VTechWorks Repository :: Browsing by Author "Capelluto, Daniel G. S."

Browsing by Author "Capelluto, Daniel G. S."

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Actin polymerization dynamics at the leading edge
Hu, Xiaohua (Virginia Tech, 2012-10-05)
Actin-based cell motility plays crucial role throughout the lifetime of an organism. While the dendritic nucleation model explains the initiation and organization of the actin network in lamellipodia, two questions need to be answered. In this study, I reconstructed cellular motility in vitro to investigate how actin filaments are organized to coordinate elongation and attachment to leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel beads or thin glass nanofibers coated with N-WASP WCA domains. During sustained motility, physiological concentrations of Mg²⁺ generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg²⁺ abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, filament capping, or actin shell formation. Addition of other types of crosslinkers restored both comet tail attachment and particle motility. We propose a model in which polycation-induced filament bundling sustains processive barbed end attachment to the leading edge. I lowered actin, profilin, Arp2/3, and CP concentrations to address the generation of actin filament orientation during the initiation of motility. In the absence of CP, Arp2/3 nucleates barbed ends that grow away from the nanofiber surface and branches remain stably attached to nanofiber. CP addition causes shedding of short branches and barbed end capture by the nanofiber. Barbed end retention by nanofibers is coupled with capping, indicating that WWCA and CP bind simultaneously to barbed ends. In pull-down assays, saturating CP addition only blocks WWCA binding to barbed end by half. Labeled WWCA bound to barbed ends with an affinity of 14 pM and unlabeled WWCA with an affinity of 75 pM. CP addition increased WWCA binding slightly at low CP concentrations and decreased WWCA binding to 50% at high CP concentrations. Molecular models of CP and WH2 domains bound respectively to the terminal and penultimate actin subunit showed no overlap and that CP orientation might blocks WWCA dissociation from the penultimate subunit. Simultaneous binding of CP and WWCA to barbed ends is essential to the establishment of filament orientation at the leading edge.
Binding properties of adaptor proteins Tollip and Tom1
Brannon, Mary Katherine (Virginia Tech, 2015-07-02)
Adaptor proteins, like Tollip and Tom1, facilitate cellular cargo sorting through their ubiquitin-binding domains. Tollip and Tom1 bind to each other through their TBD and GAT domains, respectively, whereas Tollip interacts with phosphatidylinositol-3-phosphate (PtdIns(3)P)-containing endosomal membranes. Tom1 and Tollip interaction and association with endosomes is proposed to be involved in the lysosomal degradation of polyubiquitinated cargo. Through cellular, biochemical, and biophysical techniques, we have further characterized the association of Tom1 with Tollip. Mutations in the binding interface of the Tom1 GAT and Tollip TBD complex leads to a subcellular mis-localization of both proteins, indicating that Tom1 may serve to direct Tollip to specific cellular pathways. It was determined that Tom1 inhibits the binding of Tollip to PtdIns(3)P and inhibition was reversed when mutations in the binding interface of the Tom1 GAT and Tollip TBD were present. Furthermore, it was established that, upon the binding of Tollip TBD to Tom1 GAT, ubiquitin is inhibited from binding to Tom1 GAT. It was also demonstrated that Tom1 GAT, but not Tollip TBD, can weakly bind to PtdIns(3)P. Consequently, we propose that association of Tom1 may serve to direct Tollip for involvement in specific cell signaling pathways. Gaining insight into the function of Tom1 and Tollip may lead to their use as therapeutic targets for increasing the efficiency of cargo trafficking and also for patients recovering from various cardiac injuries.
Biochemical and Microscopic Characterization of INFT-1: an Inverted Formin in C. elegans
Li, Ying (Virginia Tech, 2011-03-14)
Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins.
