood.

This study aims to determine how Pam16 interacts with Tim44 and the functional consequences of the interaction. Our previous genetic and co‐immunoprecipitation analyses implicated distinct regions in Pam16 and Tim44, both in their N‐termini, that may be the sites of interaction. Alterations in these regions not only caused Pam16 to dissociate from the translocon, but also led to temperature‐sensitive growth defects, demonstrating the importance of this interaction. We now report site‐specific crosslinking demonstrating that Pam16 and Tim44 are in close proximity to each other. When a photoactivatable crosslinker was incorporated at several positions in these regions, Pam16 could be crosslinked to Tim44, or vice versa. Altogether, these results suggest that Pam16 directly interacts with Tim44 via these regions.

In addition to crosslinking to Tim44, we found that the extreme N terminus of Pam16 also crosslinked to Tim50, an essential translocon subunit that facilitates movement of the preprotein from the translocon of the outer membrane to that of the inner membrane. Tim50 is a transmembrane protein with an essential intermembrane space domain and a matrix‐exposed region whose function is unknown. Taken together, these results support the idea of Pam16 as an essential tether in the import motor, mediating complex interactions with subunits that are necessary for mitochondrial protein import.
Structure and Stability of C19orf10 as Assessed by Circular Dichroism
Authors: Bortnov Valeriu , Annis Douglas S. , McCaslin Darrell R. , Mosher Deane F.
Published: November 2023

Human C19orf10 is a poorly characterized 142‐residue protein that is present in numerous tissues and widely expressed in cultured cells. Homologues of this protein are encoded in organisms ranging from humans to slime molds. C19orf10 and its homologues contain an N‐terminal signal sequence to direct the protein into the endoplasmic reticulum (ER) and a KxEL sequence at the C‐terminus that may result in its retention in the ER. A recent publication (Korf‐Klingebiel et al., Nature Medicine 21, 140–149, 2015) provided evidence that C19orf10 in monocytes is involved in tissue repair and promotion of angiogenesis after myocardial infarction. Although this suggests that C19orf10 acts as a paracrine signaling factor after secretion, the conserved KxEL sequence suggests functions in the ER that have yet to be investigated. The structure of C19orf10 is not known nor does its sequence suggest any known structural motif. We sought to characterize the C19orf10 structure using circular dichroism (CD) spectroscopy. Recombinant human C19orf10 fused to a C‐terminal poly‐histidine tag was produced as a secreted protein from baculovirus‐infected insect cells. The purified protein was soluble in dilute acetic acid (pH 4.5) as well as in phosphate‐buffered dilute saline (pH 7.5). Spectra at 56°C and below were nearly identical under both buffer conditions and were dominated by an increase in molar ellipticity from 217 nm down to 202 nm with a small negative signal above 217 nm, suggestive of the presence of β‐strands and β‐turns. At 70°C in neutral buffer, the positive signal was replaced by a broad negative peak centered at ~217 nm that did not recover on return to lower temperature. Under acidic conditions the positive signal also became negative with an inflection near 220 nm but continuing downward with decreasing wavelength; however, the spectra recovered upon return to lower temperature. Although the spectra at the two pHs were not identical at high temperature, these results were suggestive of dramatic temperature‐induced changes in structure under both conditions. This data provides evidence that (1) C19orf10 has a global fold that is stable over a pH range from 4.5 to 7.5 and (2) the fold can be regenerated only at acidic pH after heat denaturation. This characterization of recombinant C19orf10 secreted using the quality control mechanism for correct folding in the ER of eukaryotic cells should facilitate further structural studies by serving as a guide to optimize and validate refolding protocols for C19orf10 produced in more robust bacterial expression systems.
An Automated Method for the Correction of Unsubstantiated Ramachandran Outliers in Protein Structures
Authors: Smith Connor R. , Alaniz Jacob A. , West Korbin H. J. , Weiss Charles J. , Novak Walter R.P.
Published: November 2023

Ramachandran outliers are amino acid residues with phi and psi dihedral angles that result in energetically unfavorable conformations. While some of these conformations are supported by the data and provide key information regarding protein structure and functionality, it is currently not known how many of these outliers are actually supported by the data and how many are simply errors in the structural model. Unfortunately, in the Protein Data Bank, there exists a large number of protein structures that contain an excessive number of these outliers (>0.2%). We have developed an algorithm capable of the automated correction of unsubstantiated Ramachandran outliers. This program was built with the Python programming language using the capabilities of the protein determination software PHENIX for outlier detection and refinement.

