bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure activity relationship study of the Cleistriosides and Cleistetrosides for antibacterial/anticancer activity
Authors: Shi Pei
Published: June 2021

The de novo asymmetric syntheses of several partially acylated dodecanyl tri‐ and tetra‐rhamnoside oligosaccharides have been achieved by using the divergent route. The desired sugar absolute stereochemistry is achieved by the use of highly enantioselective Noyori reduction and a diastereoselective palladium‐catalyzed glycosylation.1In addition; all remaining triol functionalities of the sugar moieties were established via corresponding post‐glycosylation transformations with the use of only three or less protecting groups.

As one of the primary components of bacterial cell‐walls,2 oligosaccharides and the rhamnan in particular, play important biological roles, such as cell‐cell interaction, cell adhesion, and immunogenic recognition.3,4 Since the bacterial cell wall oligo‐rhamnose‐containing structurally motifs are not present in mammalian cells,5 they are potentially interesting targets for the development of anti‐bacterial agents.

With these ten oligosaccharides in hand, biological activities were measured against several bacteria strains and NCI‐H460 human lung cancer epithelial cell line. This study for the first time reports anticancer activity and comprehensively the antibacterial activity for this class of natural products. In addition, seven of these oligosaccharides were found to have potent activities against the NCI panel of 60 cell lines. Interestingly, the observed varieties of antibacterial and anticancer activities suggest that the degree of acylation and location of the specific acetate groups might play an important role on the underlying modes of action.6
Global deletion of stearoyl‐CoA desaturase‐2 enhances glucose tolerance
Authors: O'Neill Lucas , Ntambi James
Published: June 2021

Stearoyl‐CoA desaturase (SCD) catalyzes the rate‐limiting step in the synthesis of monounsaturated fatty acids (MUFAs) from saturated fatty acids. There are four SCD isoforms in mouse (SCD1–4) and two in human (hSCD1 and 5). Here we begin to elucidate the function of stearoyl‐CoA desaturase‐2 (SCD2) in whole body metabolism. SCD2 is highly expressed in the brain, immune cells, and pancreas. Depending on the genetic background, prenatal loss of SCD2 is either embryonic lethal or 70% lethal within 24 hours after birth. Our microarray data comparing SCD2KO embryos to WT embryos showed that ~20% of the genes down regulated are pancreatic. In a follow up study within the pancreas, it was shown that SCD2 is predominately expressed in the alpha and beta cells. We report that global loss of SCD2 in mice confers enhanced glucose tolerance and lowers both fasting and fed plasma glucose levels. We hypothesize this is attributed to enhanced insulin secretion. Moreover, we propose a mechanism where endogenously synthesized MUFAs in beta cells act as antagonists for insulin secretion. This work was funded by NIH.
Identification of Putative Trafficking Motifs in System xc−
Authors: Lang Sara , Georges Anne , Chase Leah
Published: June 2021

System xc−is a heterodimeric plasma membrane transporter, comprised of xCT and CD98, that facilitates the stoichiometric exchange of extracellular cystine for intracellular glutamate. System xc− serves to protect the cell from oxidative stress, and brief exposure of cells to oxidative agents leads to an increase in trafficking of xCT to the plasma membrane. However, little is known about the mechanisms which govern the constitutive and regulated trafficking of xCT. In the present study, we sought to identify the role of the C‐terminus of the light chain, xCT, in the observed constitutive trafficking behavior of system xc−. To do this, we created two constructs, myc‐xCT and HA‐CD98, and used site‐direct mutagenesis to disrupt putative trafficking motifs within the C‐terminus of xCT. When these mutants were expressed in COS‐7 cells, we found that the C‐terminus of xCT is critical for the proper trafficking of xCT, and further identified a specific motif within the C‐terminus that, when disrupted, alters basal surface expression of xCT. We hypothesize that the motif is a tyrosine‐based, clathrin‐dependent endocytic motif that contributes to the constitutive internalization of xCT. The key amino acid of the motif, a tyrosine, is conserved in 6 of 7 members of the SLC7 family and is hypothetically located in a position accessible to intracellular proteins. This research was supported by NSF‐RUI #0848564.
cAMP‐stimulated Expression of DGKθ Requires Steroidogenic Factor‐1 and Sterol Regulatory Element Binding Protein
Authors: Cai Kai , Sewer Marion
Published: June 2021

Diacylglycerol kinase theta (DGKθ) plays a key role in cortisol production in the adrenal cortex by generating phosphatidic acid (PA), an agonist of the nuclear receptor steroidogenic factor‐1 (SF‐1). In an attempt to identify the role of cAMP signaling in DGKθ gene expression regulation, we found that dibutyryl cAMP (Bt2cAMP) increases the mRNA and protein expression of DGKθ in H295R human adrenocortical cells. Reporter gene assays using 1.5 kB of the DGKθ promoter fused to the luciferase gene identified two regions of the promoter that were required for cAMP responsiveness. Moreover, overexpression of SF‐1 and sterol regulatory element binding protein 1 (SREBP1) increased the DGKθ transcriptional activity and mutation of the cAMP responsive regions abolished the ability of SF‐1 and SREBP1 to activate DGKθ luciferase activity. Chromatin immunoprecipitation assays revealed that Bt2cAMP promoted the recruitment of SF‐1 and SREBP1 to the endogenous DGKθ promoter. Finally, silencing SF‐1 or SREBP1 reduced both basal and cAMP‐stimulated DGKθ gene expression and diminished cAMP‐stimulated PA production. Taken together, we identify SF‐1 and SREBP1 as regulators of DGKθ transcription and PA production in human adrenocortical cells.
Expression and Characterization of Recombinant Neutral Sphingomyelinase 2 in Escherichia coli: Evidence of Sensitivity to Redox State
Authors: Dotson P. Patrick , Nikolova‐Karakashian Mariana
Published: June 2021

Neutral sphingomyelinases are a family of lipid‐metabolizing enzymes that hydrolyze sphingomyelin to ceramide. Neutral sphingomyelinase 2 (nSMase‐2), in particular, was identified as the main cytokine‐inducible sphingomyelinase. Several recent studies have correlated nSMase‐2 activity with the state of oxidative stress. Depletion of cellular GSH, NADH oxidase, or exposure to agents that generate reactive oxygen species have been linked to upregulation of nSMase‐2 activity in various mammalian cells. The mechanisms for activation of nSMase‐2 during oxidative stress conditions, however, remain not completely understood.

