Mapping protein energy landscapes with amide hydrogen exchange and mass spectrometry: I. A generalized model for a two-state protein and comparison with experiment

Protein amide hydrogen exchange (HDX) is a convoluted process, whose kinetics is determined by both dynamics of the protein and the intrinsic exchange rate of labile hydrogen atoms fully exposed to solvent. Both processes are influenced by a variety of intrinsic and extrinsic factors. A mathematical formalism initially developed to rationalize exchange kinetics of individual amide hydrogen atoms is now often used to interpret global exchange kinetics (e.g., as measured in HDX MS experiments). One particularly important advantage of HDX MS is direct visualization of various protein states by observing distinct protein ion populations with different levels of isotope labeling under conditions favoring correlated exchange (the so-called EX1 exchange mechanism). However, mildly denaturing conditions often lead to a situation where the overall HDX kinetics cannot be clearly classified as either EX1 or EX2. The goal of this work is to develop a framework for a generalized exchange model that takes into account multiple processes leading to amide hydrogen exchange, and does not require that the exchange proceed strictly via EX1 or EX2 kinetics. To achieve this goal, we use a probabilistic approach that assigns a transition probability and a residual protection to each equilibrium state of the protein. When applied to a small protein chymotrypsin inhibitor 2, the algorithm allows complex HDX patterns observed experimentally to be modeled with remarkably good fidelity. On the basis of the model we are now in a position to begin to extract quantitative dynamic information from convoluted exchange kinetics.     

Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N₂ bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N₂ bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N₂ sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, “semi-unfolded” ↔ “native” ↔ “globally unfolded” → “aggregated”. This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS.     

Compressing the free energy range of substructure stabilities in iso‐1‐cytochrome c

Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 ± 0.4 kcal mol⁻¹ to unfold the least stable substructure compared with 5.05 ± 0.30 kcal mol⁻¹ for global unfolding of iso-1-cytochrome c. Thus, substructure stabilities of iso-1-cytochrome c span only ∼1.2 kcal mol⁻¹ compared with ∼8 kcal mol⁻¹ for horse cytochrome c. Consistent with the less cooperative folding thus expected for the horse protein, the guanidine-HCl m-values are ∼3 kcal mol⁻¹M⁻¹ versus ∼4.5 kcal mol⁻¹M⁻¹ for horse versus yeast cytochrome c. The tight free energy spacing of the yeast cytochrome c substructures suggests that its folding has more branch points than for horse cytochrome c. Studies on a variant of iso-1-cytochrome c with an H26N mutation indicate that the least and most stable substructures unfold sequentially and the two least stable substructures unfold independently as for horse cytochrome c. Thus, important aspects of the substructure architecture of horse cytochrome c, albeit compressed energetically, are preserved evolutionally in yeast iso-1-cytochrome c.     

Mutual conformational adaptations in antigen and antibody upon complex formation between an Fab and HIV-1 capsid protein p24

BACKGROUND: Elucidating the structural basis of antigen-antibody recognition ideally requires a structural comparison of free and complexed components. To this end we have studied a mouse monoclonal antibody, denoted 13B5, raised against p24, the capsid protein of HIV-1. We have previously described the first crystal structure of intact p24 as visualized in the Fab13B5-p24 complex. Here we report the structure of the uncomplexed Fab13B5 at 1.8 A resolution and analyze the Fab-p24 interface and the conformational changes occurring upon complex formation. RESULTS: Fab13B5 recognizes a nearly continuous epitope comprising a helix-turn-helix motif in the C-terminal domain of p24. Only 4 complementarity-determining regions (CDRs) are in contact with p24 with most interactions being by the heavy chain. Comparison of the free and complexed Fab reveals that structural changes upon binding are localized to a few side chains of CDR-H1 and -H2 but involve a larger, concerted displacement of CDR-H3. Antigen binding is also associated with an 8 degrees relative rotation of the heavy and light chain variable regions. In p24, small conformational changes localized to the turn between the two helices comprising the epitope result from Fab binding. CONCLUSIONS: The relatively small area of contact between Fab13B5 and p24 may be related to the fact that the epitope is a continuous peptide rather than a more complex protein surface and correlates with a relatively low affinity of antigen and antibody. Despite this, a significant quaternary structural change occurs in the Fab upon complex formation, with additional smaller adaptations of both antigen and antibody.     

