Inherited thrombocytopenia associated with a variant in the FLI1 binding site in the 5' UTR of ANKRD26.

Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.

A novel RNF125 variant associated with Tenorio syndrome alters ubiquitin chain binding.

A key signalling pathway required for clearance of viruses from host cells relies on the receptor protein, retinoic acid-inducible gene I (RIG-I). The activity of RIG-I is tightly controlled, and once bound to viral dsRNA, addition of lysine 63-linked ubiquitin chains activates signalling. Meanwhile, the addition of lysine 48-linked ubiquitin chains to RIG-I is required to terminate signalling when the infection has been resolved. Really interesting new gene (RING) finger protein 125 (RNF125) is the E3 ligase responsible for addition of the ubiquitin chains that terminate signalling, with disruption of its function associated with Tenorio syndrome. Here we describe a novel RNF125 gene variant in an individual with clinical symptoms including intellectual disability, macrocephaly and congenital heart disease, consistent with Tenorio syndrome. The newly identified Tenorio syndrome-associated variant [(NM_017831.4):c.670G>C p.Glu224Gln] is the first to be found in the ubiquitin interaction motif (UIM) of RNF125. While the E3 ligase activity of this RNF125 variant is retained, it has an impaired ability to interact with lysine 63-linked ubiquitin chains. The function of the UIM in RNF125 is uncertain; however, this study suggests that the UIM binds lysine 63-linked ubiquitin chains, and that this interaction is required for the normal function of RNF125.

Altered conformational dynamics contribute to species-specific effects of cytochrome c mutations on caspase activation.

Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.

A novel frameshift GP1BB mutation causes autosomal dominant macrothrombocytopenia with decreased vWF receptor expression but normal platelet aggregation.

GP1bß is a component of the von Willebrand factor (vWF) receptor complex that is necessary for platelet formation and activation. A novel frameshift variant in GP1BB has been identified in a family with macrothrombocytopenia. The variant leads to a protein that is 101 amino acids longer than wild type with loss of the transmembrane domain. As there is no defect in platelet aggregation, the family are classified as heterozygous carriers of a Bernard-Soulier syndrome-related mutation. The levels of the vWF receptor on platelets are reduced to 50% of the controls, with the presence of large platelets but normal platelet aggregation demonstrating that decreased vWF receptor expression impacts proplatelet formation but not platelet function.

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity.

Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.

Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1.

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.

Megakaryocytes from CYCS mutation-associated thrombocytopenia release platelets by both proplatelet-dependent and -independent processes.

Thrombocytopenia Cargeeg is a rare autosomal dominant disorder and one of three thrombocytopenias caused by mutation of cytochrome c (Online Mendelian Inheritance in Man entry THC4). Our previous observations of platelet-like structures in the marrow space and early platelet production in vitro suggested that the low platelet phenotype in Thrombocytopenia Cargeeg subjects is caused by premature release of platelets into non-vascular regions of the bone marrow. We now show that two processes of platelet release occur in Thrombocytopenia Cargeeg subjects. Circulating platelets have a normal marginal microtubule coil, and cultured megakaryocytes derived from peripheral blood cells of Thrombocytopenia Cargeeg subjects form proplatelets normally and release platelets containing a marginal microtubule coil, consistent with effective platelet release via the proplatelet mechanism. In contrast, platelet-like structures within the extravascular bone marrow space have the dimensions of platelets but lack the marginal microtubule coil, suggesting abnormal proplatelet-independent platelet release. The mechanism of extravascular platelet release remains unclear. The failure to recapitulate this mechanism in vitro implies that the phenotype is not simply an intrinsic property of CYCS mutation-carrying megakaryocytes, but is dependent on the interaction between these cells and their environment.

The role of peroxiredoxin 1 in redox sensing and transducing.

Peroxiredoxin 1 is a member of the ubiquitous peroxiredoxin family of thiol peroxidases that catalyse the reduction of peroxides. In recent years eukaryotic peroxiredoxins have emerged as a critical component of cellular redox signalling, particularly in response to alterations in production of hydrogen peroxide. Peroxiredoxins are exquisitely sensitive to oxidation by hydrogen peroxide making them key peroxide sensing enzymes within cells. Evidence gathered over the last decade suggests that in addition to sensing the redox signal, peroxiredoxins have a major role in transducing this signal to downstream signalling proteins, ultimately contributing to regulation of diverse cellular processes including proliferation, differentiation and apoptosis. In this review we present the three current models for the sensing and signal transducing roles of peroxiredoxins, with a specific focus on mammalian peroxiredoxin 1. The evidence for each mechanism is discussed and areas for future work are identified.

Enhancing the peroxidase activity of cytochrome c by mutation of residue 41: implications for the peroxidase mechanism and cytochrome c release.