In this study, we investigate whether nSMase‐2 is a redox sensitive enzyme. A recombinant mouse nSMase‐2 construct was overexpressed in an Escherichia coli strain whereby recombinant proteins were produced that, based on in vitro activity assays, were functionally active. Interestingly, we demonstrate that nSMase‐2 exists in multimeric forms that are sensitive to reducing agents, which could correspond to cysteine‐mediated oligomerization between nSMase‐2 molecules. To directly test this, 17 of the 22‐ cysteine residues within nSMase‐2 were gradually mutated to serine. Gradual mutagenesis resulted in a loss of nSMase‐2 multimers with cysteine residues at position C561 and C617, within the catalytic domain, being identified as being involved in nSMase‐2 oligomerization. Furthermore, we show that the cysteine‐mediated nSMase‐2 oligomerization may be modulated through the thiol‐reducing enzyme thioredoxin. Together, these results suggest that nSMase‐2 is a redox‐sensitive enzyme and the redox state of specific cysteine residues may regulate nSMase‐2 activity.
Searching the extracellular domain of CD36 for fatty acid binding by Surface Plasmon Resonance
Authors: Jay Anthony , Chen Alexander , Hamilton James A.
Published: June 2021

Obesity has become a major public health concern and represents a predisposition factor for the development of cardiovascular‐related disease, ischemic stroke, and non‐insulin dependent diabetes mellitus. Deciphering the molecular mechanisms of ligand binding and subsequent signaling of plasma membrane “scavenger” proteins such as CD36, will provide new insights for therapeutic interventions for lipotoxicity, inflammation, and disease associated with obesity. Upon CD36 ligand binding and activation, inflammatory signaling often occurs, leading to an increase in proinflammatory cytokines. Localized fatty acid (FA) binding at the plasma membrane has been postulated to occur in a single site in the extracellular‐loop of the two‐pass transmembrane receptor CD36, which is also a well‐established oxLDL binding domain. FA binding to CD36 has been poorly characterized; furthermore, lower affinity binding sites might be present in the large, glycosylated domain. Here we report novel studies of FA binding to CD36 by the biophysical method Surface Plasmon Resonance (SPR). After covalently linking CD36 to an SPR chip surface, various aqueous FA's were pulsed across the surface for a time period of 60s. For comparison, human serum albumin (HSA) was studied in parallel experiments. This method detected FA binding to CD36 at a lower capacity than to HSA, but with similar rapid association and dissociation.
Spatial and temporal regulation of the Nedd4 family ubiquitin ligases through phospholipid binding
Authors: Scott Jordan , Stahelin Robert V.
Published: June 2021

The Nedd4 family of HECT ubiquitin ligases are essential regulators of cellular polarity, ion channel activity, motility, and inflammatory signaling. HECT family ubiquitin ligases employ a catalytic cysteine residue to target cellular substrates for mono or poly ubiquitination. The Nedd4 family has nine members each with an N‐terminal C2 domain. Little is known about how their cellular localization and catalytic activity are regulated by lipid binding. These domains bind phospholipids and are required for the localization of some family members to the plasma membrane. Using surface plasmon resonance we have investigated the specific lipid binding properties of the Nedd4 family C2 domains. We find that several of these domains bind to phosphoinositides with nanomolar affinity. We have used vesicular sedimentation assays and lipid blots to confirm these binding results. It is likely that phosphoinositide binding enables these proteins to be regulated spatially and temporally in cells. We are currently employing confocal microscopy and fluorescent C2 constructs to determine how cellular lipid binding regulates the ability of these ubiquitin ligases to target cellular substrates under normal and phospholipid‐modulated conditions. This research is supported by the American Heart Association (12PRE11960030) and the NIH (T32GM075762).
The iron transport inhibitor ferristatin II induces degradation of transferrin receptor‐1
Authors: Byrne Shaina L , Buckett Peter D , Sanford Jack , Chen Juxing , Enns Caroline , Wessling‐Resnick Marianne
Published: June 2021

Ferristatin II, a polyaromatic compound previously proposed to be an iron uptake inhibitor, induces degradation of transferrin (Tf) receptor‐1 (TfR1) in HeLa cells within 4 h of treatment. Dose response experiments established an IC50 of 12 μM for inhibition of 55Fe‐Tf uptake, consistent with receptor degradation. Ferristatin II‐induced TfR1 degradation was blocked by nystatin, a cholesterol‐depleting agent that disrupts lipid raft membrane microdomains. siRNA knockdown of flotillin, a protein component of lipid rafts, also blocked TfR1 degradation by ferristatin II. Constitutive trafficking of TfR1 through clathrin‐mediated endocytosis occurs with or without ligand. In contrast, Tf antagonized TfR1 degradation by ferristatin II, an effect that was abrogated for the Tf binding mutant G647A TfR1. Combined, these data indicate that ferristatin II‐induced trafficking and degradation of TfR1 occurs through a lipid raft pathway gated in a ligand dependent manner.

