Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation

Cyanobacteria, algae, and plants oxidize water to the O₂ we breathe, and consume CO₂ during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO₂/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast’s lumen, resulting in a light-driven production of O₂ and CO₂. Depletion of from the media leads, in the absence of added buffers, to a reversible down-regulation of O₂ production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO₂/ in photosynthetic water oxidation.Oxygenic photosynthesis in cyanobacteria, algae, and higher plants leads to the reduction of atmospheric CO₂ to energy-rich carbohydrates. The electrons needed for this process are extracted in a cyclic, light-driven process from water that is split into dioxygen (O₂) and protons. This reaction is catalyzed by a penta-µ-oxo bridged tetra-manganese calcium cluster (Mn₄CaO₅) within the oxygen-evolving complex (OEC) of photosystem II (PSII) (1–4). The possible roles of inorganic carbon, , in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO₂ and therefore has to be based on the photolysis of H₂CO₃ (“Kohlensäure”) and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6–8). Several research groups followed up on these initial results and identified two possible sites of C_i interaction within PSII (reviewed in refs. 9–12). Functional and spectroscopic studies showed that facilitates the reduction of the secondary plastoquinone electron acceptor (Q_B) of PSII by participating in the protonation of . Binding of (or ) to the nonheme Fe between the quinones Q_A and Q_B was recently confirmed by X-ray crystallography (3, 13, 14). Despite this functional role at the acceptor side, the very tight binding of to this site makes it impossible for the activity of PSII to be affected by changing the C_i level of the medium; instead inhibitors such as formate need to be added to induce the acceptor-side effect (15). Consequently, the water-splitting electron-donor side of PSII has also been studied intensively (for recent reviews, see refs. 11 and 12). Although a tight binding of C_i near the Mn₄CaO₅ cluster is excluded on the basis of X-ray crystallography (3, 14), FTIR spectroscopy (16), and mass spectrometry (17, 18), the possibility that a weakly bound affects the activity of PSII at the donor side remains a viable option (reviewed in refs. 10 and 19).In the present study using higher plant PSII membranes, we specifically evaluate a recently suggested role of weakly bound , namely, that it acts as an acceptor for, and transporter of, protons produced by water splitting in the OEC (20–22).     

Prognostic value of paroxysmal nocturnal haemoglobinuria clone presence in aplastic anaemia patients treated with combined immunosuppression: results of two‐centre prospective study

Paroxysmal nocturnal haemoglobinuria (PNH) clones are frequently detected in patients with aplastic anaemia (AA). To evaluate the prognostic role of PNH clone presence we conducted a prospective study in 125 AA patients treated with combined immunosuppressive therapy (IST). Seventy‐four patients (59%) had a PNH clone (PNH+ patients) at diagnosis, with a median clone size of 0·60% in granulocytes and 0·15% in red blood cells. The response rate at 6 months was higher in PNH+ patients than that in PNH‐ patients, both after first‐ and second‐line IST: 68% vs. 45%, P = 0·0164 and 53% vs. 13%, P = 0·0502 respectively. Moreover, 42% of PNH+ patients achieved complete remission compared with only 16% of PNH‐ patients (P = 0·0029). In multivariate logistic regression analysis, PNH clone presence (odds ratio 2·56, P = 0·0180) and baseline absolute reticulocyte count (ARC) ≥30 × 10⁹/l (odds ratio 5·19, P = 0·0011) were independent predictors of response to treatment. Stratification according to PNH positivity and ARC ≥30 × 10⁹/l showed significant distinctions for cumulative incidence of response, overall and failure‐free survival. The results of this prospective study confirmed the favourable prognostic value of PNH clone presence in the setting of IST for AA.     

