Cytosolic Distribution Research Articles

Cytosolic protein delivery using pH-responsive, charge-reversible lipid nanoparticles

Although proteins have attractive features as biopharmaceuticals, the difficulty in delivering them into the cell interior limits their applicability. Lipid nanoparticles (LNPs) are a promising class of delivery vehicles. When designing a protein delivery system based on LNPs, the major challenges include: (i) formulation of LNPs with defined particle sizes and dispersity, (ii) efficient encapsulation of cargo proteins into LNPs, and (iii) effective cellular uptake and endosomal release into the cytosol. Dioleoylglycerophosphate-diethylenediamine (DOP-DEDA) is a pH-responsive, charge-reversible lipid. The aim of this study was to evaluate the applicability of DOP-DEDA-based LNPs for intracellular protein delivery. Considering the importance of electrostatic interactions in protein encapsulation into LNPs, a negatively charged green fluorescent protein (GFP) analog was successfully encapsulated into DOP-DEDA-based LNPs to yield diameters and polydispersity index of < 200 nm and < 0.2, respectively. Moreover, ~ 80% of the cargo proteins was encapsulated into the LNPs. Cytosolic distribution of fluorescent signals of the protein was observed for up to ~ 90% cells treated with the LNPs, indicating the facilitated endocytic uptake and endosomal escape of the cargo attained using the LNP system.

Fatty acylation enhances the cellular internalization and cytosolic distribution of a cystine-knot peptide

SummaryDelivering peptides into cells could open up possibilities for targeting intracellular proteins. Although fatty acylation of peptide therapeutics improves their systemic half-life, it remains unclear how it influences their cellular uptake. Here, we demonstrate that a fatty acylated peptide exhibits enhanced cellular internalization and cytosolic distribution compared to the un-acylated version. By using a cystine-knot peptide as a model system, we report an efficient strategy for site-specific conjugation of fatty acids. Peptides modified with fatty acids of different chain lengths entered cells through clathrin-mediated and macropinocytosis pathways. The cellular uptake was mediated by the length of the hydrocarbon chain, with myristic acid conjugates displaying the highest distribution across the cytoplasm including the cytosol, and endomembranes of the ER, Golgi and mitochondria. Our studies demonstrate how fatty acylation improves the cellular uptake of peptides, and lay the groundwork for future development of bioactive peptides with enhanced intracellular distribution.

Establishment of chemically oligomerizable TAR DNA-binding protein-43 which mimics amyotrophic lateral sclerosis pathology in mammalian cells

One of the pathological hallmarks of amyotrophic lateral sclerosis (ALS) is mislocalized, cytosolic aggregation of TAR DNA-Binding Protein-43 (TDP-43). Not only TDP-43 per se is a causative gene of ALS but also mislocalization and aggregation of TDP-43 seems to be a common pathological change in both sporadic and familial ALS. The mechanism how nuclear TDP-43 transforms into cytosolic aggregates remains elusive, but recent studies using optogenetics have proposed that aberrant liquid–liquid phase separation (LLPS) of TDP-43 links to the aggregation process, leading to cytosolic distribution. Although LLPS plays an important role in the aggregate formation, there are still several technical problems in the optogenetic technique to be solved to progress further in vivo study. Here we report a chemically oligomerizable TDP-43 system. Oligomerization of TDP-43 was achieved by a small compound AP20187, and oligomerized TDP-43 underwent aggregate formation, followed by cytosolic mislocalization and induction of cell toxicity. The mislocalized TDP-43 co-aggregated with wt-TDP-43, Fused-in-sarcoma (FUS), TIA1 and sequestosome 1 (SQSTM1)/p62, mimicking ALS pathology. The chemically oligomerizable TDP-43 also revealed the roles of the N-terminal domain, RNA-recognition motif, nuclear export signal and low complexity domain in the aggregate formation and mislocalization of TDP-43. The aggregate-prone properties of TDP-43 were enhanced by a familial ALS-causative mutation. In conclusion, the chemically oligomerizable TDP-43 system could be useful to study the mechanisms underlying the droplet-aggregation phase transition and cytosolic mislocalization of TDP-43 in ALS and further study in vivo.TDP-43 is the primary protein aggregate in amyotrophic lateral sclerosis (ALS), and its mislocalization from nucleus is a hallmark of ALS pathology whose mechanisms remain unclear. In this study, the authors report a novel chemically oligomerizable TDP-43 system. Induction of TDP-43 oligomerization mimics ALS pathology such as mislocalization of TDP-43 and recruitment of critical proteins into the aggregates.