This work is supported by NIH grant DK064750 to M.W‐R., DK054488 to C.E., S.L.B is supported by NHGRI/NIEHS Training Grant T32ES016645.
The Disruption of the Mitotic Cell Cycle Exit by Inositolless Death in S. cerevisiae
Authors: Hanson Barbara Ann , O'Connor Paul , Frandina John , Pittari Joseph , Munezero Jean , Croglio Vincent
Published: June 2021

A rapid decline in cell viability and in Clb2 cyclin are the early changes that occur when an inositol‐requiring mutant of S. cerevisiae (MC6A) undergoes inositolless death. Another change is the appearance of polymerized tubulin in nonbudding cells. One explanation for the presence of polymerized tubulin is that the cells enter the cell cycle but do not progress through anaphase to the depolymerization of the spindle microtubules. This may be due to the decline in Clb2p and other key regulatory cell cycle proteins. In this study, the levels of mRNA for the genes: Securin (Pds1), Separin (Esp1) and a major subunit (Apc1) of the Anaphase‐promoting complex, were measured by RT‐PCR using RNA extracted from cells grown with and without inositol for 0, 3, 6, and 24 h after the start of inositolless death. The proteins also were measured by western blots of protein extracts from these cells. The mRNA levels for Pds1 and Apc1 declined to 55% and 60% of control levels at 3 h and 38% and 45% at 24h. The mRNA levels for Esp1 declined the most rapidly to to 40% of control levels at 3h and to 15% at 24h. It may be the rapid reduction in Esp1 mRNAs and the resulting low levels of the Esp1 protease prevent the sister chromatid separation and the progression through anaphase to spindle microtubule depolymerization. (Funded by Canisius College)
Artemisia princeps var. orientalis induces cell cycle arrest accompanied by upregulation of p21CIP1 and caspase‐dependent apoptosis in human breast carcinoma cells
Authors: Choi Eun‐Jeong , Kim Gun‐Hee
Published: June 2021

The aim of the present study was to investigate the anticancer activity and mechanism of action of methanol extract from Artemisia princeps var. orientalis (APME). First, the antiproliferative activity of APME against T47D cells was at various concentrations (5–200 μg/mL) and treatment times (24–72 h) determined by MTT assay. APME significantly inhibited cell proliferation in a dose‐dependent manner, regardless of treatment times. Under the same condition, cytotoxicity (LDH release) of APME was only appeared at 72 h. In addition, treatment with the IC50 concentration of APME for 24 h caused cell cycle arrest and apoptosis. APME arrested cells in both G1 and G2/M phases of the cell cycle, a response that was related to the induction of CDK1, cyclin B, p21Cip1 expression, and repression of CDK2 and cyclin A. The induction of p21Cip1 by APME caused activation of p53 and modulation of its downstream targets Bcl‐2 and Bax. Subsequently, caspase‐8 and ‐9 were cleaved in cells treated with APME. Activation of these caspases in turn led to increased activation of caspase‐3. The results of this study suggest that APME produce anticancer effects in T47D cells through cell cycle arrest associated with the induction of p21Cip1 and apoptosis mediated by p53 and caspase‐depedent pathways.

This research was supported by Basic Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012–0006811)
mir‐27b regulates expression of fibrotic genes by targeting Gremlin 1
Authors: Graham Julie R , Williams Cara , Yang Zhiyong
Published: June 2021

Fibrosis is a chronic disease that causes excessive deposition of scar tissue in the afflicted organs. TGF‐β plays a central role in this process by stimulating the production of extracellular matrix proteins such as collagen. MicroRNAs (miRNAs) have been implicated in the control of both fibrosis and TGF‐β signaling. The goal of the present study is to identify and characterize new miRNAs that regulate TGF‐β‐induced fibrosis. TGF‐β‐mediated fibrotic responses in A549 lung epithelial cells were used for functional profiling of miRNAs by screening a library of miRNA inhibitors for its effects on TGF‐β‐induced type I collagen expression. The inhibition of one miRNA in particular, miR‐27b, caused a significant increase in collagen expression. Additional experiments showed that increasing miR‐27b levels resulted in a significant decrease in collagen expression. Computational analysis revealed that a predicted target of this miRNA is Gremlin 1, a BMP antagonist known to play an important role in fibrosis. A549 cells treated with TGF‐β exhibited an increase in Gremlin 1 expression and a corresponding decrease in miR‐27b expression, suggesting that TGF‐β regulates Gremlin 1 via miR‐27b. Modulating miR‐27b levels affected Gremlin 1 expression. Subsequent experiments using a luciferase reporter construct that contained either the intact or the mutated 3′UTR of Gremlin 1 confirmed that Gremlin 1 is a direct target of miR‐27b. We have also shown that reducing Gremlin 1 levels with either an siRNA or miR‐27b results in a similar reduction of TGF‐β‐induced fibrotic genes such as type I collagen, CTGF and PAI‐1. In summary, we have demonstrated that miR‐27b targets Gremlin 1, and that this regulation is likely to play a critical role in TGF‐β‐mediated fibrosis.
The conformation of active receptor‐bound arrestin
Authors: Gurevich Vsevolod , Kim Miyeon , Vishnivetskiy Sergey A , Van Eps Ned , Alexander Nathan S , Cleghorn Whitney M , Zhan Xuanzhi , Hanson Susan M , Morizumi Takefumi , Ernst Oliver P , Meiler Jens , Hubbell Wayne L
Published: June 2021

Visual arrestin‐1 binds light‐activated phosphorylated rhodopsin (P‐Rh*) to terminate G‐protein signaling. To map conformational changes upon binding to the receptor, pairs of spin labels were introduced in arrestin‐1 and double electron–electron resonance was used to monitor inter‐spin distance changes upon P‐Rh* binding. We found that the relative position of the N‐and C‐domains remains largely unchanged, contrary to expectations of a “clam‐shell” model. A loop implicated in P‐Rh* binding that connects b‐strands V and VI (the “finger” loop,” residues 67–79) moves toward the P‐Rh* in the complex, but does not assume a fully extended conformation. A striking and unexpected movement of a loop containing residue 139 away from the adjacent finger loop was observed, which appears to facilitate P‐Rh* binding. We detected smaller movements of distal loops containing residues 157 and 344 at the tips of the N‐and C‐domains, which correspond to “plastic” regions of arrestin‐1 that have distinct conformations in monomers of the crystal tetramer. Remarkably, the loops containing residues 139, 157, and 344 have high flexibility in both free and P‐Rh*‐bound arrestin‐1.