Support or Funding Information

This research is supported by the Wabash College Haines Biochemistry Fund.
Complex structure of the disulfide bond‐dimerized PDZ‐RhoGEF and CXCR2 PDZ‐binding motif: A new mode of PDZ dimerization
Authors: Spellmon Nicholas S , Holcomb Joshua , Niu Andrea , Choudhary Vishakha , Brunzelle Joseph , Li Chunying , Yang Zhe
Published: November 2023

Eukaryotic cellular mobility is critically dependent on actin organization and remodeling that are masterfully governed through the Rho signaling pathway activated by Rho guanine exchange factors (RhoGEF). PDZ‐RhoGEF is one unique member of the RhoGEF family which possesses a PDZ domain that is known to nucleate cell surface factors to perpetuate signaling for actin organization and cell mobility. However, the role of the PDZ domain of PDZ‐RhoGEF in signaling transduction pathways for cellular mobility is still not well understood. In this study, we identified PDZ‐RhoGEF binds directly to a novel binding partner CXCR2, a G protein coupled receptor which is known to initiate the Rho signaling program for chemotaxis, haptotaxis, cellular growth and tumor invasion. In order to understand the molecular mechanism of binding, we solved the crystal structure of the chimeric complex between the PDZ domain of PDZ‐RhoGEF and the PDZ‐binding motif of CXCR2. This complex structure reveals that the CXCR2 peptide forms an antiparallel β‐sheet against PDZ‐RhoGEF with the carboxyl‐terminal leucine packaging into a deep hydrophobic pocket within the PDZ domain. Unexpectedly, we found a disulfide bond linking the PDZ domain dimer which secures a parallel binding mode of the CXCR2 ligands to the PDZ monomers. To our knowledge, this is the first PDZ dimer structure covalently linked through a disulfide bond and may be a unique feature to PDZ‐RhoGEF family. This new type of dimerization may be important for stabilizing the CXCR2 macromolecular complex and thereby provide swift signaling for cellular mobility and cancer invasion.

Support or Funding Information

National Heart, Lung, and Blood Institute
The Conformation of Apolipoprotein E4 on Discoidal and Spherical High Density Lipoproteins Using Chemical Crosslinking and Fluorescence Spectroscopy
Authors: Bala Noor , Taiwo Kehinde , Narayanaswami Vasanthy
Published: November 2023

Human apolipoprotein E (apoE) is 34‐kDa 299 residue exchangeable apolipoprotein that plays a critical role in lipid transport and cholesterol metabolism in the plasma and brain. The APOE gene polymorphism results in three different alleles ɛ2, ɛ3, and ɛ4, which produce the common protein isoforms, apoE2, apoE3, and apoE4, respectively. Whereas apoE3 is considered to be the anti‐atherogenic protein, apoE4 is considered a risk factor for developing Alzheimer's disease. ApoE associates mainly with vLDL but is associated with HDL‐like particles in the brain. During brain cholesterol metabolism, apoE associates with HDL to form discoidal nascent HDL which are remodeled into spherical HDL by the action of lecithin cholesterol acyltransferase (LCAT). In this study, we aim to monitor the conformational changes apoE4 undergoes during LCAT‐mediated conversion of reconstituted discoidal‐HDL (rHDL) to spherical‐HDL using chemical crosslinking and N‐(1‐pyrene)maleimide as a fluorescence probe. Single cysteine mutants of apoE4 were expressed in E. coli, purified by affinity chromatography, and reconstituted with POPC or POPC/cholesterol. The Cys residues serve as the site for attachment for the fluorescent probe or crosslinker. Discoidal rHDL (apoE4/POPC) was chemically cross‐linked using a Cys‐specific crosslinker. SDS‐PAGE revealed monomeric and dimeric bands in variants with Cys at positions 29 and 256. The dimers suggest that the Cys residues on the apoE4 mutant molecules were at a crosslinkable distance when bound to rHDL. Other residues along the length of apoE4 were not at a crosslinkable distance when bound to rHDL. To obtain spherical rHDL, recombinant human LCAT was expressed using Chinese hamster ovary cells and its specific activity was determined to be 0.8 nmol/h/mg protein using POPC/apoAI as substrate. We are currently in the process of confirming the conformation of apoE4 around the discoidal and spherical particles by an independent approach using spatially–sensitive fluorescence probes that allow us to measure the proximity of two Cys residues on two different apoE4 molecules on rHDL. It is anticipated that the combination of cross‐linker distance constraints and the excimer fluorescence emission spectra of the seven apoE4 mutants on HDL would yield an understanding of the organization of apoE4 on these particles. The significance of this study is that it offers an innovative approach to obtain insight into the structure and organization of apoE on large lipoprotein complexes. Further, it allows us to identify potential differences between apoE3 and apoE4 from a structural perspective and determine distinguishing features that contribute to the role of apoE4 in developing AD.