Cationic Glycopolymers for DNA Delivery: Cellular Internalization Mechanisms and Biological Characterization
McLendon, Patrick Michael (Virginia Tech, 2009-10-20)
Understanding the biological mechanisms of polymeric DNA delivery is essential to develop vehicles that perform optimally. In this work, the cellular internalization mechanisms of poly(glycoamidoamine) (PGAA) DNA delivery polymers were investigated. Polymer:DNA complexes interact with cell-surface glycosaminoglycans (GAGs) in a manner independent of electrostatic interactions. Desulfation and GAG removal leads to decreased uptake. Individual polyplexes appear to have differing affinities for specific GAGs, as polyplex dissociation occurs in a charge-independent manner, and may influence binding. Internalization occurs through close interactions with GAGs, as GAGs accumulate on polyplex surfaces, resulting in negatively-charged polyplexes and decompaction of intact polyplexes is observed upon interaction with GAG. PGAA polyplexes enter cells via a complex, multifaceted internalization route. Pharmacological inhibition of endocytosis and visualization by confocal microscopy reveal that internalization occurs primarily through an actin and dynamin-dependent mechanism. Caveolae/raft-mediated endocytosis appears to be the predominant internalization mechanism, with clathrin-mediated endocytosis also significantly involved. Internalization occurs to a smaller degree via macropinocytosis and direct membrane penetration. Caveolae-mediated, but not clathrin-mediated, internalization leads to transgene expression, suggesting a targeting opportunity based on uptake mechanisms. PEGylation of PGAA polyplexes was achieved to minimize polyplex aggregation in serum. Polyplex size increased in serum, but PEGylation prevented further polyplex growth over time compared to non-PEGylated polymers. Specific targeting of hepatocytes through end-modification of PEG with galactose was unsuccessful, likely due to inaccessibility of targeting groups. Further hepatocyte targeting efforts focused on malonate-based polymers with clickable linkages for facile linkage of targeting groups. Despite favorable surface presentation of galactose, receptor-specific internalization of polyplexes was unsuccessful, as competitive inhibition in HepG2 cells resulted in significant polyplex internalization derived from nonspecific membrane interactions. Chemical modification of vehicles allows systematic study of structure-function properties leading to efficient intracellular delivery. Increasing G4 molecular weight generally increases toxicity and decreases transgene expression in HeLa cells. Incorporating galactose into a lanthanide-chelating polymer facilitated efficient cellular internalization that was visualized by two-photon microscopy. Increased gene expression was observed that correlated to increasing galactose, suggesting that polymer degradation increases gene expression. Also studied were branched peptides targeted to HIV-1 TAR, which displayed high biocompatibility and favorable internalization profiles in mammalian cells.
Circadian Control of Cell Cycle Progression
Santos, Carlo Steven (Virginia Tech, 2009-03-31)
Tumorigenesis is the result of uncontrolled cell growth due to the deregulation of cell cycle checkpoints 1. Period 2 (Per2) is a tumor suppressor that oscillate in expression in a 24-hour cycle 2, 3. Here, we show that Per2 interacts with the tumor suppressor protein p53. Both G1 and G2 checkpoint pathways involve a p53 dependent pathway which can trigger the cell to go through cell arrest or programmed cell death4. Understanding all the mitigating factors involved in regulating cell cycle progression under DNA damage can offer a better idea in how cells become immortal. Initially discovered through screening of a human liver cDNA library, the novel interaction between p53-Per2 was further documented using co-precipitation. Interestingly, under genotoxic stress conditions, p53 and Per2 were not found to bind which leads us to suspect that Per2 does not affect active p53 which may possibly be due to post translational modifications of its active state. Furthermore we investigated p53's ability to act as a transcription factor in the presence of Per2, showing that the Per2-p53 complex prevents p53 from binding to DNA. This implies that the tetramerization of p53 may also be another factor in Per2's ability to bind to p53. A truncated p53 lacking the last 30 amino acids that theoretically increase p53's ability to form a tetramer showed a drastic reduction in binding to Per2 5, 6. On the other hand, p53 lacking the tetramerization domain showed binding similar to wildtype. Consequently we speculate that the ability of Per2 to modulate p53 and act as a tumor suppressor protein may be dependent on either the post translational modifications of p53 or its oligomeric state.
