bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phosphomimetic Constructs of Akt/PKB do not Mimic Activated Akt in Cells
Authors: Kunkel Maya T. , Newton Alexandra C. 
Published: November 2023

Akt/protein kinase B is a serine/threonine kinase of the AGC family of kinases which plays a key role in signaling downstream of phosphatidylinositol 3‐kinase. Akt is activated following localization to PIP3 via its PH domain, and once recruited to the plasma membrane, Akt is phosphorylated at Thr308 within its activation loop by its upstream kinase PDK1 followed by phosphorylation at the C‐terminal hydrophobic site, Ser473. Here we show that phosphomimetic substitutions at these positions do not mimic the global signaling output of phosphorylated Akt. Specifically, we use live cell imaging with a FRET‐based Akt activity reporter, B Kinase Activity Reporter (BKAR), to show that constructs of Akt with Asp at position 308, or Asp at both Thr308 and Ser473, are neither basally active nor activatable following stimulation of COS cells with agonists such as EGF. In contrast, wild‐type Akt is robustly activated following EGF stimulation. Consistent with the lack of activity of Akt‐T308D revealed by live cell imaging, Western blot analysis using a phospho‐specific antibody to Akt consensus substrate phosphorylation sites also reveals no basal or EGF‐stimulated phosphorylation by the phosphomimetic construct. Although some previous reports indicate that substitution with Asp at both sites yields an active kinase, our data reveal that Asp is a poor phosphomimetic at the key regulatory sites of Akt, thus one should exercise caution in the use of such constructs to examine the biology of Akt signaling.Support or Funding InformationThis work was supported by NIH P01 DK5444.
Cancer‐Associated Fusion Proteins Reveal Inhibitory Role of Protein Kinase C's Processing Phosphorylations
Authors: Baffi Timothy R. , Ngoc Van An‐Angela , Antal Corina E. , Newton Alexandra C. 
Published: November 2023

Since the discovery that the Philadelphia translocation event produces the oncogenic Bcr‐Abl fusion gene that sustains chronic myelogenous leukemia (CML), gain‐of‐function fusion proteins have emerged as novel cancer drivers and potential therapeutic targets. Recently, several fusion proteins involving translocations in the N‐terminal regulatory region of the tumor suppressor Protein Kinase C (PKC) were identified in cancer. Here, we show that the fusion proteins are not processed by phosphorylation, an event that accompanies the maturation of wild‐type PKC, yet are, surprisingly, constitutively and maximally active, independent of membrane‐targeting second messengers. This constitutive activity is not a result of the various proteins to which PKC is fused, but instead results from deletion of the autoinhibitory pseudosubstrate in the fusion protein: constructs of PKC deleted in just the pseudosubstrate are also not phosphorylated yet constitutively and maximally active in cells. Because unphosphorylated PKC is unstable and sensitive to degradation, it is not clear whether these fusion proteins exist in appreciable levels in the tumors in which their mRNA was identified. Importantly, however, characterization of these fusions reveals the unexpected finding that phosphorylation serves as a mechanism to maintain it in an autoinhibited state. Characterizing the functional consequences of fusion proteins in cancer is not only critical to understanding their contribution to oncogenic signaling, but also to elucidating the underlying biochemical regulatory mechanisms of relevant enzymes such as PKC.Support or Funding InformationThis work was supported by the National Institutes of Health Grant GM43154 and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).
Oscillatory Control of Interferon Regulatory Factor 3
Authors: Gregory Shana , Ng Chen Seng , Hoffmann Alexander 
Published: November 2023