Bypass of the pre-60S ribosomal quality control as a pathway to oncogenesis

Ribosomopathies are a class of diseases caused by mutations that affect the biosynthesis and/or functionality of the ribosome. Although they initially present as hypoproliferative disorders, such as anemia, patients have elevated risk of hyperproliferative disease (cancer) by midlife. Here, this paradox is explored using the rpL10-R98S (uL16-R98S) mutant yeast model of the most commonly identified ribosomal mutation in acute lymphoblastic T-cell leukemia. This mutation causes a late-stage 60S subunit maturation failure that targets mutant ribosomes for degradation. The resulting deficit in ribosomes causes the hypoproliferative phenotype. This 60S subunit shortage, in turn, exerts pressure on cells to select for suppressors of the ribosome biogenesis defect, allowing them to reestablish normal levels of ribosome production and cell proliferation. However, suppression at this step releases structurally and functionally defective ribosomes into the translationally active pool, and the translational fidelity defects of these mutants culminate in destabilization of selected mRNAs and shortened telomeres. We suggest that in exchange for resolving their short-term ribosome deficits through compensatory trans-acting suppressors, cells are penalized in the long term by changes in gene expression that ultimately undermine cellular homeostasis.Ribosomopathies are a family of congenital diseases that are linked to genetic defects in ribosomal proteins or ribosome biogenesis factors. They are characterized by pleiotropic abnormalities that include birth defects, heart and lung diseases, connective tissue disorders, anemia, ataxia, and mental retardation (reviewed in ref. 1). Although each ribosomopathy presents a unique pathological spectrum, the inherited forms are characterized by bone marrow failure and anemia early in life, followed by elevated cancer risk by middle age. For example, although childhood anemia is one of the cardinal symptoms of the genetically inherited disease Diamond–Blackfan anemia, these patients have a fivefold higher lifetime risk of cancer than the general population and a 30- to 40-fold higher risk of developing acute myeloid leukemia, osteosarcoma, or colon cancer (reviewed in refs. 2, 3). Similarly, patients with X-linked dyskeratosis are predisposed to myeloid leukemia and a variety of solid tumors (4), whereas patients with 5q− syndrome are at higher risk of developing acute myeloid leukemia (reviewed in ref. 5). In the genetically tractable zebrafish model, heterozygous loss-of-function mutations in several ribosomal proteins cause development of peripheral nerve sheet tumors (6). Somatically acquired mutations in ribosomal proteins are also implicated in cancer: ∼10% of children with T-cell acute lymphoblastic leukemia (T-ALL) were found to harbor somatic mutations in the ribosomal protein of the large subunit (LSU) 10, 5, and 22 (RPL10, RPL5, and RPL22) (7). [Note that the proteins encoded by these genes are also named uL16, uL18, and eL22, respectively, under the newly proposed uniform ribosomal protein nomenclature (8).] A separate study identified heterozygous deletions in the region of chromosome 1p that contains RPL22 (eL22) in an additional 10% of patients with T-ALL (9). The model of ribosomal proteins as targets for somatic mutations in cancer is further supported by the finding that two ribosomal protein genes (RPL5/uL18 and RPL22/eL22) are included in the list of 127 genes identified as significantly mutated in cancer in the context of the first Cancer Genome Atlas pan-cancer analysis in 12 tumor types (10).Ribosomopathies present an intriguing paradox: Although patients initially present with hypoproliferative disorders (e.g., anemias, bone marrow failure), those who survive to middle age often develop hyperproliferative diseases (i.e., cancers). The link between ribosome defects and hypoproliferative disease phenotypes has been extensively studied: The current working hypothesis is that impaired ribosome biogenesis activates a “ribosomal stress” cascade, activating the cellular TP53 pathway and resulting in cell cycle arrest and cell death (11). However, activation of TP53 does not explain why ribosomal defects are associated with hyperproliferative diseases, particularly cancer. Mutations in the ribosomal protein gene RPL10/uL16 were recently identified in patients with T-ALL (7). The T-ALL–associated RPL10/uL16 mutations occurred almost exclusively in residue arginine 98 (R98), with the exception of one patient harboring the Q123P mutation, which lies adjacent to R98 within the rpL10/uL16 3D structure (Fig. 1). Both residues are at the base of an essential flexible loop in rpL10 that closely approaches the peptidyltransferase center in the catalytic core in the ribosome (12). In addition to its role in catalysis (13, 14), rpL10/uL16 plays an important role in the late stages of 60S subunit biogenesis. After initial production of the separate ribosomal subunits in the nucleus, immature and functionally