First insight in trace element distribution in the intestinal cytosol of two freshwater fish species challenged with moderate environmental contamination

Cytosolic distribution of six essential elements and nonessential Cd among biomolecules of different molecular masses was investigated in the intestine of brown trout (Salmo trutta) from the karst Krka River and Prussian carp (Carassius gibelio) from the lowland Ilova River. Fish were sampled at two locations (reference and contaminated) and in two seasons (autumn and spring). Analyses were conducted by size exclusion high performance liquid chromatography and high resolution inductively coupled plasma mass spectrometry. Although studied salmonid and cyprinid fish have different biological characteristics, obtained profiles often showed mostly similar patterns in both species. Specifically, Cd and Cu were dominantly bound to metallothioneins in both species, but the same association was not observed for Zn, whereas Mo distribution was similar in the intestine of both fish species with two well shaped and clear peaks in HMM (100–400 kDa) and VLMM (2–8 kDa) range. In brown trout, Se was mostly associated with biomolecules of very low molecular masses (VLMM, <10 kDa), whereas significant additional elution in HMM region (30–303 kDa) was observed only in Prussian carp. Iron binding to VLMM biomolecules (1.8–14 kDa) was observed only in brown trouts, and of Zn in Prussian carps. Cobalt was mostly bound to HMM biomolecules (85–235 kDa) in brown trout and to VLMM biomolecules (0.7–18 kDa) in Prussian carp. Comparison of intestinal profiles with previously published data on liver and gills revealed some similarities in distribution, but also organ-specific differences due to the different function and composition of each organ. As so far there is no published data on intestinal trace metal distribution, the obtained results represent the novel findings, and the key point for the exact identification of specific metal-binding biomolecules which could eventually be used as biomarkers of metal exposure or effects.

Leaf Plasmodesmata Respond Differently to TMV, ToBRFV and TYLCV Infection.

Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata’s structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells.

MAVS splicing variants associated with TRAF3 and TRAF6 in NF-κB and IRF3 signaling pathway in large yellow croaker Larimichthys crocea

Mitochondrial antiviral signaling protein (MAVS) acts as an essential adaptor in host RIG-I-like receptors (RLRs) mediated antiviral signaling pathway. In the present study, two MAVS transcript variants, the typical form and a splicing variant, namely Lc-MAVS_tv1 and Lc-MAVS_tv2 were characterized in large yellow croaker (Larimichthys crocea). The putative Lc-MAVS_tv1 protein contains 512 aa, with an N-terminal CARD domain, a central proline-rich region, and a C-terminal transmembrane (TM) domain, whereas Lc-MAVS_tv2 contains 302 aa and lacks the C-terminal TM domain due to a premature stop in the 102 bp intron fragment insertion. Lc-MAVS_tv1 was identified as a mitochondrion localized protein whereas Lc-MAVS_tv2 exhibited an entire cytosolic distribution. Quantitative real-time PCR revealed that Lc-MAVS_tv1 mRNA was broadly expressed in examined organs/tissues and showed extremely higher level than that of Lc-MAVS_tv2, and both of them could be up-regulated under poly I:C, LPS, PGN, and Pseudomonas plecoglossicida stimulation in vivo. Interestingly, overexpression of Lc-MAVS_tv2 could induce the activation of NF-κB but not IRF3, and Lc-MAVS_tv2 co-transfected with Lc-MAVS_tv1 induced a significantly higher level of NF-κB and IRF3 promoter activity. In addition, Lc-MAVS_tv2 overexpression could enhance TRAF3 and TRAF6 mediated NF-κB activation, but suppress TRAF3 and TRAF6 mediated IRF3 activation, implying that the splicing variant Lc-MAVS_tv2 may function as an important regulator in MAVS mediated signaling pathway.