Innate immune responses to pathogen exposure are coordinated by two transcription factors, Nuclear Factor‐kappaB (NF‐κB) and Interferon Regulatory Factor 3 (IRF3). Prior work has established that NF‐κB activity shows complex temporal dynamics, and several inflammatory stimuli trigger oscillations of its nuclear activity. Indeed, oscillatory control of NF‐κB has been a focus for the research community to understand the underlying negative feedback control by IκBα and its physiological function. To our surprise, recent studies in our laboratory have revealed that IRF3 is also oscillatory in response to lipopolysaccharide (LPS) stimulation. However, those preliminary studies employed an overexpressed GFP‐IRF3 fusion protein. In this study, we sought to determine whether the endogenous IRF3 also shows oscillatory dynamics or whether the prior observation may be an artifact of the exogenous fusion protein. To this end cytosolic and nuclear fractions were collected from macrophages and Western Blots were performed. After improving the reliability of the Western blot protocol our data confirm the single cell results in that IRF3 is rapidly translocated to the nucleus within 30 min following LPS stimulation, is then exported back to the cytoplasm by 90 min, and relocated to the nucleus by 2 hrs. The importance of this finding is that it suggests the hypothesis that different stimuli may trigger different IRF3 oscillatory dynamics that may result in different innate immune responses. Future studies will address that hypothesis.Support or Funding InformationThis investigation was supported in part by a MARC Undergraduate Student Training in Academic Research (U‐STAR) National Research Service Award (NRSA) Institutional Research Training Grant (2 T34 GM008663) from the National Institutes of Health, National Institute for General Medical Sciences.
Sorting Nexin 9 Differentiates Ligand‐Activated Smad3 from Smad2 for Nuclear Translocation and TGFβ Signaling
Authors: Kang Jeong‐Han , Wilkes Mark C. , Repellin Claire E. , Andrianifahanana Mahefatiana , Yin Xueqian , Leof Edward B. 
Published: November 2023

The primary mediator of transforming growth factor β (TGFβ) responses are the Smad proteins. Here, we identify sorting nexin 9 (SNX9) as being required for Smad3 (but not Smad2) nuclear translocation and cell stimulation. SNX9 principally interacts with phosphorylated (p) Smad3 and promotes more rapid nuclear delivery from that observed independent of ligand. Although SNX9 does not bind Imp7, Impb, Nup153 or Nup214, it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates recruitment of pSmad3/SNX9 to the nuclear pore, heterodimerization of Imp8 with Impb, and nuclear translocation of pSmad3, but not SNX9. The demonstration that Smad3 is regulated in a distinct manner from Smad2 provides the opportunity to develop intervention strategies to enhance or dampen specific aspects of the cellular response to TGFβ.Support or Funding InformationThis work was supported by Public Health Service grants GM‐055816 and GM‐054200 from National Institutes of General Medical Sciences.
Induction of Browning of White Adipocyte Formation by Ganoderma tsugae Extract
Authors: Tseng Hsiu‐Hsueh , Yeh Wan‐Chun , Tu Yun‐Chen , Yang Bi‐Fen , Lai Yen‐Ting , Kao Ming‐Ching , Liu Jah‐Yao 
Published: November 2023

Ganoderma, also known as Lingzhi or Reishi, is the top grade of Traditional Chinese Medicine (TCM) and has gained considerable attention in oriental countries for its potential functions of promoting the health benefit, regulating the vital energy, postponing the old and feeble, and extending life span that were mentioned in the TCM books. However, the metabolic health benefits by regulating the vital energy from Ganoderma remain to be experimentally confirmed. Adipose tissue is viewed as one major lipid metabolic and energy‐modulated organ, and is metabolically adaptive to external stimuli. Recently, browning of white adipocyte upon stimulation has been considered as a key aspect in energy metabolism due to its energy expenditure characteristics. The current study aims to investigate the direct role of Ganoderma extracts on adipocyte formation and lipid metabolism as well as regulation of energy homeostasis. To examine the relevance of these effects mediated by Ganoderma, in vitro and in vivo assays were performed after treatment with Ganoderma extracts. Here, we demonstrate that a quality assured extract of Ganoderma tsugae (GTE), a popular species of Lingzhi locally cultivated in Taiwan, had no obviously cytotoxic effect but contributed to adipogenesis accompanied with the changes of morphological features (lipid droplet and mitochondria) in 3T3‐L1 adipocytes. Moreover, the histological analysis of white fat‐pad tissues from mice during the development of obesity showed browning characteristics after treatment with GTE. Subsequently, western blotting further confirmed the browning of white adipocyte formation, including up‐regulation of adipogenic‐, mitochondrial‐ and energy‐related proteins induced by GTE. In addition, GTE treatment not only reduced lipogenesis and lipid droplet fusion but also promoted lipid mobilization. Taken together, our findings reveal for the first time that GTE promotes metabolic health benefit and regulates the vital energy, as mentioned in the traditional Chinese Materia Medica, by modulating intracellular lipid homeostasis and induction of browning of adipocyte formation. This study provides new perspectives to elucidate the role and physiological effects of Ganoderma via current biotechnological techniques.Support or Funding InformationThis study was supported by the Ministry of Science and Technology (MOST 104‐2320‐B‐039‐024), R.O.C.
Increased Endurance Exercise Capacity Secondary to Liver Fatty Acid‐Binding Protein Ablation
Authors: Xu Heli , Gajda Angela , Zhou Yin Xiu , Fatima Anam , Agellon Luis B. , Storch Judith 
Published: November 2023