inactive pre-60S subunits are exported to the cytoplasm, where they undergo additional maturation events (15), including incorporation of rpL10/uL16, before they can associate with mature 40S subunits and engage in protein synthesis (16). Among the critical set of final 60S maturation steps is the release of the antiassociation factor Tif6, followed by release of Nmd3, the primary export adaptor for the pre-60S subunit in yeast and in humans (17, 18). Tif6 release requires the tRNA structural mimic Sdo1p (19) and the GTPase Efl1, a paralog of eukaryotic elongation factor 2 (eEF2) (20). We have suggested that structural rearrangements of the internal loop of rpL10/uL16 coordinate this final maturation process, resulting in a test drive of the pre-60S subunit to ensure that only properly functioning subunits are allowed to enter the pool of translationally active ribosomes (13, 21). Defective ribosomes carrying mutations in rpL10/uL16 specifically fail in this test drive, leading to their degradation through a molecular pathway that is yet to be characterized. Beyond 60S maturation, rpL10/uL16 plays an important role in coordinating intersubunit rotation and controlling allosteric rearrangements within the ribosome, helping to ensure the directionality and fidelity of protein synthesis (13).Open in a separate windowFig. 1.Localization of rpL10 and the loop in the LSU. (A) rpL10/uL16 in the context of the crown view of the LSU. (B) Close-up view of rpL10/uL16 and the local environment. The flexible loop structure is indicated by dashed red lines, and the positions of R98 and Q123 are indicated. rpL10/uL16 is situated between helices 38 and 89, and it is located in close proximity to several functional centers of the LSU, including the peptidyltransferase center (PTC), aa-tRNA accommodation corridor, and elongation factor binding site. Images were generated using PyMOL.rpL10/uL16 is highly conserved among eukaryotes: The yeast and human proteins are interchangeable, and residue 98 is invariantly an arginine (22). Human RPL10/uL16 is located on the X chromosome, and is therefore expressed as a single-copy gene in males. Thus, the haploid yeast model is an excellent mimic of the situation in the cells of a patient with T-ALL. Yeast cells expressing rpl10-R98S, rpl10-R98C, and rpl10-H123P (corresponding to Q123 in human rpL10/uL16) as the sole forms of rpL10/uL16 displayed proliferative defects. Further, polysome profiling revealed increased ratios of free 60S and 40S subunits vs. monosomes, markedly reduced polysomes, and the presence of halfmers in these mutants, suggesting defects in both ribosome biogenesis and subunit joining (7). Tif6 and Nmd3 both accumulated in the cytoplasm in the mutant cells, indicating a defect in their release from the cytoplasmic 60S (7). Thus, all of the rpl10/uL16 mutations appeared to affect 60S biogenesis at the Efl1-dependent quality control step. Consistent with the yeast-based observations, mouse lymphoid cells expressing rpl10-R98S displayed slower proliferation rates than cells expressing WT RPL10/uL16 and conferred defective polysome profiles (7).The studies presented in the current report use the yeast rpl10-R98S mutant to elucidate the structural, biochemical, and trans-lational fidelity defects that may lead to carcinogenesis. This mutant perturbs the structural equilibrium of ribosomes toward the “rotated state.” At the biochemical level, this underlying structural defect alters the affinity of mutant ribosomes for a specific set of trans-acting ligands. In turn, the biochemical defects affect translational fidelity, promoting elevated rates of −1 programmed ribosomal frameshifting (−1 PRF) and impaired recognition of termination codons. Globally increased rates of −1 PRF result in a decreased abundance of cellular mRNAs that harbor operational −1 PRF signals (23, 24). These −1 PRF signal-containing mRNAs include EST1, EST2, STN1, and CDC13, which play central roles in yeast telomere maintenance (23). In rpl10-R98S cells, the steady-state abundances of these mRNAs are decreased, resulting in telomere shortening. A spontaneously acquired trans-acting mutant suppresses the ribosome biogenesis defects of the rpl10-R98S mutant, thereby reestablishing high levels of ribosome production and cell proliferation. Importantly, however, suppression of the biogenesis and growth impairment defects fails to suppress the profound structural, biochemical, and translational fidelity defects of rpL10-R98S ribosomes. These findings suggest that suppression of the growth defect results from bypassing the test drive. Although the suppressor mutation enables cells to grow at normal rates, genetic suppression comes at the cost of releasing functionally defective ribosomes into the translationally active pool. We propose two different but not mutually exclusive models for how somatically acquired rpL10/uL16 mutations may promote cancer: (i) Mutant ribosomes may drive altered gene expression programs, promoting T-ALL, or (ii) the suppressor mutations may themselves be the drivers of T-ALL.     