A monoclonal antibody fab fragment crystallized with and without a peptide epitope from HIV

The Fab fragment of CB 4-1, a monoclonal murine antibody against HIV protein p24, has been produced. It forms a complex with a synthetic antigen, an epitope of p24 made up of 11 amino acids, with the binding constant k_d = 3.6 × 10^?9 M. Crystals of hexagonal and orthorhombic space group has been obtained by cocrystallization of the Fab with the epitope and crystallization without the epitope, respectively. In either case, the crystals are suitable for X-ray structural analysis. Crystals of the Fab fragment cocrystallized with the peptide have the space group P 6₃22 with cell dimensions of a = b = 105 Å, c = 297 Å. Fab crystals without the epitope are in space group C 222 with cell dimensions a = 110.1 Å, b = 110.2 c Å = 150.1 Å. © 1993 Wiley-Liss, Inc.     

Chimeric fab fragments of antibodies to recombinant Ebola virus glycoprotein

Previously, we have determined the nucleotide and amino acid sequences of the variable domains of three mouse monoclonal antibodies specific to the individual epitopes of the Ebola virus glycoprotein: GPE118 (IgG), GPE325 (IgM) and GPE534 (IgG) [1]. In the present paper, chimeric Fab fragments of Fab118, Fab325, and Fab534 antibodies were obtained based on the variable domains of murine antibodies by attaching CH1 and CL constant regions of human kappa-IgG1 to them. The recombinant chimeric Fab fragments were synthesized in the heterologous expression system Escherichia coli, isolated and purified using metal chelate affinity chromatography. The immunochemical properties of the obtained Fab fragments were studied by immunoblotting techniques as well as indirect and competitive ELISA using recombinant Ebola virus proteins: EBOV rGPdTM (recombinant glycoprotein of Ebola hemorrhagic fever virus without the transmembrane domain), NP (nucleoprotein) and VP40 (structural protein). The identity of recombinant chimeric Fab fragments, as well as their specificity to the recombinant glycoprotein of Ebola hemorrhagic fever virus (EBOV GP) was proved. The results of indirect ELISA evidence the absence of immunological cross-reactivity to NP and VP40 proteins of Ebola virus. The dissociation constants of the antigen-antibody complex K _d equal to 5.0, 1.0 and 1.0 nM for Fab118, Fab325 and Fab534, respectively, were determined; they indicate high affinity of the obtained experimental samples to EBOV GP. The epitope specificity of Fab fragments was studied using a panel of commercial neutralizing antibodies. It was found that all studied antibodies to EBOV GP are targeted to different epitopes, while the epitopes of the recombinant chimeric Fab fragments and original murine monoclonal antibodies (mAbs) coincide. All the obtained and studied mAbs to EBOV GP are specific to epitopes that coincide or overlap the epitopes of three commercial neutralizing mAbs to Ebola virus: epitopes Fab118 and Fab325 overlap the epitope of the known commercial mAb h13F6; Fab325 epitope also overlaps mAb c6D8 epitope; Fab534 epitope is located near mAb KZ52 conformational epitope, in the formation of which amino acid residues of GP1 and GP2 domains of EBOV GP are involved.     

Barley beta‐glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H₂O₂-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O₂⁻) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O₂⁻, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure.     

Recognition of defined epitopes by affinity‐purified anti‐immunoglobulin Fab autoantibodies isolated from HIV‐infected humans

Infection of humans with HIV‐1 has previously been independently shown to result in the generation of autoantibodies (AAbs) reactive with immunoglobulin Fab fragments (Heidelberg), and with autoantibodies to T‐cell receptors (TCRs) (Tucson). Here, we carry out epitope mapping studies of affinity‐purified AAbs to Fab fragments prepared from individual HIV‐positive patients for their capacity to bind recombinant constructs and peptide‐defined epitopes modeling TCR and Ig light chains. Some affinity‐purified autoantibodies reacted strongly with TCRs expressed by intact T‐cells, and recombinant V_α/V_β constructs as well as with certain synthetic peptide epitopes. The binding reactions of affinity‐purified AAbs of individual patients were distinct, and the AAb preparations consisted of populations of polyclonal lgs as reflected in specificity and isotype. AAb pools from individual patients all bound particular regions of TCR and Ig chains defined by comprehensive peptide synthesis including the CDR1 and Fr3 segments of the variable domains and the joining segment/switch peptide. In addition, other reactivities to restricted regions of α, β and λ light chains were documented. These results substantiate the cross‐reactivity of TCR and Ig–Fab determinants, and are consistent with the hypothesis that autoantibodies arising as a consequence of HIV infection can have an immunomodulatory role. Copyright © 1999 John Wiley & Sons, Ltd.     

Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry

In the double-stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid-bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N-terminal helix-loop-helix (H-L-H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid-bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.     

Biochemical comparison of Anopheles gambiae and human NADPH P450 reductases reveals different 2'-5'-ADP and FMN binding traits

NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2′, 5′-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k _cat) of 105 s⁻¹ and 88 s⁻¹, respectively, for mosquito and human CPR. However, the inhibitory effects of 2′,5′-ADP on activity were different; the IC₅₀ value of AgCPR for 2′, 5′ –ADP was significantly higher (6–10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2′, 5′- ADP. This was confirmed by isothermal titration calorimetry where binding of 2′,5′-ADP to AgCPR (K _d = 410±18 nM) was ∼10 fold weaker than human CPR (K _d = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K_d = 83±2.0 nM) than the equivalent human domain (K_d = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC₅₀ = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of ‘smart’ insecticides and synergists that selectively target mosquito CPR.     

Conformational dynamics of partially denatured myoglobin studied by time-resolved electrospray mass spectrometry with online hydrogen-deuterium exchange

This study demonstrates the use of electrospray mass spectrometry in conjunction with rapid online mixing ("time-resolved" ESI-MS) for monitoring protein conformational dynamics under equilibrium conditions. The hydrogen/deuterium exchange (HDX) kinetics of mildly denatured myoglobin (Mb) at pD 9.3, in the presence of 27% acetonitrile, were studied with millisecond time resolution. Analytical ultracentrifugation indicates that the average protein compactness under these solvent conditions is similar to that of native holomyoglobin (hMb). The mass spectrum shows protein ions in a wide array of charge and heme binding states, indicating the presence of multiple coexisting conformations. The experimental approach used allows the HDX kinetics of all of these species to be monitored separately. A combination of EX1 and EX2 behavior was observed for hMb ions in charge states 7+ to 9+, which predominantly represent nativelike hMb in solution. The EX1 kinetics are biphasic, indicating the presence of two protein populations that undergo conformational opening events with different rate constants. The EX2 kinetics observed for nativelike hMb are biphasic as well. All other charge and heme binding states represent non-native protein conformations that are involved in rapid interconversion processes, thus leading to monoexponential EX2 kinetics with a common rate constant. Burst phase labeling for these non-native proteins occurs at 125 sites. In contrast, the nativelike protein conformation shows burst phase labeling only for 88 sites. A kinetic model is developed which is based on the assumption of three distinct (un)folding units in Mb. The model implies that the free energy landscape of the protein exhibits a major barrier. The crossing of this barrier is most likely associated with slow, cooperative opening/closing events of the heme binding pocket. Rapid conformational fluctuations on either side of the barrier give rise to the observed EX2 kinetics. Simulated HDX kinetics based on this model are in excellent agreement with the experimental data.     

Kinetics and thermodynamics of metal‐binding to histone deacetylase 8

Histone deacetylase 8 (HDAC8) was originally classified as a Zn(II)-dependent deacetylase on the basis of Zn(II)-dependent HDAC8 activity in vitro and illumination of a Zn(II) bound to the active site. However, in vitro measurements demonstrated that HDAC8 has higher activity with a bound Fe(II) than Zn(II), although Fe(II)-HDAC8 rapidly loses activity under aerobic conditions. These data suggest that in the cell HDAC8 could be activated by either Zn(II) or Fe(II). Here we detail the kinetics, thermodynamics, and selectivity of Zn(II) and Fe(II) binding to HDAC8. To this end, we have developed a fluorescence anisotropy assay using fluorescein-labeled suberoylanilide hydroxamic acid (fl-SAHA). fl-SAHA binds specifically to metal-bound HDAC8 with affinities comparable to SAHA. To measure the metal affinity of HDAC, metal binding was coupled to fl-SAHA and assayed from the observed change in anisotropy. The metal K_D values for HDAC8 are significantly different, ranging from picomolar to micromolar for Zn(II) and Fe(II), respectively. Unexpectedly, the Fe(II) and Zn(II) dissociation rate constants from HDAC8 are comparable, k_off ∼0.0006 s⁻¹, suggesting that the apparent association rate constant for Fe(II) is slow (∼3 × 10³ M⁻¹ s⁻¹). Furthermore, monovalent cations (K⁺ or Na⁺) that bind to HDAC8 decrease the dissociation rate constant of Zn(II) by ≥100-fold for K⁺ and ≥10-fold for Na⁺, suggesting a possible mechanism for regulating metal exchange in vivo. The HDAC8 metal affinities are comparable to the readily exchangeable Zn(II) and Fe(II) concentrations in cells, consistent with either or both metal cofactors activating HDAC8.     