Support or Funding Information

This project is supported by a grant from the National Institutes of Health #GM105561.
A DISCRETE SERCA N‐DOMAIN LOOP PLAYS A ROLE IN PUMP STRUCTURAL DYNAMICS AND FUNCTIONAL REGULATION
Authors: Raguimova Olga N , Smolin Nikolai , Bovo Elisa , Zima Aleksey V , Robia Seth L
Published: November 2023

The sarco/endoplasmic reticulum calcium ATPase (SERCA) and its inhibitor phospholamban (PLB) are major regulators of calcium handling in cardiomyocytes. We have previously hypothesized that a compact structure of SERCA is the signature of a high efficiency catalytic pathway. Previous molecular dynamics (MD) simulations of SERCA spontaneous movements showed that a SERCA headpiece open‐to‐closed transition was initiated by an interaction between the A‐domain and the beta 5,6‐loop of the N‐domain. Here, we used additional MD simulations to identify specific residues of the loop that facilitate SERCA headpiece disorder‐to‐order transition. Fluorescence resonance energy transfer (FRET) measurements between fluorescently tagged A‐ and N‐domains of SERCA confirmed that mutation of key loop residues reduced the rate of SERCA headpiece closure during calcium cycling. As a functional consequence of loop mutations, SERCA ATPase activity was impaired by 60% without a reduction in calcium sensitivity. Calcium transport by SERCA was also reduced by loop mutations, as measured by a low affinity calcium sensor (R‐CEPIA1er) in the ER of HEK cells. Interestingly, MD simulations of a SERCA‐PLB complex model showed that PLB phosphorylation stabilizes the SERCA headpiece in an ordered conformation via an interaction with N‐loop. Thus our data suggest that the beta 5,6‐loop of SERCA N‐domain is important for stabilization of the SERCA compact conformation during calcium transport and plays a role in functional regulation by PLB phosphorylation. This small discrete structure represents a potential target for rational design of novel therapeutics to modulate cardiac function.
Conformational Changes in Palladin Actin‐Binding Domains Measured by Fluorescent Resonance Energy Transfer
Authors: Womack Samuel , Vattepu Ravi , Beck Moriah Rene
Published: November 2023

The fundamental goal of our research is to understand how cell motility in both normal and metastatic cells is regulated by palladin, an actin binding protein that plays a key role in remodeling the actin cytoskeleton. Our recent work has established that palladin regulates actin dynamics and organization by enhancing the polymerization rate and stabilizing filaments in an orthogonal meshwork. Palladin family members all utilize immunoglobulin‐like domains to bind and crosslink actin filaments. Various isoforms of palladin contain from three to five immunoglobulin‐like domains that have been implicated in a variety of protein‐protein interactions. Our previous results indicate that the Ig3 domain of palladin is directly involved in binding and crosslinking of actin and also promotes actin polymerization, whereas the Ig4 domain of palladin does not interact with actin directly. Nevertheless a tandem Ig3–4 domain protein exhibits dramatically more efficient actin binding, crosslinking, and polymerization than the isolated Ig3 domain. Recently we established that the Ig3–4 domains of palladin undergo actin‐induced dimerization, which likely results in a conformational change. NMR chemical shift measurements indicate that interactions between the tethered Ig3 and Ig4 domains are limited to the interface and lead to no more than marginal structural changes. To further narrow the gap in our understanding of palladin's role in actin assembly, we need to examine these interactions in the cellular context and demonstrate that palladin can bind directly to monomeric actin. Our current efforts are aimed at combining fluorescence resonance energy transfer (FRET) and confocal microscopy to analyze the interactions of palladin and G‐actin within cells and with isolated proteins. These experiments will also enable us to investigate the dynamic intra‐ and intermolecular interactions through the use of sophisticated fluorescent probes. Our first goal was to use site‐directed mutagenesis to replace six cysteines with alanine in two tandem immunoglobulin‐like domains of palladin before confirming that these mutations do not interfere with actin binding and bundling functions of palladin. We will share our results that make use of a newly synthesized Pacific Blue fluorescent trivalent nickel complex that we attach to the C‐terminal His6‐purification tag on our palladin constructs. We have also introduced a unique cysteine residue at the N‐terminus of palladin to allow conjugation to fluorescein maleimide so that we can track conformational changes that occur in the tandem actin‐crosslinking domains of palladin using FRET.