Support: NIH Grants EY011500, GM077561, GM081756 (VVG), GM080403, MH090192 (JM), EY005216, EY00331, and Jules Stein Professorship Endowment (WLH), Anne and Max Tanenbaum Chair in Neuroscience and Canada Excellence Research Chair program (OPE).
Identification and Expression Analysis of Two Homologs from Xenopus laevis of the Tumorhead Putative Binding Protein, FBXO30
Authors: Ayala‐Figueroa Jesus Manuel , Flores Noelia , Nuñez Dariana , Zbinden Theodor , Traverso Edwin
Published: June 2021

Tumorhead (TH) is a maternal factor that regulates cell proliferation during early embryogenesis in Xenopus laevis. To understand how TH functions at the molecular level, we have been studying its relationship with the novel F‐Box containing protein FBXO30, found in a two‐hybrid screen for TH binding proteins. Using primers based on the sequence we obtained, along with primers based on the 5′ and 3′ UTRs of the Xenopus tropicalis FBXO3O mRNA, we obtained RT‐PCR products with total RNA samples from eggs and embryos at early developmental stages. Using this approach, we have uncovered the presence of two FBXO30 homolog genes in X. laevis, FBXO30‐A and FBXO30‐B. The predicted FBXO30‐A full length protein sequence is 91% and 63% identical to its counterparts from Xenopus tropicalis and Homo sapiens, respectively. These proteins contain very conserved Traf‐like zinc finger‐containing domains at their N‐terminus, and F‐Box domains at their C‐terminus, while the internal part of the proteins diverge extensively. By RT‐PCR, we have found that FBXO30‐A and FBXO30‐B are maternal factors as their messages are present in the unfertilized egg. The FBXO30‐A mRNA persists during the cleavage stages, but decreases after the mid‐blastula transition and is barely detected once gastrulation starts. Our studies show the presence of two homologs of FBXO30 in X. laevis that are maternally expressed, which could be key regulators of early development working with TH to promote cell proliferation.
Retardation of cell proliferation by low‐temperature atmospheric plasma exposure on C2C12 myoblast
Authors: Nakai Naoya , Kawano Fuminori , Ohira Yoshinobu
Published: June 2021

Physical plasma is a partially ionized gas, containing radicals, electrons, positive and negative ions, and various excited atoms. In the present study, we investigate the effect of low‐temperature atmospheric plasma exposure on proliferation of C2C12 myoblast. The low‐temperature atmospheric pressure gas‐plasma was generated through an electrical discharge in an argon gas. Plasma exposure increased the phosphorylation of extracellular‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) in an electrical power‐and exposure time‐dependent manner. One min of plasma exposure increased the phosphorylation of ERK and JNK at 30 min after the exposure. However, phosphorylation of ERK and JNK was markedly decreased less than control levels at 4 h after the plasma exposure, and returned to the control levels at 24 h. One min of plasma exposure every 24 h inhibited the cell proliferation without cell death. Cell numbers in plasma exposure groups were 42.2% and 32.9% when compared to the control groups at 24 h and 48 h after the plasma exposure, respectively. In conclusion, low‐temperature atmospheric plasma exposure temporarily increased the phosphorylation of ERK and JNK and retarded proliferation of C2C12 myoblast. This work is supported by Grant‐in‐Aid for Challenging Exploratory Research (23650405 to N.N.) from the Japan Society for the Promotion of Science (JSPS).
AKAP79 Interacts with the Axon Guidance Receptor Robo2
Authors: Samelson Bret , Colledge Marcie , Scott John
Published: June 2021

A‐kinase anchoring proteins (AKAPs) are scaffolds that bring together complexes containing protein kinase A (PKA) and other signaling molecules. They localize these complexes to specific regions of the cell to regulate numerous signaling pathways. AKAP79 is a neuronal scaffold that coordinates PKA, protein kinase C (PKC), and protein phosphatase 2B (PP2B) signaling at the plasma membrane. The anchoring of these enzymes via AKAP79 is important for the function of numerous receptors.

We have identified an interaction between AKAP79 and the axon guidance receptor Robo2 in a mass spectrometry screen. The Roundabout (Robo) family of receptors is involved in both the repulsion of axonal growth cones during pathfinding as well as the stimulation of dendritic branching. This interaction was confirmed by performing GST‐Robo2 pulldowns from mouse brain lysate as well as immunoprecipitations from HEK293 cells overexpressing Robo2 and AKAP79. Further studies demonstrated that the Robo2‐AKAP79 interaction is disrupted by calcium. In addition, we performed in vitro kinase assays to determine whether Robo2 was a substrate for any AKAP79 anchored kinases. We discovered that Robo2 can be phosphorylated by both PKA and PKC. Based on these findings, we hypothesize that the AKAP79 signaling complex may regulate the essential developmental functions of Robo2.