Conversion of the 2 Cl−/1 H+ antiporter ClC‐5 in a NO3−/H+ antiporter by a single point mutation

Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl⁻/1 H⁺. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO₃⁻/H⁺ antiporter atClCa, led to coupled NO₃⁻/H⁺ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO₃⁻/H⁺ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism.     

Beneficial effects of resistance exercise on glycemic control are not further improved by protein ingestion

Purpose

To investigate the mechanisms underpinning modifications in glucose homeostasis and insulin sensitivity 24 h after a bout of resistance exercise (RE) with or without protein ingestion.

Methods

Twenty-four healthy males were assigned to a control (CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). Muscle biopsy and blood samples were obtained at rest for all groups and immediately post-RE (75% 1RM, 8×10 repetitions of leg-press and extension exercise) for EX and EX+PRO only. At 24 h post-RE (or post-resting biopsy for CON), a further muscle biopsy was obtained. Participants then consumed an oral glucose load (OGTT) containing 2 g of [U-¹³C] glucose during an infusion of 6, 6-[²H₂] glucose. Blood samples were obtained every 10 min for 2 h to determine glucose kinetics. EX+PRO ingested an additional 25 g of intact whey protein with the OGTT. A final biopsy sample was obtained at the end of the OGTT.

Results

Fasted plasma glucose and insulin were similar for all groups and were not different immediately post- and 24 h post-RE. Following RE, muscle glycogen was 26±8 and 19±6% lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for EX and EX+PRO (75.1±2.7 and 75.3±2.8 mmol·L⁻¹∶120 min, respectively) compared with CON (90.6±4.1 mmol·L⁻¹∶120 min). Plasma insulin response was 13±2 and 21±4% lower for EX and CON, respectively, compared with EX+PRO. Glucose disappearance from the circulation was ∼12% greater in EX and EX+PRO compared with CON. Basal 24 h post-RE and insulin-stimulated PAS-AS160/TBC1D4 phosphorylation was greater for EX and EX+PRO.

Conclusions

Prior RE improves glycemic control and insulin sensitivity through an increase in the rate at which glucose is disposed from the circulation. However, co-ingesting protein during a high-glucose load does not augment this response at 24 h post-exercise in healthy, insulin-sensitive individuals.     

Identification of a novel antigen cross‐presenting cell type in spleen

Antigen-presenting cells (APC), like dendritic cells (DC), are essential for T-cell activation, leading to immunity or tolerance. Multiple DC subsets each play a unique role in the immune response. Here, a novel splenic dendritic-like APC has been characterized in mice that has immune function and cell surface phenotype distinct from other, described DC subsets. These were identified as a cell type continuously produced in spleen long-term cultures (LTC) and have an in vivo equivalent cell type in mice, namely ‘L-DC’. This study characterizes LTC-DC in terms of marker phenotype and function, and compares them with L-DC and other known splenic DC and myeloid subsets. L-DC display a myeloid dendritic-like phenotype equivalent to LTC-DC as CD11c^loCD11b^hiMHC-II⁻CD8α⁻ cells, distinct by high accessibility and endocytic capacity for blood-borne antigen. Both LTC-DC and L-DC have strong antigen cross-presentation ability leading to strong activation of CD8⁺ T cells, particularly after exposure to lipopolysaccharide. However, they have weak ability to stimulate CD4⁺ T cells in antigen-specific responses. Evidence is presented here for a novel DC type produced by in vitro haematopoiesis which has distinct antigen-presenting potential and reflects a DC subset present also in vivo in spleen.     