Click Chemistry on DNA and Targeting RNA structure with Peptide Boronic Acids
Crumpton, Jason B. (Virginia Tech, 2012-04-20)
The utilization of click chemistry to perform inter- and intramolecular ligation on DNA has become ubiquitous in the literature. Advances in copper (I) stabilizing ligands that prevent DNA degradation via redox pathways have provided nucleic acid researchers access to the efficiency and quantitative nature of the click reaction. The majority of ligation procedures in the literature are performed in solution after DNA assembly and modification with alkyne reporter groups. However, without specialty alkyne reagents that can be sequentially and selectively deprotected, the solution phase method requires that the click reaction be performed on all DNA-attached alkynes simultaneously. Therefore, the variability of the azide reagent is limited to a singular R group. However, performing the click reaction on DNA during synthetic elongation (immediately after each alkyne installation) allows for the possibility of performing multiple click reactions with variable azide reagents. Unfortunately, most solid phase click procedures require long reaction times or the utilization of microwave irradiation to accelerate the reaction. The development of methods for the ligation of azides to alkynes without the use of microwave irradiation on solid phase is potentially very useful. Herein, we report a simple, efficient, and robust solid phase synthetic method for the ligation of azido-diamondoids to the alkyne-modified phosphate backbone of DNA with click chemistry using [Cu(CH₃CN)₄]PF₆ without stabilizing ligand. Interestingly, it was found that as the size of diamondoid increased, a corresponding increase in melting temperature of hybridized duplexes was observed. The developed method has the potential to complement existing DNA ligation procedures for applications in biotechnology and diagnostics. Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries. It is well known that RNA ligands incorporating basic and intercalating moieties display high RNA affinity. Unfortunately, these ligands are also often plagued by promiscuous binding to off-target substrates. Due to the potential utility of RNA ligands in biology and medicine, it is imperative to elucidate RNA binders which display high specificity as well as affinity. Boronic acid peptides promise unique RNA binding motifs through the interaction between the empty p-orbital of boron and the 2'-hydroxyl group of RNA. Herein, we describe the incorporation of lysine and phenylalanine boronic acid analogues into a branched peptide combinatorial library in an effort to impart increased selectivity towards the HIV-1 Rev Response Element (RRE). We were able to easily select and deconvolute 6 resulting "hit" peptides from 65,536 unique library members by high throughput screening and de novo sequencing. Although we were unable to evaluate peptide selectivity towards RRE due to general insolubility in aqueous media, we demonstrated the efficient deconvolution of a branched peptide library that incorporates boronic acids.
Development of Building Blocks - Thermostable Enzymes for Synthetic Pathway Biotransformation (SyPaB)
Sun, Fangfang (Virginia Tech, 2012-04-25)
Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes that microbes cannot do. One of the largest challenges is the high cost and instability of enzymes and cofactors. To overcome this obstacle, strong motivations have driven intensive efforts in discovering, engineering, and producing thermostable enzymes. In this project, ribose-5-phosphate isomerase (RpiB), one of the most important enzymes in the pentose phosphate pathway, was cloned from a thermophile Thermotoga maritima, and heterologously expressed in Escherichia coli, purified and characterized. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 80°C and pH 6.5-8.0. It had a half lifetime of 71 h at 60°C, resulting in its turn-over number of more than 2 x108 mol of product per mol of enzyme. Another two thermostable enzymes glucose-6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) and their fusion proteins G6PDH-DI and DI-G6PDH were cloned from Geobacillus stearothermophilus, heterologouely expressed in E. coli and purified through its His-tag. The individual proteins G6PDH and DI have good thermostability and reactivity. However, the presence of DI in fusion proteins drastically decreased G6DPH activity. However, a mixture of G6PDH and a fusion protein G6PDH-DI not only restored G6PDH activity through the formation of heteromultimeric network but also facilitated substrate channeling between DI and G6PDH, especially at low enzyme concentrations. My researches would provide important building blocks for the on-going projects: high-yield hydrogen production through cell-free enzymatic pathways and electrical energy production through enzymatic fuel cells.
Disabled-2 regulates platelet heterotypic and homotypic aggregation through sulfatide binding
Welsh, John Douglas (Virginia Tech, 2010-03-29)
At the site of vascular injury platelet aggregation serves to stem blood flow, initiate the inflammatory response, and stimulate wound healing. Platelets become stimulated, release their granule contents, and become adherent to one another. Platelet granules contain important clotting factors and regulators of aggregation. Disabled-2 (Dab2) is a negative regulator of platelet aggregation released from platelet α-granules. Dab2 binds to the αIIbβ3 integrin, through the PTB domain, and blocks fibrin binding to the integrin which serves as the major cause of platelet-platelet interactions. Dab2 is also capable of binding to sulfatides, through the N-PTB region, expressed on the outer leaflet of adjacent cells. Dab2-sulfatide binding decreases Dab2's ability to interact with the αIIbβ3 integrin, however sulfatides activate and stimulate platelet-platelet and platelet-leukocyte aggregation. Sulfatide addition to platelets stimulates increased αIIbβ3 integrin and P-selectin expression through stimulation of continued platelet degranulation, and these surface receptors mediate platelet heterotypic and homotypic aggregation. Here, we show that Dab2 N-PTB binding of sulfatides serves to increase the inhibitory affect of Dab2. Sulfatide stimulation of platelet degranulation can be blocked by the addition of N-PTB. Inhibition of sulfatide induced αIIbβ3 integrin and P-selectin expression result in decreased platelet-platelet aggregation under flow. N-PTB also blocks sulfatide induced platelet-leukocyte interactions and aggregation. Experimental data supports the hypothesis that Dab2-sulfatide binding serves to increase the inhibition of platelet aggregation.