Support or Funding Information

This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418 and P20 GM103638 and an undergraduate research grant from Wichita State University.
Conformational Motions Impacting Function in an Enzyme Superfamily
Authors: Narayanan Chitra , Bernard David N , Bafna Khushboo , Choudhary Om P , Chennubhotla Chakra S , Agarwal Pratul K , Doucet Nicolas
Published: November 2023

Correlation between conformational dynamics and enzyme function has been well established for discrete enzyme systems. However, approaches for characterizing dynamical properties across diverse sequence homologs within a family and their correlation with enzyme activity remain challenging. Members of the pancreatic‐type ribonuclease (ptRNase) superfamily share similarities in structure and fold, but display large variations in conformational dynamics, catalytic efficiencies, and tissue specific biological activities, making them ideal model systems for probing the relationship between conformational motions and function. As a step towards determining the relationship between dynamics, catalytic mechanism and catalytic efficiency for various members of this broad vertebrate family, we have performed the systematic characterization of the intrinsic dynamics of over twenty RNases with experimentally solved structures over a wide range of time‐scales by integrating molecular dynamics simulations and NMR relaxation dispersion experiments. Our results show distinct patterns of dynamical variations between the canonical RNases clustered on taxonomic groups. We show that conformational motions on the catalytically relevant micro‐ to milli‐second timescale are significantly different for RNases sharing a common fold. Interestingly, sequences sharing similar conformational exchange on the catalytic timescale also share similar biological functions. These results suggest that selective pressure for the conservation of specific atomic‐scale dynamical behaviors, among other factors, may potentially impact distinct biological functions within the same fold. Further experiments are required to characterize this correlation between conserved dynamical properties and biological function.
Identifying and Ameliorating Complex Collagen Misfolding Defects
Authors: Shoulders Matthew D
Published: November 2023

Collagen is the most abundant protein in our bodies, constituting roughly one‐third of the proteome. In keeping with collagen's status as the molecular scaffold for human life, the maintenance of collagen proteostasis is one of the most critical roles of the endoplasmic reticulum (ER) proteostasis network, which is responsible for folding, modifying, processing, and quality controlling nascent collagen proteins. Dysregulated collagen proteostasis leads to numerous currently incurable diseases, including osteogenesis imperfecta. Here, we use a mechanistic biochemistry approach to decipher a cysteine‐based code for collagen heterotrimer assembly in the ER, a process that is critical for the maintenance of bone and tissue health. Moreover, we show that pharmacologic manipulation of the unfolded protein response, which maintains ER proteostasis, is able to resolve collagen folding and secretion defects that underpin osteogenesis imperfecta. These studies provide new insights into how cells solve the collagen proteostasis problem, with potentially broad implications for complex protein folding in the ER and for efforts to modulate the proteostasis network to therapeutically address the collagenopathies.

Support or Funding Information

NIH NIAMS 1R03AR067503
Protein Aggregation in Ehrlichia chaffeensis during Infection of Mammalian Cells
Authors: Zolkiewski Michal , Kuczynska‐Wisnik Dorota , Cheng Chuanmin , Ganta Roman R.
Published: November 2023

Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected ticks to humans and several other mammals. Human monocytic ehrlichiosis (HME) caused by E. chaffeensis occurs mainly in the U.S.A, but also has been reported in other parts of the world. The severity of HME varies from asymptomatic to fatal. To establish infections in the host cells, E. chaffeensis needs the capability to evade the host defenses, but it is still not clear how the pathogen responds to and overcomes the host‐induced stress. We have previously shown that the expression of the molecular chaperone clpB in E. chaffeensis is strongly upregulated immediately after infection of mammalian cells. ClpB is an ATP‐dependent heat‐shock protein with a unique disaggregase activity that reactivates aggregated proteins under cellular stress conditions. Induction of clpB in E. chaffeensis during its replication in mammalian cells suggests that the ClpB activity supports the pathogen survival inside the host, but no link between protein aggregation and a pathogen life cycle has been established so far. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the mammalian cell environment induced misfolding and aggregation of a significant fraction of E. chaffeensis proteins. The size of the aggregated fraction of E. chaffeensis proteins increased during the first 48 h post infection. The 48‐h post infection window corresponds to the upregulated production of the clpB mRNA. To disrupt the activity of ClpB in E. chaffeensis, we incubated the infected cells with up to 0.5 mM guanidinium chloride (GuHCl), which inhibits the ClpB ATPase activity in vitro. 0.5 mM GuHCl had no impact on the host cells, whereas the viability of the pathogen was reduced by ~80% in the presence of the inhibitor. Importantly, we found that the size of the aggregated protein fraction in E. chaffeensis increased significantly in cultures supplemented with 0.5 mM GuHCl. We also found that ClpB accumulated with protein aggregates formed in E. chaffeensis in the presence of GuHCl. Thus, we discovered that an exposure of E. chaffeensis to the stressful host environment during infection results in aggregation of the pathogen's proteins. Moreover, we showed that the molecular chaperone ClpB supports the survival of E. chaffeensis during the infectious stage by reducing the amount of protein aggregates. Altogether, our studies establish a novel link between the protein aggregation control and the pathogen survival.

Support or Funding Information

This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (grant P20 GM103418), the National Institute of Allergy and Infectious Diseases (grant R01 AI070908 to RG and R56 AI121366 to MZ), and the Center of Excellence for Vector‐Borne Diseases at Kansas State University.