This work was supported in part by NIH grant GM48231.
Hepatocyte Nuclear Factor‐4 Phosphorylation Mediated by Inflammatory Response
Authors: Wang Zhongyan , Salih Erdjan , Burke Peter A.
Published: June 2021

Hepatocyte nuclear factor (HNF)‐4, a liver‐enriched transcription factor, regulates a large number of liver‐specific genes and plays an important role in liver development and maintenance of liver phenotype. While HNF‐4 function is regulated by phosphorylation, only a limited number of phosphorylation sites have been identified, and its roles and dynamic changes in response to inflammatory stimulation are not explored. Utilizing the cytokine treated HNF‐4 enriched HepG2 cells, we have quantitatively analyzed HNF‐4 phosphorylation by using the combined stable isotopic variants of dithiothreitol with mass spectrometry. Twelve serine/threonine phosphorylation sites in the diverse functional domains of HNF‐4 were identified and quantified after cytokine exposure. Cytokine treatment (IL‐6, IL‐1β and TNF‐α) causes a dynamic change in HNF‐4 phosphorylation in a peptide‐and time‐dependent manner. Protein kinase A (PKA) significantly reduces HNF‐4 binding activity, which mimics the repressive effect of cytokines, and a PKA inhibitor partially restores the reduced HNF‐4 binding activity induced by cytokines. These results suggest that cytokines mediate the phosphorylation/dephosphorylation levels of HNF‐4, and a PKA signaling pathway involves in the modification of HNF‐4 function during the inflammatory response.
Discovery of a bis‐phosphokinetic motif in a regulatory region of the transcription factor, BCL11B
Authors: Filtz Theresa Marie , Vogel Walter K. , Gafken Philip R. , Leid Mark
Published: June 2021

BCL11B is a haploinsufficient tumor suppressor and transcription factor that is absolutely required for thymocyte development. Stimulation of thymocytes through the MAP kinase pathways alters the phosphorylation and sumoylation of BCL11B on 19 of 23 previously identified phosphorylated residues and 2 sumoylated residues. Composite modifications of BCL11B post‐stimulation include hyperphosphorylation at 5 min, dephosphorylation at 30 min and subsequent increased sumoylation at 60 min. We sought to define the kinetics of individual sites of BCL11B phosphorylation and sumoylation to better understand the role of these modifications in integrating extracellular signals to nuclear function in developing thymocytes. Using quantitative mass spectrometry to analyze BCL11B from natively‐expressing thymocytes, we identified four distinct phosphokinetic patterns among the 19 sites and the sumoylation kinetics of one site. Further, we have identified two peptides of bis‐phosphorylated residues with the motif TPPXXSP in which stimulation‐dependent dephosphorylation of threonine is accompanied by a bimodal regulation of serine. The bis‐phosphorylated peptides are within a previously identified protein‐protein interaction and putative sumoylation‐regulatory domain of BCL11B. (This work was supported by the NIH, grant R15 GM096243, and the OSU College of Pharmacy.)
Phosphorylation‐dependent interaction of PRIP with Akt
Authors: Sugiyama Goro , Nagano Koki , Otani Takahito , Gao Jing , Takeuchi Hiroshi , Hirata Masato
Published: June 2021

Phospholipase C (PLC)‐related but catalytically inactive protein (PRIP) is a protein with a domain organization similar to PLC‐δ1. We have reported that PRIP interacts with the catalytic subunits of protein phosphatases, PP1c and PP2Ac, depending on the phosphorylation level of PRIP. We also found that Akt was precipitated along with PRIP by anti‐PRIP antibody from neuronal cells. In this study, we investigated the interaction between PRIP and Akt.

Co‐immunoprecipitation assay was performed using COS7 cells expressing glutathione S‐transferase (GST)‐fused PRIP. Both endogenous Akt and PP2Ac were co‐precipitated along with PRIP by GST antibody, and the precipitation was up‐regulated when the cells were stimulated with insulin. GST‐pull down experiments using recombinant GST‐Akt and his‐tagged (His‐) PRIP or the deletion mutant of PRIP (74–298) showed the direct binding of Akt to 74–298 residues in RPIP. Pretreatment of PRIP with Akt in the presence of ATP increased this interaction. Akt phosphorylated PRIP, but not the mutant PRIP whose Ser‐96 was replaced with Ala (S96A). Pre‐treatment of S96A mutant with Akt did not increase the binding to Akt, indicating that phosphorylation of PRIP on Ser‐96 increases the binding of Akt to PRIP. These results indicate that PRIP modulates Akt signaling by the regulation of complex formation between Akt and protein phosphatases.
Two unique phosphorylation‐driven signaling pathways crosstalk in Staphylococcus aureus: STK1 meets GraR
Authors: Fridman Michael , Golemi‐Kotra Dasantila
Published: June 2021

The Stk1/Stp1 and GraSR signal transduction pathways are two distinct pathways in Staphylococcus aureus that rely on reversible phosphorylation processes in transducing external stimuli intracellularly. Our study shows for the first time that these two systems interact in S. aureus. GraR undergoes phosphorylation by Stk1 at three threonine residues in the DNA‐binding domain. Phosphorylation by Stk1 depends on the structural integrity of GraR and amino acid sequences flanking the phosphorylation sites. Its homolog in Bacillus subtilis, BceR, which shares 56% sequence identity with GraR, does not undergo phosphorylation. In addition, an unrelated response regulator of S. aureus, VraR, does not undergo phosphorylation by Stk1. These findings provide strong evidence that GraR is a target of Stk1. Stk1‐dependent phosphorylation of GraR increases its DNA‐binding affinity to one of the GraR‐target promoters. The crosstalk between these two otherwise independent pathways may facilitate S. aureus adaptation to diverse environmental cues and mediate its virulence.