Editorial: Signaling Proteins for Endosomal and Lysosomal Function
Capelluto, Daniel G. S.; Conde, Cecilia B.; Tumbarello, David A.; van den Bogaart, Geert (Frontiers, 2021-12-16)
How Oomycete and Fungal Effectors Enter Host Cells and Promote Infection
Kale, Shiv D. (Virginia Tech, 2011-04-05)
The genus Phytophthora contains a large number of species that are known plant pathogens of a variety of important crops. Phytophthora sojae, a hemibiotroph, causes approximately 1-2 billion dollars (US) of lost soybean world-wide each year. P. infestans, the causative agent of the Irish potato famine, is responsible for over 5 billion dollars (US) worth of lost potato each year. These destructive plant pathogens facilitate pathogenesis through the use of small secreted proteins known as effector proteins. A large subset of effector proteins is able to translocate into host cells and target plant defense pathways. P. sojae Avr1b is able to suppress cell death triggered by BAX and hydrogen peroxide. The W-domain of Avr1b is responsible for this functionality, and is recognized by the Rps1b gene product to induce effector triggered immunity. These oomycete effector proteins translocate into host cells via a highly conserved N-terminal motif known as RXLR-dEER without the use of any pathogen encoded machinery. In fungi an RXLR-like motif exists, [R,K,H] X [L,F,Y,M,~I] X, that is able to facilitate translocation without pathogen encoded machinery. Both functional RXLR and RXLR-like motifs are able to bind phosphatidylinositol-3-phosphate (PtdIns- 3-P) to mediate entry into host cells. The use of novel inhibitory mechanisms has shown effector entry can be blocked either by sequestering PtdIns-3-P on the outer leaflet of plant and animal cells or by competitive inhibition of the binding pocket of the RXLR or RXLR-like motifs.
Ligand Binding Reveals a Role for Heme in Translationally-Controlled Tumor Protein Dimerization
Lucas, Andrew T.; Fu, Xiangping; Liu, Jingjing; Brannon, Mary K.; Yang, Jianhua; Capelluto, Daniel G. S.; Finkielstein, Carla V. (PLOS, 2014-11-14)
The translationally-controlled tumor protein (TCTP) is a highly conserved, ubiquitously expressed, abundant protein that is broadly distributed among eukaryotes. Its biological function spans numerous cellular processes ranging from regulation of the cell cycle and microtubule stabilization to cell growth, transformation, and death processes. In this work, we propose a new function for TCTP as a “buffer protein” controlling cellular homeostasis. We demonstrate that binding of hemin to TCTP is mediated by a conserved His-containing motif (His76His77) followed by dimerization, an event that involves ligand-mediated conformational changes and that is necessary to trigger TCTP's cytokine-like activity. Mutation in both His residues to Ala prevents hemin from binding and abrogates oligomerization, suggesting that the ligand site localizes at the interface of the oligomer. Unlike heme, binding of Ca2+ ligand to TCTP does not alter its monomeric state; although, Ca2+ is able to destabilize an existing TCTP dimer created by hemin addition. In agreement with TCTP's proposed buffer function, ligand binding occurs at high concentration, allowing the “buffer” condition to be dissociated from TCTP's role as a component of signal transduction mechanisms.
Membrane binding properties of Disabled-2
Alajlouni, Ruba (Virginia Tech, 2011-04-21)
Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the °IIb? integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes.
Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif
Zhao, Xiaolin; Xiong, Wen; Xiao, Shuyan; Tang, Tuo-Xian; Ellena, Jeffrey F.; Armstrong, Geoffrey S.; Finkielstein, Carla V.; Capelluto, Daniel G. S. (Nature Publishing Group, 2017-02-22)
Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.