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4‐phosphatase‐1

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P₃ and PtdIns(3,4)P₂, direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P₂ is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 (^−/−MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to ^+/+MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser⁴⁷³ and Thr³⁰⁸-Akt phosphorylation and activation of Akt-dependent signalling in ^−/−MEFs, relative to ^+/+MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed ^−/−MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.     

Latent HIV-1 infection of resting CD4⁺ T cells in the humanized Rag2⁻/⁻ γc⁻/⁻ mouse

Persistent human immunodeficiency virus type 1 (HIV-1) infection of resting CD4⁺ T cells, unaffected by antiretroviral therapy (ART), provides a long-lived reservoir of HIV infection. Therapies that target this viral reservoir are needed to eradicate HIV-1 infection. A small-animal model that recapitulates HIV-1 latency in resting CD4⁺ T cells may accelerate drug discovery and allow the rational design of nonhuman primate (NHP) or human studies. We report that in humanized Rag2^−/− γ_c^−/− (hu-Rag2^−/− γ_c^−/−) mice, as in humans, resting CD4⁺ T cell infection (RCI) can be quantitated in pooled samples of circulating cells and tissue reservoirs (e.g., lymph node, spleen, bone marrow) following HIV-1 infection with the CCR5-tropic JR-CSF strain and suppression of viremia by ART. Replication-competent virus was recovered from pooled resting CD4⁺ T cells in 7 of 16 mice, with a median frequency of 8 (range, 2 to 12) infected cells per million T cells, demonstrating that HIV-1 infection can persist despite ART in the resting CD4⁺ T cell reservoir of hu-Rag2^−/− γ_c^−/− mice. This model will allow rapid preliminary assessments of novel eradication approaches and combinatorial strategies that may be challenging to perform in the NHP model or in humans, as well as a rigorous analysis of the effect of these interventions in specific anatomical compartments.     

Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell

The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl⁻) in regulation of lysosomal acidification [intra-lysosomal pH (pH_lys)] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl⁻ condition elevated pH_lys and reduced the intra-lysosomal Cl⁻ concentration ([Cl⁻]_lys) via reduction of cytosolic Cl⁻ concentration ([Cl⁻]_c), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G₀/G₁ arrest without induction of apoptosis. We also studied effects of direct modification of H⁺ transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H⁺-ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na⁺/H⁺ exchanger (NHE)] elevated pH_lys and decreased [Cl⁻]_lys associated with inhibition of cell proliferation via induction of G₀/G₁ arrest similar to the culture under a low Cl⁻ condition. However, unlike low Cl⁻ condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl⁻]_c compared with low Cl⁻ condition. These observations suggest that the lowered [Cl⁻]_c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl⁻ is a key factor of lysosome acidification and autophagy.     

Dusp5 negatively regulates IL‐33‐mediated eosinophil survival and function

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5^−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5^−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-X_L expression that results in enhanced eosinophil survival. In addition, Dusp5^−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival.     

Wild-type p53-induced phosphatase 1 (Wip1) forestalls cellular premature senescence at physiological oxygen levels by regulating DNA damage response signaling during DNA replication

Wip1 (protein phosphatase Mg²⁺/Mn²⁺-dependent 1D, Ppm1d) is a nuclear serine/threonine protein phosphatase that is induced by p53 following the activation of DNA damage response (DDR) signaling. Ppm1d^−/− mouse embryonic fibroblasts (MEFs) exhibit premature senescence under conventional culture conditions; however, little is known regarding the role of Wip1 in regulating cellular senescence. In this study, we found that even at a representative physiological concentration of 3% O₂, Ppm1d^−/− MEFs underwent premature cellular senescence that depended on the functional activation of p53. Interestingly, Ppm1d^−/− MEFs showed increased H2AX phosphorylation levels without increased levels of reactive oxygen species (ROS) or DNA base damage compared with wild-type (Wt) MEFs, suggesting a decreased threshold for DDR activation or sustained DDR activation during recovery. Notably, the increased H2AX phosphorylation levels observed in Ppm1d^−/− MEFs were primarily associated with S-phase cells and predominantly dependent on the activation of ATM. Moreover, these same phenotypes were observed when Wt and Ppm1d^−/− MEFs were either transiently or chronically exposed to low levels of agents that induce replication-mediated double-stranded breaks. These findings suggest that Wip1 prevents the induction of cellular senescence at physiological oxygen levels by attenuating DDR signaling in response to endogenous double-stranded breaks that form during DNA replication.