This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Ministry of Economic Development and Innovation (Ontario, Canada).
Structures of the G‐beta–CCT and PhLP1–G‐beta–CCT complexes reveal a molecular mechanism for G protein beta subunit folding and beta‐gamma dimer assembly
Authors: Plimpton Rebecca , Cuellar Jorge , Lai Chun Wan Jeffrey , Taylor Ryan , Carrascosa Jose L , Prince John T , Valpuesta Jose , Willardson Barry M
Published: June 2021

G protein signaling depends on the ability of the individual subunits of the G protein heterotrimer to assemble into a functional complex. Formation of the G protein beta‐gamma dimer is particularly challenging because it is an obligate dimer of individual subunits which are unstable on their own. Recent studies have revealed an intricate chaperone system that brings the G‐beta and G‐gamma subunits together. This system includes the cytosolic chaperonin containing TCP‐1 (CCT) and a co‐chaperone phosducin‐like protein 1 (PhLP1). The mechanism of CCT‐substrate interactions is currently a key focus in the field. Two key intermediates in the G‐beta‐gamma assembly process, the G‐beta– CCT and the PhLP1–G‐beta–CCT complexes, have been isolated and their structures determined by cryo‐electron microscopy. We employed chemical crosslinking to identify the binding interactions that occur in formation of these two complexes. Our analysis reveals G‐beta interacts with CCT in a quasi‐native state mostly through hydrophobic residues of CCT‐beta and PhLP1 binding promotes final folding and repositioning of G‐beta for release from CCT and assembly with G‐gamma. This research was funded by the NIH.
Control of plasma membrane lipid asymetry at the bud neck: septin‐bound protein kinase Gin4 locally controls flippase function
Authors: Roelants Françoise M , Su Brooke , von Wulffen Joachim , Thorner Jeremy
Published: June 2021

In the yeast plasma membrane, phospholipids are asymmetrically distributed between the inner and outer leaflets. The outer leaflet contains predominantly phosphatidylcholine (PtdCho) and sphingolipids, whereas the inner leaflet is enriched in phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEth), phosphatidylinositol (PtdIns), and a phosphoinositide (PtdIns4,5P2). Five genes encode type 4 P‐ATPase implicated in translocating PtdSer, PtdEth and PtdCho. In particular, Dnf1 and Dnf2 catalyze inward movement of PtdEth and PtdSer from the outer to the inner leaflet. The action of these flippases depends on activating phosphorylation by Fpk1, a protein kinase located at the cell cortex. Using an assay that monitors Fpk1 activity in vivo, we found that Fpk1 is hyper‐active in cells lacking the protein kinase Gin4 that had previously been implicated in assembly of the septin ring at the bud neck. We found, instead, that Gin4 phosphorylates Fpk1 at multiple sites, which we mapped. After mutating the phospho‐acceptor residue to Ala in these sites, we found by several biochemical and phenotypic criteria that such an Fpk111A mutant is hyper‐active. Thus, Gin4 acts as a negative regulator of Fpk1 and, thus, negatively regulates indirectly flippase function. These observations suggest that control of plasma membrane asymmetry may be the primary function of Gin4 and may be necessary for optimal cytokinesis.
Characterization of BI‐D1870, the ATP‐competitive small molecule inhibitor of p90 Ribosomal S6 Kinase (RSK)
Authors: Jones Lauren S
Published: June 2021

Acute myeloid leukemia (AML) is a cancer caused by the accumulation of immature, abnormal myeloid cells in the bone marrow due to deregulated differentiation, rapid cell growth and the inhibition of apoptosis. Flt3‐ITD (FMS‐Like Tyrosine Kinase 3‐internal tandem duplication) mutants are commonly found in acute leukemia and they constitutively activate pathways such as MAPK and STAT5. RSK (P90 Ribosomal S6 Kinase) is a ser/thr kinase activated by MAPK that has been shown to regulate diverse cellular processes such as cell proliferation and survival through the phosphorylation of pro‐apoptotic proteins such as BAD. RSK also plays an active role in nuclear signaling by phosphorylating the cyclic AMP response element binding protein (CREB) and IκB. We examined the effects of the ATP‐competitive, small molecule RSK inhibitor, BI‐D1870 on MV‐411 AML cells. BID1870 inhibits the N‐terminal AGC kinase domain of RSK. After treating MV‐411 cells with 10μM BI‐D1870 for 24 hours, pRSK(T359/S363) phosphorylation was completely inhibited the mRNA expression of the transcription factor, c‐Myc was decreased dramatically. In combination with the novel IKKβ kinase inhibitor, BAY 65–1942, that blocks phosphorylation of IκBα in BCR‐ABL‐expressing myeloid cells, MCL‐1, an anti‐apoptotic member of the BCl‐2 family of proteins, was completely depleted, whereas neither compound alone affected MCL‐1. The depletion of MCL‐1 may have a role in the apoptosis of cells as the BI‐D1870/BAY 65–1942 combination induced cell death greater than either inhibitor alone. Thus BI‐D1870 in combination with other kinase inhibitors may have potential for novel anti‐AML therapies.
Investigating inactive conformations of protein kinases
Authors: Hari Sanjay B , Perera B Gayani K , Leonard Stephen E , Maly Dustin J
Published: June 2021

Protein kinases are found in countless signal transduction pathways and play key roles in the regulation of intracellular signaling events. Kinases exist in equilibria between catalytically active and inactive states. While evolutionary pressure limits kinases to one active state conformation, several inactive conformations have been described. The MAP kinase p38alpha has been shown to adopt a specific inactive conformation that appears to be inaccessible to other MAP kinases despite extensive structural homology. Seeking to understand the reason for this disparity, we found two residue positions that sensitize MAP kinases to small molecule inhibitors that are selective for specific inactive conformations. These ligands inhibit the activities of engineered mutants of p38delta and Erk2 more potently than their wild‐type counterparts by several orders of magnitude. We have determined a crystal structure of inhibitor‐sensitive Erk2 bound to one of these ligands in the active site and found that the inhibitor disrupts a key salt bridge necessary for catalysis, rendering the kinase inactive. The ability to manipulate kinases in a conformationally‐dependent manner could allow the targeted study of kinases in scaffolding complexes and other noncatalytic roles.