Molecular Biophysics Symposium, November 6, 2014
Capelluto, Daniel G. S. (Virginia Bioinformatics Institute, Virginia Tech, 2014-11-06)
Virginia Tech hosted the first Molecular Biophysics Symposium focusing on structural biology studies of proteins. The event pursued two goals. First, it was designed to enrich the interaction among biophysical research groups located in southwest Virginia. Second, the symposium offered undergraduate students, interested in continuing their graduate studies in biophysics, the opportunity to meet with local and invited biophysicists and structural biologists. Four speakers were selected from submitted abstracts by graduate students. We also encouraged undergraduate and graduate students and postdoctoral fellows to participate in poster presentations.
Molecular Mechanisms Governing Persistent Induction of Pro-Inflammatory Genes by Lipopolysaccharide
Glaros, Trevor Griffiths (Virginia Tech, 2011-07-26)
Low dose endotoxemia is caused by several health conditions including smoking, alcohol abuse, high fat diets, and aging. Several studies have correlated low dose endotoxemia with increased risks of atherosclerosis, diabetes, and Parkinson's disease. Unlike high doses of endotoxin which induce a strong but transient induction of pro-inflammatory mediators, low doses of endotoxin result in a mild but chronic induction of pro-inflammatory genes. The central hypothesis of our study was that if low doses of endotoxin are capable of inducing mild prolonged inflammation, then a unique signaling circuit must be utilized. In the first study, the molecular mechanisms for the persistent induction of lipocalin 2 (LCN2) in response to 100 ng/mL of lipopolysaccharide (LPS) in kidney fibroblasts was examined. It appears that the intracellular signaling network responsible for the persistent induction of LCN2 requires both activator protein-1 (AP-1) and CCAAT/enhancer binding protein delta (C/ebpδ). Interleukin-1 receptor-associated kinase 1 (IRAK-1) is critical for LCN2 expression. In the second study, the molecular mechanisms governing the persistent induction of interleukin 6 (IL-6) upon a 50 pg/mL challenge of LPS in macrophages was examined. At this dose, only the persistent activation of cJun N-terminal kinase (JNK) and C/ebpδ was observed. IL-6 transcription requires the transient recruitment of activating transcription factor 2 (ATF2) and the persistent recruitment of C/ebpδ to the IL-6 promoter. In the third study, the molecular mechanisms that mediate LPS-induced priming was examined. The results demonstrate that macrophages are able to sense their prior history of exposure to LPS that result in either a priming or tolerance phenotype upon a secondary challenge of LPS. Results suggest that this sensing mechanism involves cross-talk between IRAK-1 and phosphoinositide-3-kinase (PI3K). Collectively, these studies indicate that JNK and C/ebpδ are the primary players responsible for the persistent expression of pro-inflammatory genes during low dose endotoxemia. IRAK-1 is a key intracellular signaling kinase that mediates signaling at low doses of LPS. IRAK-1 is not only critical for low dose induced expression, but also for LPS-induced priming. This research has revealed a novel signaling pathway that could provide new molecular targets for drug development against chronic inflammatory diseases.
Opening the Debate: How to Fulfill the Need for Physicians’ Training in Circadian-Related Topics in a Full Medical School Curriculum
Selfridge, Julia M.; Moyer, Kurtis E.; Capelluto, Daniel G. S.; Finkielstein, Carla V. (Ubiquity Press, 2015)
Background: Circadian rhythms are daily changes in our physiology and behavior that are manifested as patterns of brain wave activity, periodic hormone production, recurring cell regeneration, and other oscillatory biological activities. Their importance to human health is becoming apparent; they are deranged by shift work and jet-lag and in disparate conditions such as insomnia, sleep syndromes, coronary heart attacks, and depression, and are endogenous factors that contribute to cancer development and progression. Discussion: As evidence of the circadian connection to human health has grown, so has the number of Americans experiencing disruption of circadian rhythms due to the demands of an industrialized society. Today, there is a growing work force that experiences night shift work and time-zone shifts shaping the demands on physicians to best meet the needs of patients exposed to chronic circadian disruptions. The diverse range of illness associated with altered rhythms suggests that physicians in various fields will see its impact in their patients. However, medical education, with an already full curriculum, struggles to address this issue. Summary: Here, we emphasize the need for incorporating the topic of circadian rhythms in the medical curriculum and propose strategies to accomplish this goal.