Mathematical model pertaining to the effect of buffer over cytosolic calcium concentration distribution

The study of calcium dynamics is an important aspect as it regulates various processes viz metabolism, secretion etc. and also play the key role of second messenger. In the present work our main focus is over the variation of the cellular calcium with the various buffers after solving the advection-diffusion equation for the cellular calcium with inclusion of the calcium-binding buffers also. The presence of buffer lower the cellular calcium level and prevents its toxicity within cell. Buffers play key role in lowering the concentration level of cytosolic calcium, by binding with the intracellular free calcium ions. The model is incorporating association and dissociation rates, diffusion coefficients and buffer concentration. The fractional model for this is developed by using the Caputo-Fabrizio operator. The corresponding boundary and initial conditions are also framed accordingly. The integral transform technique is applied to obtain the analytical solution and simulation has been done to observe the effect of buffer on the distribution of calcium concentration.

Cooperative function of synaptophysin and synapsin in the generation of\xa0synaptic vesicle-like clusters in non-neuronal cells

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.

Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons

Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson’s disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200–1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.

Mathematical modelling of cytosolic calcium concentration distribution using non-local fractional operator

&lt;p style='text-indent:20px;'&gt;The aim of this paper is to study the calcium profile governed by the advection diffusion equation. The mathematical and computational modeling has provided insights to understand the calcium signalling which depends upon cytosolic calcium concentration. Here the model includes the important physiological parameters like diffusion coefficient, flow velocity etc. The mathematical model is fractionalised using Hilfer derivative and appropriate boundary conditions have been framed. The use of fractional order derivative is more advantageous than the integer order because of the non-local property of the fractional order differentiation operator i.e. the next state of the system depends not only upon its current state but also upon all of its preceeding states. Analytic solution of the fractional advection diffusion equation arising in study of diffusion of cytosolic calcium in RBC is found using integral transform techniques. Since, the Hilfer derivative is generalisation of Riemann- Liouville and Caputo derivatives so, these two are also deduced as special cases. The numerical simulation has been done to observe the effects of the fractional order of the derivatives involved in the differential equation representing the model over the concentration of calcium which is function of time and distance. The concentration profile of calcium is significantly changed by the fractional order.&lt;/p&gt;

Surface Chemistry- and Intracellular Trafficking-Dependent Autophagy Induction by Iron Oxide Nanoparticles.

Autophagy is a cellular self-clearance process for maintaining regular cytoplasmic function, and modulation of autophagy can influence cytotoxicity, apoptosis, and clearance of toxic amyloid fibril. In a recent work, functional nanoparticles are used to modulate autophagy. However, the role of nanoparticle uptake mechanisms and their intracellular processing on autophagy is vaguely understood. Here, we show that autophagy is influenced by nanoparticle surface chemistry-directed intracellular trafficking and localization. In particular, we have designed iron oxide nanoparticles functionalized with arginine/arginine methyl ester/octyl/oleyl/cholesterol with a high cell uptake property. We found that autophagy is induced by octyl/oleyl functionalization without appreciable cell death. Further study shows that enhanced cytosolic delivery over membrane localization and increased intracellular aggregation over homogeneous cytosolic distribution lead to autophagy induction via intracellular reactive oxygen species generation. The observed result can be used to design functional nanoparticles/nanodrugs for modulating cellular autophagy that can be used in various biomedical applications.