Supported by NIH (R01GM086858) and American Heart Association (11PRE7260025)
Epitope guided engineering of monobody binders for in vivo inhibition of Erk‐2 signaling
Authors: Park Sheldon , Mann Jasdeep K , Wood Jordan F , Stephan Anne Fleur , Tzanakakis Emmanuel S , Ferkey Denise M
Published: June 2021

Although the affinity optimization of protein binders is straightforward, engineering epitope specificity is more challenging. Targeting a specific surface patch is important because the biological relevance of protein binders depends on how they interact with the target. Epitope specific binders are particularly useful to test hypotheses motivated by biochemical and structural studies. We used yeast display to engineer monobodies that bind a defined surface patch on the mitogen activated protein kinase (MAPK), Erk‐2. The targeted area (“CD” domain) is known to control the specificity and catalytic efficiency of phosphorylation by the kinase by binding a linear peptide (“D” peptide) on substrates and regulators. A binder that disrupts the docking interaction should thus be useful for regulating Erk‐2 signaling in vivo. Although the CD domain constitutes only a small percentage of the surface area of the enzyme (~ 5%), sorting a yeast displayed monobody library with wild type (wt) Erk‐2 and a rationally designed mutant led to isolation of high affinity clones with desired epitope specificity. The engineered binders inhibited the activity of Erk‐2 in vitro and in mammalian cells. Furthermore, they specifically inhibited the activity of Erk‐2 orthologs in yeast and suppressed a mutant phenotype in round worms caused by overactive MAPK signaling. The study therefore shows that positive and negative screening can be used to bias the evolution of epitope specificity and predictably design inhibitors of biologically relevant protein‐protein interaction.
The effect of TBBPA on MAP3Ks in human natural killer cells
Authors: Celada Lindsay J. , Whalen Margaret M.
Published: June 2021

Natural killer (NK) cells are part of the innate immune system. Upon encountering a tumor cell, virally‐infected cell, or antibody‐coated cell, the NK cell releases cytotoxic granules which lead to the lysis of the target cell. Tetrabromobisphenol A (TBBPA) is an organic brominated flame retardant (BFR) known to have harmful effects on the environment. It is utilized as a heat resistant chemical in various industrial and commercial applications in order to retard the burning of textiles, buildings, plastics, among other things. Significant levels of TBBPA have been found in human serum, human milk and adipose tissue. Human exposure can result from the inhalation of polluted air and/or the ingestion of contaminated food. A previous study demonstrated that a twenty‐four hour exposure of NK cells to 5μM of TBBPA resulted in a >;95% decrease in cytotoxic function and exposure to 2.5 μM of TBBPA resulted in a 76% decrease in lytic function. TBBPA also caused a decrease in NK cell binding capacity by 70% at 5 μM. We have shown that exposureto this chemical leads to the activation of specific Mitogen‐Activated‐Protein‐Kinases(MAPKs) and MAPK‐Kinases (MAP2Ks) in human NK cells. The objective of this study is to examine whether there also exists a TBBPA‐induced activation of MAP3Ks, the upstream regulator MAP2Ks. Our results show significant activating phosphorylations of MAP3Ks after exposure of human NK cells to TBBPA.
Structural mechanism of trimeric PR70 PP2A holoenzyme: insights into Cdc6 dephosphorylation
Authors: Wlodarchak Nathan Joseph , Guo Feng , Satyshur Kenneth A , Jiang Li , Jeffrey Philip D , Sun Tingwan , Stanevich Vitali , Mumby Marc C , Xing Younga
Published: June 2021

Protein phosphatase 2A (PP2A) regulates many essential aspects of cellular function via diverse families of heterotrimeric holoenzymes. A regulatory subunit from the B” family plays a role in regulating the cellular level of Cdc6, a cell cycle regulator crucial for precise control of DNA replication licensing. No previous structural information for the B” family has been reported, and elucidating the interactions necessary for holoenzyme formation is critical in understanding how PP2A interacts with specific substrates. Here we report the high‐resolution three‐dimensional structure of the PR70 holoenzyme, and we show the B” subunit is an elongated EF‐hand calcium‐binding protein that stabilizes a remarkably compact holoenzyme conformation. The second EF‐hand of PR70 directly contacts the PP2A scaffold subunit and is buttressed by a protruding hydrophobic motif. A PR70 helix near the Cdc6‐binding region directly contacts the catalytic subunit, and this contact is required for the full activity of PR70 holoenzyme in dephosphorylation of the N‐terminal destruction motif of Cdc6. In contrast to PR70 holoenzyme, the holoenzyme involving the B’/B56/PR61 subunit is nearly inactive toward Cdc6. Our studies revealed important structural bases for the B” family of PP2A holoenzymes and mechanisms for precise control of Cdc6 dephosphorylation.