Functional conservation despite structural divergence in ligand-responsive RNA switches

An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.Internal ribosome entry site (IRES) elements provide an alternative mechanism for translation initiation by directing the assembly of functional ribosomes directly at the start codon in a process that does not require 5′ cap recognition or ribosomal scanning and that is independent of many host initiation factors (1–4). The genomes of Flaviviridae and Picornaviridae contain elements that share similarity with the archetypical hepatitis C virus (HCV) IRES in overall domain organization, but not sequence or details of secondary structure (5). The HCV IRES adopts a complex architecture of four independently folding domains (Fig. 1A) (6). Domain II is nearly 100% conserved in clinical isolates (7) and has analogous counterparts in other viral IRES elements, all of which display some secondary structure similarity, but significant sequence variation in the subdomain IIa-like internal loop (Fig. 1B). Domain II has been shown to promote stable entry of HCV and classic swine fever virus (CSFV) mRNA at the decoding groove of the 40S subunit (8–10) and is required for initiation factor removal before ribosomal subunit joining (11), as well as adjustment of initiator tRNA orientation (12). The transition from initiation to elongation stages of translation depends critically on domain II (13). Recently, direct interaction of HCV domain II with initiator tRNA has been demonstrated (14). In HCV, subdomain IIa folds into an L-shaped motif (15) (Fig. 1C) that introduces a 90° bend in domain II (16) and directs the IIb hairpin toward the E-site at the ribosomal subunit interface (17, 18).Open in a separate windowFig. 1.Structures and ligands of viral IRES. (A) The IRES in the 5′ UTR of the HCV genome. The location of subdomain IIa is highlighted by an orange box. The viral genome encodes structural (S) and nonstructural (NS) proteins and contains a structured 3′ UTR. (B) Secondary structure predictions of domain II motifs in viral IRES elements from HCV and other flaviviruses, including CSFV and BVDV, as well as picornaviruses such as AEV and SVV. Non-Watson–Crick base pairs are indicated by the ○ symbol. Sequence conservation is indicated in red. (C) Crystal structure of the subdomain IIa RNA from HCV. (D) Benzimidazole (1) and diaminopiperidine (2) inhibitors of IRES-driven translation that target the HCV subdomain IIa. (E) Crystal structure of the HCV subdomain IIa RNA in complex with inhibitor 1.The HCV IRES subdomain IIa is the target for viral translation inhibitors (Fig. 1D) that bind to the internal loop and block translation by capturing distinct conformational states of the RNA (7). Structure analysis revealed that benzimidazole inhibitors such as compound 1 (19, 20) interact with an extended architecture of IIa in which the stems flanking the internal loop are coaxially stacked on both sides of the ligand-binding pocket (Fig. 1E) (21). In contrast, diaminopiperidine compounds such as 2 bind and lock the IIa RNA in a bent conformation that corresponds to the ligand-free state (22). Conformational capture of the subdomain IIa switch by ligands in solution was demonstrated by FRET experiments and established as a mechanism of IRES inhibition (23). On the basis of these findings, it was proposed that subdomain IIa may be the target for a cognate biological ligand whose adaptive recognition by the RNA motif may facilitate ribosome release from the IRES-bound complex (7).Here, we have explored potential candidates for a cognate ligand of the subdomain IIa switch and investigated the structural and functional conservation of similar ligand responsive switch motifs in other IRES RNAs.     