Abstract 518: Phosphorylation of MK5 at Threonine-182 in the Activation Loop is Mediated by P38α/β in Cardiac Fibroblasts

MAP kinase-activated protein kinase-5 (MK5) is a protein serine/threonine kinase involved in fibroblast function. MK5 is activated by phosphorylation at threonine-182 (Thr182): p38α/β, ERK3, and ERK4 have been implicated. We examined the phosphorylation of MK5 in adult cardiac ventricular fibroblasts. In serum-starved cardiac myofibroblasts (fibroblasts maintained on a plastic substrate), phospho-MK5 Thr182 (pThr182) immunoreactivity was predominantly nuclear. In response to serum, sorbitol, angiotensinII, TGFβ, or H 2 O 2 , pThr182 immunoreactivity both increased in intensity and relocated to the cytoplasm and the perinuclear region. In each case, the p38α/β inhibitor, SB203580, prevented both the increase in intensity and redistribution of pThr182 immunoreactivity. Incontrast, siRNA-mediated knockdown of ERK3 resulted in a diffuse cytosolic distribution of pThr182 immunoreactivity but failed to attenuate the increase in intensity. On Phos-tag PAGE, the electrophoretic mobility of phosphorylated proteins is reduced. Phos-tag PAGE resolved MK5 immunoreactivity from actively dividing myofibroblasts into several slower-migrating bands that were absent following 1) pretreatment with phosphoprotein phosphatase or 2) including EDTA in the Phos-tag gels. In serum-stimulated myofibroblasts, SB203580 reduced both the abundance of lower-mobility forms of MK5 on Phos-tag PAGE and the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. When fibroblasts were maintained on a compliant (8-kPa) substrate, and hence quiescent, the lower mobility forms of MK5 immunoreactivity were less abundant relative to myofibroblasts. Furthermore, in whole heart lysates from micesacrificed 8 weeks after constriction of the transverse aorta (TAC), Phos-tag PAGE revealed banding patterns consistent with increased MK5 phosphorylation relative to sham hearts. Taken together, these observations suggest: 1) p38α/β are the primarymediators of MK5 phosphorylation at Thr182 in cardiac fibroblasts, 2) ERK3 may be responsible for targeting activated MK5 to specific cytosolic sites, 3) Thr182 is not the only site at which MK5 is phosphorylated in vivo , and 4) MK5 phosphorylation increases with fibroblast activation.

Abstract 546: S-palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of Gc-1 in Cardiomyocytes

Objectives: The nitric oxide (NO) receptor soluble guanylyl cyclase (GC-1) can localize to cell membrane microdomains, including caveolae. In failing hearts, GC-1 relocalizes away from caveolae and becomes oxidized. How this occurs is unknown. We hypothesized that S-palmitoylation, a reversible, enzyme-catalyzed, post-translational modification that enhances membrane affinity of protein substrates, mediates caveolae-localization and modulates redox regulation of GC-1. Methods: Adult male C57BL/6 mice were subjected to transverse aortic constriction (TAC) versus thoracotomy (Sham). Modified acyl-biotin exchange (ABE) assays were performed to assess S-palmitoylation of GC-1 in failing TAC vs non-failing Sham hearts. Using bioinformatics, we identified 5 conserved cysteines in the GC-1α subunit as potential S-palmitoylation sites. We created cysteine-serine mutants of GC-1α at each and all 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). Mutants and WT GC-1α were expressed in neonatal rat cardiomyocytes (NRCM) and their palmitoylation status compared by ABE. We assessed localization and oxidation of GC-1α (CS* vs WT) by confocal microscopy and redox Western, respectively. Results: In TAC hearts, S-palmitoylation of GC-1α was half of that in Sham ( p =0.02). Likewise, palmitoyl-transferase ZDHHC16 was downregulated in TAC vs Sham by 31% ( p =0.01) at the transcript level and by 56% ( p =0.03) at the protein level. In NRCMs, S-palmitoylation of CS* was markedly diminished (6.9%, p &lt;0.001), while that of C15S, C176S, and C497S were modestly diminished (23.8%, p=0.03; 23.2%, p=0.01; 22.8%, p=0.01), compared to WT (54.7%). Moreover, S-palmitoylation of WT but not CS* was decreased by palmitoylation inhibitor 2-BP (17.8%, p&lt;0.0001). Confocal microscopy showed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed increased oxidation of CS* vs WT upon H 2 O 2 treatment (1.7-fold increase, p=0.008). Conclusions: Our data support that GC-1α is S-palmitoylated at C15, C176, and C497. S-palmitoylation of GC-1α at these 3 cysteines appears to protect GC-1α against oxidation and to mediate its membrane localization.