Two fatty acid‐binding proteins (FABPs) are abundantly expressed in proximal enterocytes, intestinal FABP (IFABP, FABP2) and liver FABP (LFABP, FABP2), both with high affinity for binding long chain fatty acids. We previously showed that LFABP and IFABP knockout mice have divergent phenotypes in response to high‐fat feeding, with LFABP−/− mice gaining more weight and body fat, and IFABP−/− mice remaining lean relative to wildtype (WT) mice. Interestingly, despite their obese phenotype, LFABP−/− mice showed signs of being metabolically healthy, with a similar glucose tolerance to WT mice regardless of their markedly greater fat mass and an increased endurance exercise performance. To explore this surprising exercise phenotype, here we focused on possible metabolic alterations in the skeletal muscle secondary to LFABP ablation. The results showed higher mitochondrial enzyme activities in LFABP−/− mouse muscle, along with an increased triglyceride level and decreased AMPK activity, indicating that the mice have abundant energy availability at rest, compared with WT mice. Upregulation of PGC1α and PPARα mRNA levels was also found, suggesting an induction of mitochondrial biogenesis and an adjustment in lipid utilization. These results suggest that although LFABP is not normally expressed in muscle, its ablation in liver and intestine appears to contribute to a higher mitochondrial content and efficiency in energy utilization in the skeletal muscle, leading to the higher endurance exercise capacity in the LFABP−/− mice.Support or Funding InformationThis work was supported by National Institutes of Health Grant DK‐38389 from NIDDK, and by funds from the New Jersey Agricultural Experiment Station (to J. S.).
Sirtuin 3 is a Novel Regulator of Mitochondrial Ceramide Metabolism in Stroke
Authors: Gudz Tatyana I. , Riley Christopher , Keffler Jarryd , Yu Jin , Kindy Mark , Macklin Wendy , Lombard David , Novgorodov Sergei 
Published: November 2023

Experimental evidence supports the role of mitochondrial ceramide accumulation as a cause of mitochondrial dysfunction and brain injury after stroke. Herein, we report that SIRT3 regulates mitochondrial ceramide biosynthesis via deacetylation of ceramide synthase (CerS) 1, 2 and 6. Reciprocal immunoprecipitation experiments revealed that CerS1, CerS2 and CerS6, but not CerS4, are associated with SIRT3 in cerebral mitochondria. Furthermore, CerS1, 2 and 6 are hyper‐acetylated in the mitochondria of SIRT3‐null mice and SIRT3 directly deacetylates the ceramide synthases in a NAD+‐dependent manner that increases enzyme activity. Investigation of SIRT3 role in mitochondrial response to brain ischemia/reperfusion (IR) showed that SIRT3‐mediated deacetylation of ceramide synthases increased enzyme activity and ceramide accumulation after IR. Functional studies demonstrated that absence of SIRT3 rescued the IR‐induced blockade of the electron‐transport chain at the level of Complex III, decreased ROS generation and protein carbonyls in mitochondria. Importantly, Sirt3 gene ablation protected the brain from IR injury. These data support the hypothesis that IR activates SIRT3, resulting in the deacetylation of ceramide synthases and the elevation of ceramide which could inhibit Complex III, leading to increased ROS generation and brain injury. The results of these studies highlight a novel mechanism of SIRT3 involvement in modulating mitochondrial ceramide biosynthesis and suggest a critical role of SIRT3 in promoting mitochondrial dysfunction and brain injury after stroke.Support or Funding InformationNIH grant NS083544
Alterations in Acyl Phosphatidylglycerol Levels in ΔpagP, ΔpldB, and Δcls Escherichia coli leads to Phenotypic Variance
Authors: Pourmaleki Maryam , Garrett Teresa A 
Published: November 2023