The PH Domain and C-Terminal polyD Motif of Phafin2 Exhibit a Unique Concurrence in Animals
Hasan, Mahmudul; Capelluto, Daniel G. S. (MDPI, 2022-07-07)
Phafin2, a member of the Phafin family of proteins, contributes to a plethora of cellular activities including autophagy, endosomal cargo transportation, and macropinocytosis. The PH and FYVE domains of Phafin2 play key roles in membrane binding, whereas the C-terminal poly aspartic acid (polyD) motif specifically autoinhibits the PH domain binding to the membrane phosphatidylinositol 3-phosphate (PtdIns3P). Since the Phafin2 FYVE domain also binds PtdIns3P, the role of the polyD motif remains unclear. In this study, bioinformatics tools and resources were employed to determine the concurrence of the PH-FYVE module with the polyD motif among Phafin2 and PH-, FYVE-, or polyD-containing proteins from bacteria to humans. FYVE was found to be an ancient domain of Phafin2 and is related to proteins that are present in both prokaryotes and eukaryotes. Interestingly, the polyD motif only evolved in Phafin2 and PH- or both PH-FYVE-containing proteins in animals. PolyD motifs are absent in PH domain-free FYVE-containing proteins, which usually display cellular trafficking or autophagic functions. Moreover, the prediction of the Phafin2-interacting network indicates that Phafin2 primarily cross-talks with proteins involved in autophagy, protein trafficking, and neuronal function. Taken together, the concurrence of the polyD motif with the PH domain may be associated with complex cellular functions that evolved specifically in animals.
Phafins Are More Than Phosphoinositide-Binding Proteins
Tang, Tuoxian; Hasan, Mahmudul; Capelluto, Daniel G. S. (MDPI, 2023-04-30)
Phafins are PH (Pleckstrin Homology) and FYVE (Fab1, YOTB, Vac1, and EEA1) domain-containing proteins. The Phafin protein family is classified into two groups based on their sequence homology and functional similarity: Phafin1 and Phafin2. This protein family is unique because both the PH and FYVE domains bind to phosphatidylinositol 3-phosphate [PtdIns(3)P], a phosphoinositide primarily found in endosomal and lysosomal membranes. Phafin proteins act as PtdIns(3)P effectors in apoptosis, endocytic cargo trafficking, and autophagy. Additionally, Phafin2 is recruited to macropinocytic compartments through coincidence detection of PtdIns(3)P and PtdIns(4)P. Membrane-associated Phafins serve as adaptor proteins that recruit other binding partners. In addition to the phosphoinositide-binding domains, Phafin proteins present a poly aspartic acid motif that regulates membrane binding specificity. In this review, we summarize the involvement of Phafins in several cellular pathways and their potential physiological functions while highlighting the similarities and differences between Phafin1 and Phafin2. Besides, we discuss research perspectives for Phafins.
Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1
Xiong, Wen; Tang, Tuo-Xian; Littleton, Evan S.; Karcini, Arba; Lazar, Iuliana M.; Capelluto, Daniel G. S. (Springer Nature, 2019-07-26)
Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate (PtdIns5P), which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.
Protein Trafficking or Cell Signaling: A Dilemma for the Adaptor Protein TOM1
Roach, Tiffany G.; Lang, Helja K. M.; Xiong, Wen; Ryhanen, Samppa J.; Capelluto, Daniel G. S. (2021-02-26)
Lysosomal degradation of ubiquitinated transmembrane protein receptors (cargo) relies on the function of Endosomal Sorting Complex Required for Transport (ESCRT) protein complexes. The ESCRT machinery is comprised of five unique oligomeric complexes with distinct functions. Target of Myb1 (TOM1) is an ESCRT protein involved in the initial steps of endosomal cargo sorting. To exert its function, TOM1 associates with ubiquitin moieties on the cargo via its VHS and GAT domains. Several ESCRT proteins, including TOLLIP, Endofin, and Hrs, have been reported to form a complex with TOM1 at early endosomal membrane surfaces, which may potentiate the role of TOM1 in cargo sorting. More recently, it was found that TOM1 is involved in other physiological processes, including autophagy, immune responses, and neuroinflammation, which crosstalk with its endosomal cargo sorting function. Alteration of TOM1 function has emerged as a phosphoinositide-dependent survival mechanism for bacterial infections and cancer progression. Based on current knowledge of TOM1-dependent cellular processes, this review illustrates how TOM1 functions in coordination with an array of protein partners under physiological and pathological scenarios.

Browsing by Author "Capelluto, Daniel G. S."

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