A pH-sensitive fluorescent protein sensor to follow the pathway of calcium phosphate nanoparticles into cells

Calcium phosphate nanoparticles (100 nm) were fluorescently labelled with poly(ethyleneimine) (PEIATTO490LS; red fluorescence). They were loaded with a Tandem fusion protein consisting of mRFP1-eGFP (red and green fluorescence in the same molecule)that acts as smart biological pH sensor to trace nanoparticles inside cells. Its fluorescence is also coupled to the structural integrity of the protein, i.e. it is also a label for a successful delivery of a functional protein into the cell. At pH 7.4, the fluorescence of both proteins (red and green) is detectable. At a pH of 4.5-5 inside the lysosomes, the green fluorescence is quenched due to the protonation of the eGFP chromophore, but the pH-independent red fluorescence of mRFP1 remains. The nanoparticles were taken up by cells (cell lines: HeLa, Caco-2 and A549) via endocytic pathways and then directed to lysosomes. Time-resolved confocal laser scanning microscopy confirmed mRFP1 and nanoparticles co-localizing with lysosomes. The fluorescence of eGFP was only detectable outside lysosomes, i.e. most likely inside early endosomes or at the cell membrane during the uptake, indicating the neutral pH at these locations. The Tandem fusion protein provides a versatile platform to follow the intracellular pathway of bioactive nanocarriers, e.g. therapeutic proteins. The transfection with a Tandem-encoding plasmid by calcium phosphate nanoparticles led to an even intracellular protein distribution in cytosol and nucleoplasm, i.e. very different from direct protein uptake. Neither dissolved protein nor dissolved plasmid DNA were taken up by the cells, underscoring the necessity for a suitable carrier like a nanoparticle. Statement of SignificanceA pH-sensitive protein ("tandem") was used to follow the pathway of calcium phosphate nanoparticles. This protein consists of a pH-sensitive fluorophore (eGFP; green) and a pH-independent fluorophore (mRFP1; red). This permits to follow the pathway of a nanoparticle inside a cell. At a low pH inside an endolysosome, the green fluorescence vanishes but the red fluorescence persists. This is also a very useful model for the delivery of therapeutic proteins into cells. The delivery by nanoparticles was compared with the protein expression after cell transfection with plasmid DNA encoding for the tandem protein. High-resolution image analysis gave quantitative data on the intracellular protein distribution.

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes.

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

Microtubule Arrangement and FKBP51 Localization is Altered in BMPR2‐Deficient Pulmonary Artery Endothelial Cells Isolated from a Rat Model of PAH

Pulmonary arterial hypertension (PAH) is an incurable vasoconstrictive pathology of which there are limited palliative treatments. The hallmarks of PAH include vascular smooth muscle cell proliferation and endothelial dysfunction in the small pulmonary arteries. Endothelial cytoskeletal defects have been implicated in BMPR2‐associated PAH, and BMPR2‐silencing alters cytoskeletal arrangement in pulmonary artery endothelial cells (PAECs). Indeed, targeting cytoskeletal proteins, particularly microtubules, may be a novel approach to PAH treatment as microtubule disruption has been efficacious in the treatment of other inflammatory and proliferative diseases. FK506‐binding protein 51 (FKBP51), a peptidyl‐prolyl isomerase member of the immunophilin family, is a known regulator of the glucocorticoid receptor and its expression is upregulated upon glucocorticoid receptor activation. Furthermore, FKBP51 is a binding partner to Hsp90 and stabilizes microtubules in PAECs. Here, we show microtubule disruption and altered FKBP51 distribution in PAECs from a rat model of PAH. PAECs isolated from the Sugen/hypoxia rat model of PAH (PAH PAECs) or normoxic control cells were gifted from the University of South Alabama. The PAH PAECs have lower BMPR2 gene expression than normoxic controls, as shown by real‐time PCR. These cells also demonstrate decreased BMPR2 signaling compared to normoxic control PAECs, as demonstrated by lower ID‐1 expression levels upon BMP4 or BMP6 stimulation. Immunocytochemistry studies of α‐tubulin indicate microtubule disorganization in PAH PAECs, as compared to the normoxic control PAECs, which demonstrate normal perinuclear distribution of interphase microtubules. Immunocytochemical staining of FKBP51, using a novel antibody developed by our laboratory, reveals a microtubule‐like distribution of FKBP51 in normoxic control PAECs. Upon treatment with the synthetic glucocorticoid dexamethasone (DEX), FKBP51 is predominantly observed in the nucleus of normoxic control PAECs, with reduced cytosolic organization and distribution. PAH PAECs, however, showed diffuse cytoplasmic staining of FKBP51, with no change in distribution upon DEX treatment. Western blot analysis of PAEC nuclear fractions also demonstrated greater protein levels of FKBP51 upon DEX treatment, compared to DEX‐treated PAH PAECs. Taken together, these data indicate that microtubule rearrangement is evident in BMPR2‐deficient PAH PAECs, and FKBP51 dysregulation may play a role in cytoskeletal abnormalities as well as the disruption of metabolic homeostasis in these cells.Support or Funding InformationSupported by NIH1R15HL137135‐01A1