Headgroup‐acylated glycerophospholipids (GPLs) are a low abundance class of lipids involved in cell membrane function during cell division in Escherichia coli. Acyl phosphatidylglycerol (acyl PG) is a minor headgroup‐acylated GPL found in the cell membrane. Three enzymes, PagP, PldB, and At1g78690 (AT), play roles in the synthesis of acyl PG in E. coli. PagP synthesizes acyl PG in the outer membrane from phosphatidylglycerol (PG) and PG or phosphatidylethanolamine (PE). PldB synthesizes acyl PG in the inner membrane from PG and 2‐acyl lyso PE. AT, a lysoglycerophospholipid acetyltransferase, through an unknown mechanism, synthesizes GPLs by acylating lyso GPLs, which leads to an accumulation of acyl PG when overexpressed in E. coli. This study aims to characterize the cellular phenotype of E. coli with altered levels of acyl PG. Strains of wild‐type (WT) MG1655 E. coli with pagP and pldB deletions, ΔpagP and ΔpldB, were transformed with an arabinose induced empty pBAD33.1 plasmid, or pBAD33.1 containing PagP, AT, or PldB. A triple cardiolipin synthase (cls) knockout mutant, BKT12, and its WT control, W3110, were transformed similarly. Cellular morphology, temperature sensitivity, lipid composition, cellular permeability, and minimum inhibitory concentration to a variety of antibiotics was determined for each strain. Compared to the WT controls, overexpression of PldB leads to an elongation of cell length in BKT12, MG1655, ΔpagP, and ΔpldB. No strains showed altered temperature sensitivity. Overexpression of PagP leads to an altered growth rate and the presence of an additional unidentified lipid. Additionally, PagP overexpression leads to an increase in membrane permeability in all strains. There was no change in the minimum inhibitory concentrations upon expression of PagP, AT, or PldB. In the future, a ΔpldBΔpagP strain will be constructed and similarly analyzed to help us better understand the role of headgroup‐acylated GPLs in the cell.Support or Funding InformationThis work was supported by National Science Foundation Research at Undergraduate Institutions grant #1152463 to T. A. G.
Toward a Functional, ChemoProteomic Interrogation of Kinome and Nucleotide Binding Space
Authors: Kozarich John W 
Published: November 2023

Protein kinases represent the single largest mammalian enzyme family with ~ 518 members in the human proteome and have been implicated in a wide array of complex cellular functions and pathways, ranging from metabolic regulation to tumorigenesis. The physiological functions of the majority these enzymes remain unknown. The central role of protein kinases in signal transduction has generated interest in targeting these enzymes for a wide range of therapeutic indications.We have reported a method for identifying and quantifying protein kinases in any biological sample from any species. The ActivX Activity Based Proteomics technology for kinase relies on acylphosphate nucleotide probes, prepared from biotin and ATP or ADP that react covalently at the ATP binding sites of virtually all known protein kinases. Biotinylated peptide fragments from labeled proteomes are captured, sequenced and identified using a mass‐spectrometry based analysis platform to determine the kinases present and their relative levels. Direct competition between the probes and inhibitors can be measured to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.In this presentation, the development of the acylphosphate nucleotide probes and the scope of the reaction with the kinome and other nucleotide binding proteins will be discussed. In addition, other issues that speak to the breadth and versatility of this method will be addressed. The proteome‐specific kinase selectivity by drugs will also be demonstrated. Finally, the potential of the approach in other areas of nucleotide binding space, such as the GTP‐dependent enzymes, will be demonstrated.
Structural basis of distinct salicylic acid glucosylation in Arabidopsis thaliana by two homologous enzymes: implications for plant stress response
Authors: Thompson Alayna Michelle George , Iancu Cristina V , Dean John , Choe Jun‐yong 
Published: November 2023

