Early-stage dynamics of chloride ion–pumping rhodopsin revealed by a femtosecond X-ray laser

Chloride ion–pumping rhodopsin (ClR) in some marine bacteria utilizes light energy to actively transport Cl⁻ into cells. How the ClR initiates the transport is elusive. Here, we show the dynamics of ion transport observed with time-resolved serial femtosecond (fs) crystallography using the Linac Coherent Light Source. X-ray pulses captured structural changes in ClR upon flash illumination with a 550 nm fs-pumping laser. High-resolution structures for five time points (dark to 100 ps after flashing) reveal complex and coordinated dynamics comprising retinal isomerization, water molecule rearrangement, and conformational changes of various residues. Combining data from time-resolved spectroscopy experiments and molecular dynamics simulations, this study reveals that the chloride ion close to the Schiff base undergoes a dissociation–diffusion process upon light-triggered retinal isomerization.

Chloride ion (Cl⁻) concentration in some bacterial cells is regulated by rhodopsin proteins, generally known as halorhodopsin, or hR. These proteins use light energy to pump Cl⁻ into cells (1, 2). Light is harvested by a molecule of retinal, covalently linked to an essential lysine residue in the seventh transmembrane helix of GPCR-like (G protein–coupled receptor) proteins. Light activation causes retinal to isomerize from the all-trans to the 13-cis configuration. This change triggers subsequent conformational changes throughout the rhodopsin molecule and releases chloride into the cytoplasm. Retinal thermally relaxes to the all-trans configuration within milliseconds and is then ready for the next photocycle. Cl⁻ ions are transported from the extracellular (EC) side to the cytoplasmic (CP) side during each photocycle (3, 4).Light-driven ion-pumping rhodopsin can be used to develop artificial solar energy harvesting and optogenetics (5–8), but the molecular mechanism must be understood in detail for such applications. Despite the importance of hR, our current experimental data concerning the structure and dynamics of the protein remain very limited. A related protein, proton (H⁺)-pumping bacteriorhodopsin (bR) discovered in the early 1970s, has been extensively studied by multiple methods, including time-resolved spectroscopy, crystallography, mutagenesis, and computer simulation (9–12). In particular, recent studies using time-resolved serial femtosecond crystallography (TR-SFX) methods performed at X-ray free-electron laser (XFEL) facilities allow three-dimensional (3D) visualization of retinal isomerization and associated local conformational changes. These changes are accompanied by movement of protons from a donor aspartate group to an acceptor aspartate (13–15). However, the central component of this process, the transported H⁺, is difficult to observe by X-ray crystallography and could not be directly traced in bR TR-SFX studies. Recently, a breakthrough was reported in a study on the sodium-pumping rhodopsin KR2 (K. eikastus rhodopsin 2), in which electron density signals of Na⁺ uptake were observed at Δt = 1 ms after laser illumination (16).Cl⁻, a strong X-ray scatterer, can be directly observed from electron density maps. These maps provide first-hand information on the movement of ions as being transported within short timescales after light activation. Furthermore, hR and bR presumably share a common molecular mechanism despite transporting ions in opposite directions. A close relationship is strongly implied by the interconversion of the function of two rhodopsins. Outward H⁺-pumping bR can be converted to an inward Cl⁻ pump by changing a single residue (D85T) (17), while hR from the cyanobacterium, Mastigocladopsis repens, is reported to pump protons after a single mutation (T74D) (18). The chloride pump can therefore serve as a system analogous to the proton transporter and provide valuable information that is difficult to obtain directly from bR.In this study, we focus on chloride ion–pumping rhodopsin (ClR) from the marine flavobacterium Nonlabens marinus S1-08T (19). The conserved DTD motif (Asp85-Thr89-Asp96) of the bR family, residues 85, 89, and 96, is replaced by an NTQ motif (Asn98- Thr102-Gln109) in ClR (Fig. 1). The sequence identity of ClR and canonical bR from Halobacterium salinarum is only 27%, but the two proteins, nevertheless, have highly similar structures, including the disposition of the retinal chromophore. ClR structures at cryogenic and room temperatures clearly reveal an architecture composed of seven transmembrane helices (TM A to G) (2, 20, 21). The retinal is covalently linked to the Nζ atom of the Lys235 located on TM-G. Anomalous diffraction signals of the Br⁻ identify a stable binding site near the protonated Schiff base (PSB) and a plausible exit site on the CP side (Fig. 1A). Buried water molecules and locations of cavities inside ClR suggest a pathway for Cl⁻ uptake on the EC side, but the molecular mechanism for light-triggered Cl⁻ pumping remains obscure. Upon light activation, the Cl⁻ tightly held near the PSB must break free from its hydrogen bonding network (Fig. 1B). It then passes through a hydrophobic region to reach the CP side (Fig. 1C). Crystal structures of ClR were previously determined with crystals under continuous illumination of visible laser light. Intriguingly, these steady-state models revealed unexpected movement of the retinal, without indication of photo-isomerization (22). Steady-state measurements, which show averages of mixed states, are thus of limited use in deciphering the molecular mechanism of light-driven Cl⁻ pumping.Open in a separate windowFig. 1.Structure of ClR and a plausible pathway of Cl⁻ transport. (A) Cross-sections of ClR with the backbone structure shown in cartoon representation. Transmembrane helices are marked using letters A through G, and the C-terminal helix H in the cytoplasm is also indicated. Surfaces are clipped to show the cross-section colored in yellow and the model being sliced and then opened about the axis near the helix E. Water molecules and Cl⁻ ions are shown as red- and green-colored spheres. Blue curves indicate the path of ion entering ClR and the principal pumping direction after passing retinal. (B) Key residues near the Cl⁻ ion and retinal, together with the NTQ motif shown in stick representation. (C) Residues that form a hydrophobic region between the retinal and the cytoplasm are highlighted in ball-and-stick representation. The red arrow points to a major barrier that Cl⁻ needs to overcome. ClR backbone is shown in cartoon representation, with residues colored based on hydrophobicity (the blue to red spectrum corresponds to the hydrophobicity scale from hydrophilic to hydrophobic).     

Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.     

Distribution of open reading frames of plasticity region of strain J99 in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica

The plasticity region of Helicobacter pylori strain J99 is a large chromosomal segment containing 33 strain-specific open reading frames (ORFs) with characteristics of a pathogenicity island. To study the diversity of the plasticity region, 22 probes corresponding to 20 ORFs inside the plasticity region and two ORFs on its boundaries were hybridized to genomic DNA isolated from clinical strains of H. pylori from patients with gastritis or gastric adenocarcinoma. Highly variable hybridization patterns were observed. The majority of the clinical strains presented a hybridization profile similar to that of J99; thus, these ORFs are not J99 strain specific. No association was found between a particular hybridization pattern and the clinical origin of the strain. Nevertheless, two single ORFs (JHP940 and JHP947) were more likely to be found in gastric cancer strains. They may be new pathogenicity markers. An in vitro expression study of these ORFs was also performed for the J99 strain, under different conditions. Thirteen ORFs were consistently expressed, six were consistently shut off, and three were expressed differentially. Most of the constitutionally expressed genes were located on the 3' part of the plasticity region. Our results show that the plasticity region, rather than being considered a pathogenicity island per se, should be considered a genomic island, which represents a large fragment of foreign DNA integrated into the genome and not necessarily implicated in the pathogenic capacity of the strain.     