This work was supported by University of Wisconsin‐Madison (Y. Xing), R01 GM096060–01 (Y. Xing), and GM86560 (M.C. Mumby), and T32 CA009135 (N. Wlodarchak).
Structural basis of PP2A phosphatase activator reveals a unique chaperone function in PP2A activation
Authors: Guo Feng , Stanevich Vitali , Wlodarchak Nathan , Li Yikun , Pallas David C. , Sengupta Rituparna , Jiang Li , Satyshur Kenneth A. , Xing Yongna
Published: June 2021

Protein Phosphatase 2A (PP2A) is one of the most important phosphatases essential for many cellular processes. PP2A Phosphatase Activator (PTPA) plays an important role in PP2A activation. The underlying mechanism remained unclear. To determine the structural basis of PP2A activation, we determined the crystal structure of the PP2A‐PTPA‐ATPγS complex at 2.8 Å and PP2A‐PTPA‐ADP:AlF4− complex at 1.9 Å. The structures reveal that PTPA possesses an unexpected chaperone function that stabilizes an active conformation of the PP2A C subunit (PP2Ac) active site required for stable loading of catalytic metal ions. PTPA and PP2Ac form an unconventional ATPase machinery. Assisted by PTPA, ATP β/γ phosphates bind directly to the PP2A active site pocket, forming a typical double‐octahedral chelation of catalytic metal ions together with metal chelating residues. This atomic architecture underlies a composite ATPase activity of the PP2A‐PTPA complex that utilizes PP2A's phosphatase active site upon activation. In summary, our studies uncover that PTPA is a robust activation chaperone with a unique mechanism in PP2A activation, and provide novel insights into chaperone‐mediated activation of signaling proteins. This work was supported by University of Wisconsin‐Madison (Y. Xing), ACS research scholar grant (Y. Xing), and R01 GM096060–01 (Y. Xing).
Tertiary Conformational Transition In Horse Haemoglobin Induced By Inositol Hexakisphosphate
Authors: Omotosho Omolola E , Okonjo Kehinde O. , Omotosho Victor T. , Rotimi Solomon O. , Chinedu Shalom N.
Published: June 2021

The red blood cell of the domestic horse contains two haemoglobin types. The two haemoglobins were separated on a column of carboxymethylcellulose. The equilibrium constant, Kequ, for the reaction of 5,5′‐dithiobis(2‐nitrobenzoate) — DTNB — with the CysF9[93]β sulfhydryl group of each haemoglobin was determined at 25°C as a function of pH. The reactivity of CysF9[93]β is affected by allosteric effectors such as the proton (H+) and inositol hexakisphosphate (inositol‐P6). Between pH 5.6 and 9.0 Kequ decreased by about two to four orders of magnitude, demonstrating that H+ is a heterotropic allosteric effector of haemoglobin with respect to its reaction with DTNB. Inositol‐P6 also decreased Kequ by about two to four orders of magnitude across the experimental pH range. CysF9[93]β exists in two tertiary conformations, r and t, in dynamic equilibrium. Krt, the equilibrium constant for the r ↔ t conformational transition, was determined for each of the two horse haemoglobins from an analysis of the pH dependence of Kequ. The calculations from the pH dependence of Kequ showed that the pKa values of the ionisable groups coupled to the DTNB reaction vary between 5.0 and 8.9. The equilibrium constants, Krt, for the r↔t tertiary structure transition, were 0.143 ± 0.05 and 0.446 ± 0.22 for the fast and slow stripped horse haemoglobins respectively. In the presence of inositol‐P6, Krt for the fast and slow were 2.219 ± 0.79 and 2.214 ± 0.83 respectively. The results show that inositol‐P6 increases the relative population of the t tertiary conformation. So, it increases the affinity of CysF9[93]β by changing the relative distribution of two protein conformations.
Novel anti‐osteoclastogenic activity of fisetin antagonizing RANKL‐induced bone resorption in murine macrophages
Authors: Kim Yunho , Kim Jung‐Lye , Kang Young‐Hee
Published: June 2021

Bone resorption and formation is imbalance resulting in more bone resorption than bone formation leading to osteoporosis. Osteoclasts were multi‐nucleated cells formed by exposure to RANKL and activating bone resorption. Fisetin is a flavonol found naturally in many fruits and vegetables including strawberries, blueberries and the skin of cucumbers. This study investigated that fisetin RANKL‐induced osteoclast differentiation and fusion in RAW 264.7 murine macrophages incubated with ≤10 μM fisetin for 5d. Fisetin inhibited the RANKL‐induced multi‐nucleated osteoclast formation by suppressing tartrate‐resistance acid phosphatase activity. Additionally, Fisetin treatment reduced cellular expression carbonic anhydrase II and integrin β3 elevated by RANKL. When fisetin was treated to RANKL‐exposed macrophages, the elevated actin‐ring formation was attenuated, indicating fisetin inhibition of osteoclast fusion and resorption. It was also found the fisetin diminished the RANKL induction of gelsolin, an actin‐binding protein that is a key regulator of actin filament assembly and disassermbly. Therefore, fisetin was a natural therapeutic agent retarding osteoclast differentiation and bone resorption
Dietary intake of purple sweet potato powder affected bone mineral content of ovariectomized rats
Authors: Wang Changzheng , Huang Lingyu , Zheng Xueli , Butler Cecil
Published: June 2021

Female SD rats (9 month old) were either ovariectomized or sham‐operated before they were randomly assigned into 6 treatment groups. Rats in group A were sham‐operated but those in other groups were all ovariectomized. Groups A and B were fed the AIN93M diet, but groups C, D and E were fed the AIN93M diet with 5%, 10% or 15% of the corn starch replaced by equal amounts of purple sweet potato powder, respectively. Stokes purple sweet potatoes were steam cooked at 200 F for 1 hr before the skin was removed, mashed, freeze‐dried and then ground into fine powder. Group A was fed ad lib, but the amounts of diets given to other groups were restricted to the average intake of Group A. The rats were fed their assigned diets for 6 weeks. The bone mineral content for group A was higher than for other groups but the group fed 10% purple sweet potato powder had bone mineral content similar to that of group A. Rats fed 15% purple sweet potato powder diet had diarrhea. These results indicate that purple sweet potato powder helped to prevent bone loss caused by ovariectomy. However, excessive consumption of purple sweet potato powder (15% of the diet) caused digestive disorders and was not protective against bone loss.