From the Cover: Global view of the protein universe

To explore protein space from a global perspective, we consider 9,710 SCOP (Structural Classification of Proteins) domains with up to 70% sequence identity and present all similarities among them as networks: In the “domain network,” nodes represent domains, and edges connect domains that share “motifs,” i.e., significantly sized segments of similar sequence and structure. We explore the dependence of the network on the thresholds that define the evolutionary relatedness of the domains. At excessively strict thresholds the network falls apart completely; for very lax thresholds, there are network paths between virtually all domains. Interestingly, at intermediate thresholds the network constitutes two regions that can be described as “continuous” versus “discrete.” The continuous region comprises a large connected component, dominated by domains with alternating alpha and beta elements, and the discrete region includes the rest of the domains in isolated islands, each generally corresponding to a fold. We also construct the “motif network,” in which nodes represent recurring motifs, and edges connect motifs that appear in the same domain. This network also features a large and highly connected component of motifs that originate from domains with alternating alpha/beta elements (and some all-alpha domains), and smaller isolated islands. Indeed, the motif network suggests that nature reuses such motifs extensively. The networks suggest evolutionary paths between domains and give hints about protein evolution and the underlying biophysics. They provide natural means of organizing protein space, and could be useful for the development of strategies for protein search and design.How are proteins related to each other? Which physicochemical considerations affect protein evolution and how? A global view of the protein universe may shed light on these fundamental questions. It could also suggest new strategies for protein search and design (1–3). However, forming a global picture of the protein universe is difficult because we have to piece it together from the many local glimpses that our empirical data and computational tools provide. In other words, a global picture needs to portray the relationships among all proteins, yet we only have evidence of such relationships among several proteins, based on the similarity between their sequences, structures, and functions. The considerable size of the Protein Data Bank (4) also complicates this task.In particular, an intensely debated question is whether protein space is “discrete” or “continuous” (2, 3, 5–10). These terms are loosely defined. Discrete implies that the global picture consists of separate, island-like, structural entities. In the hierarchical protein domains Structural Classification of Proteins (SCOP) (11) these entities are termed “folds,” and in the CATH database (12) they are called “topologies.” Alternatively, “continuous” implies that the space between these entities is generally populated by cross-fold similarities (e.g., refs. 2, 5, 6, 9, 13–15). If such similarities are abundant, then one must account for them when organizing and searching proteins (5, 8, 16). In support of the abundance of such similarities is the remarkable success of structure prediction methods that piece together predictions of protein fragments or larger protein segments (e.g., ref. 17).There are different approaches to forming a global view of the protein universe (18). The most significant efforts are the ones embodied in the hierarchical classifications CATH and SCOP. However, a hierarchy implicitly assumes that there are isolated regions in protein space. An alternative approach is to study the protein universe via maps––where domains are represented by points in two or three dimensions, placed so that the distances between them depend on the dissimilarity between their corresponding domains (e.g., refs. 19–21). By coloring the points according to domain characteristics, one can visually identify global properties of the protein universe (19, 20). However, a map representation in low-dimensional Euclidean space implicitly suggests that similarity among domains is transitive (i.e., that similarity within the pairs AB and BC implies that AC is similar too); we know that this is often not the case (6). Finally, a third approach to study protein space is via similarity and cooccurrence networks. In similarity networks, nodes typically represent protein domains and edges connect similar domains. Several successful studies of protein space capitalize on such networks (22, 23). Cooccurrence networks of protein domains, in which nodes represent domains and edges connect cooccurring domains, were also studied to better understand protein evolution (24–26).Here, we study the global nature of the protein universe using domain and motif networks (Fig. 1). To construct these networks, we identify evolutionary relationships among a representative set of SCOP domains; we relate two domains if they share a significantly sized part (denoted motif) with similar structure and sequence. Our analysis reveals that protein space is both discrete and continuous: SCOP domains of the all-alpha, all-beta, and alpha + beta classes, in which alpha and beta elements do not mix, mostly populate the discrete parts, whereas alpha/beta domains, with alternating alpha and beta segments, mostly populate the continuous ones. We also find that recurring motifs are very abundant; the motifs from the all-alpha and alpha/beta domains are the more abundant, and the more gregarious ones.Open in a separate windowFig. 1.Constructing the domain and motif networks. (A) The aligned protein segments, marked in colors, are the motifs. (B) In the domain network, edges connect domains that share similar motifs (e.g., domain d1wjga_ and d1vlua_ that share the cyan motif). (C) In the motif network, edges connect cooccurring motifs (e.g., the orange and cyan motifs cooccur in the d1vlua_ domain).     

Mechanisms and clinical significance of BIM phosphorylation in chronic lymphocytic leukemia

B-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIM(EL) and BIM(L), in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIM(EL) only. In CLL, anti-IgM-induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIM(EL) serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIM(EL)/BIM(L) phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIM(EL) phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.     

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane‐dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel‐filtered platelets, intact or loaded with calcium‐sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine‐positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine‐negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha‐granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine‐positive platelets formation several‐fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate‐limiting, for subpopulation segregation. Inhibition of mitogen‐activated protein kinase kinase (U0126) and glycoprotein IIb‐IIIa (Monafram^®) was without effect, whereas inhibitors of phosphatidylinositol 3‐kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.