L-dopa stimulates the release of [3H]gamma-aminobutyric acid in the basal ganglia of 6-hydroxydopamine lesioned rats

L-DOPA stimulated the K(+)-induced [3H]GABA (gamma-aminobutyric acid) release from slices of substantia nigra pars reticulata, entopeduncular nucleus, globus pallidus and caudate-putamen isolated from the ipsilateral side of 6-hydroxydopamine-lesioned rats, but the release from ipsilateral subthalamic slices was not affected. In substantia nigra, L-DOPA stimulation (EC50 = 1 microM) of [3H]GABA release was dose-dependently blocked (IC50 = 0.1 microM for the stimulation caused by 10 microM L-DOPA) by the D1 antagonist SCH 23390, but was not affected by (-)-sulpiride, a D2 antagonist. SCH 23390 also blocked the stimulation in the other nuclei. The DOPA decarboxylase inhibitor NSD-1015 (500 microM) did not prevent the stimulation induced by L-DOPA in all of the studied nuclei. The results suggest that L-DOPA is able to activate D1 receptors located on the terminals of striatal projections via the dopamine formed by a decarboxylation mediated by an NSD-1015-resistant enzyme. Activation of the presynaptic D1 receptors results in stimulation of GABA release.     

Bcl-x(L)-mediated changes in metabolic pathways of breast cancer cells: from survival in the blood stream to organ-specific metastasis

Bcl-x(L) protein plays a role in breast cancer dormancy, promoting survival of cells in metastatic foci by counteracting the proapoptotic signals in the microenvironment. The aim of this study was to identify phenotypes mediated by Bcl-x(L) in breast cancer cells that enhance in vivo survival of clinical metastases. 435/Bcl-x(L) or 435/Neo human breast cancer cells were injected into the inguinal mammary gland of nude mice, and tumors, metastases in lymph node, lung, and bone, and bloodstream surviving cells were examined. Proteomic analysis identified 17 proteins that were overexpressed (more than twofold) or underexpressed (less than twofold) in metastases. A protein interaction program allowed us to functionally associate peroxiredoxin 3, peroxiredoxin 2, carbonyl reductase 3, and enolase 1, suggesting a role for cellular responses to oxidative stress in metastasis organ selection. The prediction included proteins involved in redox systems, kinase pathways, and the ATP synthase complex. Furthermore, the interaction of redox proteins with enolase 1 suggests a connection between glycolysis and antioxidant pathways, enabling achievement of a high metastatic activity. In conclusion, Bcl-x(L) mediates a phenotype in which redox pathways and glycolysis are coupled to protect breast cancer metastatic cells during transit from the primary tumor to the metastatic state.     

Biological resurfacing of the knee: a review of surgical management

Lesions of the articular surfaces of the knee have been managed by various techniques over the last 50 years. Surgical management has involved: excising the damaged area, refashioning the underlying bone to produce a fibrous response, and introducing allograft, autograft and synthetic materials to encourage a repair matrix. The techniques and their pitfalls are reviewed and discussed, and suggestions made as to the direction of future studies for the repair of osteochondral lesions in the painful knee.     

Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders

Although nonmyeloablative conditioning regimen transplantations (NMTs) induce engraftment of allogeneic stem cells with a low spectrum of toxicity, graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality. In vivo T-cell depletion, using alemtuzumab, has been shown to reduce the incidence of GVHD. However, this type of maneuver, although reducing GVHD, may have an adverse impact on disease response, because NMTs exhibit their antitumor activity by relying on a graft-versus-malignancy effect. To explore the efficacy of alemtuzumab compared with methotrexate (MTX) for GVHD prophylaxis, we have compared the results in 129 recipients of a sibling NMT enrolled in 2 prospective studies for chronic lymphoproliferative disorders. Both NMTs were based on the same combination of fludarabine and melphalan, but the United Kingdom regimen (group A) used cyclosporin A plus alemtuzumab, whereas the Spanish regimen (group B) used cyclosporin A plus MTX for GVHD prophylaxis. Patients receiving alemtuzumab had a higher incidence of cytomegalovirus (CMV) reactivation (85% versus 24%, P <.001) and a significantly lower incidence of acute GVHD (21.7% versus 45.1%, P =.006) and chronic GVHD (5% versus 66.7%, P <.001). Twenty-one percent of patients in group A and 67.5% in group B had complete or partial responses 3 months after transplantation (P <.001). Eighteen patients in group A received donor lymphocyte infusions (DLIs) to achieve disease control. At last follow-up there was no difference in disease status between the groups with 71% versus 67.5% (P =.43) of patients showing complete or partial responses in groups A and B, respectively. No significant differences were observed in event-free or overall survival between the 2 groups. In conclusion, alemtuzumab significantly reduced GVHD but its use was associated with a higher incidence of CMV reactivation. Patients receiving alemtuzumab often required DLIs to achieve similar tumor control but the incidence of GVHD was not significantly increased after DLI.     