S‐Palmitoylation Mediates Caveolae Localization and Limits Cysteine Oxidation of GC‐1 in Cardiomyocytes

IntroductionThe nitric oxide (NO) receptor soluble guanylyl cyclase (GC‐1) was first identified as a cytosolic enzyme but is now known to localize to various cell membrane microdomains. We reported that in normal hearts, GC‐1 localizes to caveolae, where it exhibits enhanced NO‐responsiveness. In failing hearts, caveolae‐localization of GC‐1 is disrupted and GC‐1 outside of caveolae is oxidized. How GC‐1 dynamically localizes to and away from caveolae is unknown. We hypothesized that S‐palmitoylation, a reversible, enzyme‐catalyzed, post‐translational modification that enhances membrane affinity of protein substrates, mediates caveolae‐localization and modulates redox regulation of GC‐1.MethodsTo examine S‐palmitoylation of GC‐1 in the failing heart, we subjected adult male C57BL/6 mice to transverse aortic constriction (TAC) vs thoracotomy (Sham) and performed modified acyl‐biotin exchange (ABE) assays. Expression of palmitoyl‐transferases ZDHHC1, ‐16, and ‐24 were also assessed in mouse hearts at the transcript and protein levels. Using computational bioinformatics, we identified 5 conserved cysteines of potential S‐palmitoylation in the GC‐1α subunit. We created cysteine‐serine mutants of GC‐1α at each and all of the 5 sites (C3S, C15S, C176S, C497S, C595S, CS*). We expressed the mutants and WT GC‐1α in neonatal rat cardiomyocytes (NRCM) via adenoviral mediated gene transfer and compared their palmitoylation status by ABE. We also assessed caveolae‐localization of GC‐1α (CS* vs WT) by confocal microscopy and its oxidation by redox Western.ResultsIn TAC hearts, S‐palmitoylation of GC‐1α was diminished by 50% compared to Sham (p=0.02). Likewise, ZDHHC16 was also downregulated in TAC by 31% (p=0.01) at the transcript level and by 56% (p=0.03) at the protein level, relative to Sham. In NRCMs, S‐palmitoylation of CS* was profoundly diminished (6.9±2.0 % of WT, p&lt;0.001), while that of C15S, C176S, and C497S were each modestly diminished (23.8±8.4%, p=0.03; 23.2±6.6%, p=0.01; 22.8±6.0%, p=0.01, respectively of WT). S‐palmitoylation of C3S and C595S were similar to WT. Confocal microscopy of NRCMs revealed more diffuse cytosolic distribution of CS* compared to WT. Redox Westerns revealed a trend towards increased cysteine oxidation of CS* vs WT upon H2O2 treatment (1.5‐fold increase, p=0.05).ConclusionsOur data suggest that S‐palmitoylation of GC‐1α at C15, C176, and C497 mediates GC‐1 caveolae‐localization and may reciprocally regulate cysteine post‐translational modifications of GC‐1.Support or Funding Information5R01HL138528 to EJT.