The objective of this work is to understand SA‐glucose conjugate formation by two enzymes from Arabidopsis thaliana, UGT74F1 and UGT74F2. Salicylic acid (SA) is utilized as a signaling molecule by plants in response to various stresses. To store SA in an inactive form, conjugates are formed with small organic molecules such as glucose, and inactive SA can then be transported and stored in plant vacuoles. In the model organism Arabidopsis thaliana SA‐glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP‐glucose to SA. Despite being 77% identical with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms both SAG and salicylic acid glucose ester (SGE). SAG and SGE serve different cellular functions in terms of storage, so understanding their creation is important. As UGT74F1 and UGT74F1 are very similar, there are likely very subtle differences responsible for the observed final products. To investigate the difference between these enzymes, we began with crystallizing purified proteins. We solved the crystal structure of UGT74F2 complexed with its substrates UDP and SA, and this structure allowed the identification the residues that coordinate SA – H18, Y180, and M274. These residues are conserved between UGT74F1 and UGT74F2, but the loops containing these residues have several amino acid substitutions. In silico docking of SA to UGT74F1 (homology model) and UGT74F2 suggests that SA binding could be more constrained in UGT74F1. From our crystal structure and in silico modeling, we theorize that amino acid substitutions around the binding site are responsible for the observed activity differences between these enzymes. Some substitutions could affect hydrogen bonding: position 15 is threonine in UGT74F2, but serine in UGT74F1, while position 112 is alanine in UGT74F2 but a serine in UGT74F1. Other substitutions could drastically change the geometry of loops near the binding pocket, such as position 178 which is either a leucine (UGT74F1) or a proline (UGT74F2). For our future directions, we are investigating the involvement of these substitutions in product formation. We have produced mutant proteins and are investigating their products by enzymatic assays. Additionally, we are using in silico ligand docking, x‐ray scattering on solution phase protein, and x‐ray diffraction crystallography to investigate the subtle differences between UGT74F1 and UGT74F2.Support or Funding InformationSupported by RFUMS‐DePaul Pilot Grant Award
Seasonal Variation in Urinary Aflatoxin (AFM1) of Lactating Women from Rural Households in Southern Ethiopia
Authors: Gebreegziabher Tafere , Tsegaye Workneh , Stoecker Barbara J. 
Published: November 2023

Aflatoxins (AFB1, AFB2, AFG1 and AFG2) are highly toxic compounds produced by Aspergillus species. These contaminants can be present in a wide range of food stuffs especially in humid and hot climates. AFM1 is a metabolic byproduct of the potent carcinogen AFB1. As well as being carcinogenic, aflatoxins have been associated with impaired growth in infants and children. Despite the high likelihood of exposure to these contaminants, there have been only a few studies done in Ethiopia. Urine samples were collected from 101 lactating women during 2013 and short‐term aflatoxin exposures were assessed. Samples were collected in a dry season (January to February) and a wet season (June to August). Mean ± SD age of mothers was 23 ± 4 y. Mean maternal BMI was 21.3 ± 2.5 and mid‐upper arm circumference (MUAC) was 23.1 ± 1.9 cm. Corn and enset are the major staple foods of the study population. Corn would have been recently harvested in January to February and would have been stored for several months by June. Urine samples were analyzed for AFM1 using ELISA kits from Helica Biosystems Inc. AFM1 was detected in 58% and 68% of urine samples in the dry and wet seasons respectively. Among those who were exposed, the median (IQR) AFM1 was 75 (42, 165) pg/mL in the dry season and 152 (77, 352) pg/mL in the wet season. The difference of AFM1 between the two seasons was significant (p < 0.001). High urinary AFM1 in the dry season was correlated with high urinary AFM1 in the wet season (r = 0.381, p = 0.009) and this association was increased when urinary AFM1 was corrected for creatinine (r = 0.418, p = 0.001). Because of the humid and hot climatic conditions of the study area and poor quality of food storage the women are at high risk of dietary exposure of aflatoxin. Further studies are recommended to assess the prevalence of the problem and its related health implications.Support or Funding InformationSupported by Oklahoma State University