A protein binding the methylated 5''-terminal sequence, m7GpppN, of eukaryotic messenger RNA.

Ribosomal salt washes of Artemia salina embryos contain a protein(s) that binds [3H]m7GpppGpC and [3H]m7GpppGmpC, as measured by retention on nitrocellulose membrane filters. These oligonucleotides correspond in structure to the methylated 5'-terminal sequences (caps) present in many eukaryotic mRNAs. The cap binding protein does not bind the unmethylated counterparts of caps, e.g., [32P]GpppGpCp, or a derivative of m7GpppGmpC containing ring-opened m7G. None of the purified initiation factors IF-MP, IF-M2A, IF-M2B, IF-M3, or IF-MI binds the m7G-containing oligonucleotides.     

Immune response induction and new effector mechanisms possibly involved in protection conferred by the Cuban anti-meningococcal BC vaccine

This report explores the participation of some afferent mechanisms in the immune response induced by the Cuban anti-meningococcal vaccine VA-MENGOC-BC. The induction of delayed-type hypersensitivity in nursing babies and lymphocyte proliferation after immunization is demonstrated. The presence of gamma interferon IFN-gamma and interleukin-2 (IL-2) mRNAs but absence of IL-4, IL-5, and IL-10 mRNAs were observed in peripheral blood mononuclear cells from immunized subjects after in vitro challenge with outer membrane vesicles. In addition, some effector functions were also explored. The presence of opsonic activity was demonstrated in sera from vaccinees. The role of neutrophils as essential effector cells was shown. In conclusion, we have shown that, at least in the Cuban adult population, VA-MENGOC-BC induces mechanisms with a T-helper 1 pattern in the afferent and effector branches of the immune response.     

High frequency of altered HLA class I phenotypes in invasive breast carcinomas

We studied 105 tumor samples obtained from patients diagnosed as having breast carcinomas for HLA class I and II (DR) antigen expression, using a panel of mAbs defining HLA-monomorphic, locus-specific and allele-specific determinants. Peripheral blood lymphocytes from patients were also typed for HLA alleles. The results indicated total HLA class I losses in 55 patients (52.3%), HLA-A locus losses in four patients (3.8%), HLA-B locus losses in eight patients (7.6%), and A, B, locus losses in 10 patients (9.5%). The remaining 28 patients whose tissues reacted positively with monomorphic- and locus-specific mAbs were tested for HLA allelic losses using several anti-HLA mAbs defining A2, A3, A9, B8, B12, etc. Of these 28 patients, 16 (57%) showed one or more losses of HLA reactivity. These results indicated that in 88.5% of patients we detected a particular HLA-altered tumor phenotype. The downregulation of HLA class I antigens in breast carcinomas may thus be more frequent than previously reported, and patients without HLA class I downregulation may be the exception rather than the rule. It cannot be ruled out that HLA alterations are present in some of the 12 patients with an apparently normal HLA phenotype, as some HLA alleles could not be studied because of the lack of appropriate mAbs. These HLA alterations could represent an important step associated with tumor invasion, conferring to the tumor cells the ability to escape from T-lymphocyte recognition.