Alteration in the sensitivity to crizotinib by Na+/H+ exchanger regulatory factor 1 is dependent to its subcellular localization in ALK-positive lung cancers

BackgroundNa+/H+ exchanger regulatory factor 1 (NHERF1) is an important scaffold protein participates in the modulation of a variety of intracellular signal pathways. NHERF1 was able to enhance the effects of chemo-drugs in breast and cervical cancer cells. Anaplastic lymphoma kinase (ALK) fusion mutations are validated molecules targeted therapy in lung cancers, where crizotinib can be used as the specific inhibitor to suppress tumor progression. However, due to the less frequent occurrence of ALK mutations and the complexity for factors to determine drug responses, the genes that could alter crizotinib sensitivity are unclear.MethodsBoth ALK-translocated and ALK-negative lung adenocarcinoma specimens in tissue sections were collected for immunohistochemistry. The possible mechanisms of NHERF1 and its role in the cell sensitivity to crizotinib were investigated using an ALK-positive and crizotinib-sensitive lung adenocarcinoma cell line H3122. Either a NHERF1 overexpression vector or agents for NHERF1 knockdown was used for crizotinib sensitivity measures, in association with cell viability and apoptosis assays.ResultsThe expression level of NHERF1 in ALK-translocated NSCLC was significantly higher than that in other lung cancer tissues. NHERF1 expression in ALK positive lung cancer cells was regulated by ALK activities, and was in return able to alter the sensitivity to crizotinib. The function of NHERF1 to influence crizotinib sensitivity was depending on its subcellular distribution in cytosol instead of its nucleus localized form.ConclusionEctopically overexpressed NHERF1 could be a functional protein for consideration to suppress lung cancers. The determination of NHERF1 levels in ALK positive NSCLC tissues might be useful to predict crizotinib resistance, especially by distinguishing cytosolic or nuclear localized NHERF1 for the overexpressed molecules.

Deletion of tetraspanin CD151 alters the Wnt oncogene-induced mammary tumorigenesis: A cell type-linked function and signaling

Tetraspanin CD151 is increasingly implicated as a multifaceted mediator of cancer development and progression. Here we investigated the role of CD151 in breast cancer in the context of the Wnt oncogenic activation. Our data showed that removal of one or both of CD151 alleles in the MMTV-Wnt1 model significantly decreased the tumor-free survival of mice from 34 weeks on average to 22 weeks and 18 weeks, respectively. This effect coincided with an accelerated tumor growth and an increased number of Ki-67+ proliferative cells. Mechanistically, the CD151-deficient tumors were largely ER+, and exhibited hyperactivation of the Wnt pathway as reflected by a marked upregulation in β-catenin and Cyclin D1, and their target genes. In addition, E-cadherin displayed a cytosolic distribution and transcription factor Snail was markedly upregulated. Collectively, this data implies that CD151 suppresses the Wnt1-driven tumorigenesis, at least in part, via counteracting the epithelial-mesenchymal transition (EMT)-like program in luminal epithelial cells. Meanwhile, the proportion of tumor cells expressing CK5 or p63, the biomarkers of myoepithelial/basal cells, markedly decreased in the absence of CD151. This change was accompanied by a decreased invasiveness of tumors and their incompetence to form a long-term cell culture. Consistent with this basal cell-linked role, the CD151 downregulation impairs mammosphere formation in MCF-10A cells and the defect was rescued by re-expression of intact CD151 ORF, but not its integrin binding-defective mutant. Overall, our study suggests that CD151 is a key player in the Wnt oncogene-driven tumorigenesis and impacts breast cancer malignancy in a cell type-dependent manner.

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor.

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3 )-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. We used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRβ1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRβ1 intranuclear mobility was reduced. For TRα1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRα1 and TRβ1 population weresignificantly greater in MED1-/- cells, compared with MED1+/+ cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRα1; however, overexpression of MED13 had no effect on TRβ1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, our data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export.