The peroxidase activity of cytochrome c may play a key role in the release of cytochrome c from the mitochondrial intermembrane space in the intrinsic apoptosis pathway. Induction of the peroxidase activity of cytochrome c is ascribed to partial unfolding and loss of axial co-ordination between the haem Fe and Met80, and is thought to be triggered by interaction of cytochrome c with cardiolipin (diphosphatidylglycerol) in vivo. However, the reaction mechanism for the peroxidase activity of either native or cardiolipin-bound cytochrome c is uncertain. In the present study we analyse the peroxidase activity of human and mouse cytochrome c residue 41 variants and demonstrate that stimulation of peroxidase activity can occur without prior loss of Fe-Met80 co-ordination or partial unfolding. The effects of cardiolipin and mutation of residue 41 are not additive, suggesting that cardiolipin stimulates peroxidase activity by the same mechanism as residue 41 mutation. Consistent with this, mutation of residue 41 did not enhance apoptotic release of cytochrome c from mitochondria. We propose that mutation of residue 41, and interaction with cardiolipin, increase peroxidase activity by altering the 40-57 Ω loop and its hydrogen bond network with the propionate of haem ring A. These changes enhance access of hydrogen peroxide and substrate to the haem.

The Y49H cytochrome c variant enhances megakaryocytic maturation of K-562 cells.

Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus.

Conformational change and human cytochrome c function: mutation of residue 41 modulates caspase activation and destabilizes Met-80 coordination.

Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK (a) of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.

Insights into H2O2-induced signaling in Jurkat cells from analysis of gene expression.

Hydrogen peroxide (H2O2) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2O2 reversibly oxidizes thiol groups of cysteine residues to mediate intracellular signaling. While examples of H2O2-dependent signaling have been reported, the exact molecular mechanism(s) of signaling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2O2 was investigated to determine global effects on gene expression. With a low H2O2 concentration (10 µM) that did not induce an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 h (6803 differentially expressed genes). Of the genes with a greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signaling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2O2, and the molecular mechanisms of H2O2 signaling.

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: implications for mitochondria-specific therapies and probes.

BACKGROUND: Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential. CONCLUSIONS: Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function. METHODS: To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration. RESULTS: AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10-30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration. GENERAL SIGNIFICANCE: These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.

The proapoptotic G41S mutation to human cytochrome c alters the heme electronic structure and increases the electron self-exchange rate.

The naturally occurring G41S mutation to human (Hs) cytochrome (cyt) c enhances apoptotic activity based upon previous in vitro and in vivo studies, but the molecular mechanism underlying this enhancement remains unknown. Here, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and density functional theory (DFT) calculations have been used to identify the structural and electronic differences between wild-type (WT) and G41S Hs cyt c. S41 is part of the hydrogen bonding network for propionate 7 of heme pyrrole ring A in the X-ray structure of G41S Hs cyt c and, compared to WT, G41S Hs cyt c has increased spin density on pyrrole ring C and a faster electron self-exchange rate. DFT calculations illustrate an electronic mechanism where structural changes near ring A can result in electronic changes at ring C. Since ring C is part of the solvent-exposed protein surface, we propose that this heme electronic structure change may ultimately be responsible for the enhanced proapoptotic activity of G41S Hs cyt c.

Congenital thrombocytopenia and cytochrome C mutation: a matter of birth and death.

Thrombocytopenia (TP) Cargeeg is a unique autosomal dominant disorder, affecting a seven-generation family, caused by cytochrome C (CYCS) mutation that dysregulates platelet formation. The CYCS mutation in this disorder is a glycine 41 replacement by serine, which yields a cytochrome C variant with enhanced apoptotic pathway activity in vitro. The deregulated apoptosis in this disorder affects megakaryocytes (MK) during platelet formation, leading to early and ectopic platelet release in the bone marrow (BM). Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome C activity is not a critical regulator of physiological apoptosis in most cells. The pathophysiology of this unique inherited TP, with unaltered platelet survival and normal MK content in the BM, has implications for physiological and pathological mechanisms altering MK apoptosis, with implications for other unexplained thrombocytopenic disorders.

The Structure and Ubiquitin Binding Properties of TRAF RING Heterodimers.

Tumour necrosis factor (TNF) receptor associated factor (TRAF) family members share a common domain architecture, but play non-redundant physiological roles in cell signalling. At the N terminus, most TRAFs have a RING domain, followed by a series of Zinc finger (ZF) domains. The RING domain of TRAF6 dimerizes, and the RING homodimer together with the first ZF assembles ubiquitin chains that form a platform which facilitates activation of downstream kinases. The RING dimer interface is conserved amongst TRAF proteins, suggesting that functional heterodimers could be possible. Here we report the structure of the TRAF5-TRAF6 RING heterodimer, which accounts for the stability of the heterodimer as well as its ability to assemble ubiquitin chains. We also show that the RING domain of TRAF6 heterodimerizes with TRAF3 and TRAF2, and demonstrate that the linker helix and first ZF of TRAF2 can cooperate with TRAF6 to promote chain assembly. Collectively our results suggest that TRAF RING homo- and hetero-dimers have the potential to bridge interaction of nearby TRAF trimers and modulate TRAF-mediated signalling.

Targeting the apoptosome for cancer therapy.

Apoptosis is a programmed mechanism of cell death that ensures normal development and tissue homeostasis in metazoans. Avoidance of apoptosis is an important contributor to the survival of tumor cells, and the ability to specifically trigger tumor cell apoptosis is a major goal in cancer treatment. In vertebrates, numerous stress signals engage the intrinsic apoptosis pathway to induce the release of cytochrome c from mitochondria. Cytochrome c binds to apoptosis protease activating factor-1, triggering formation of the apoptosome, a multisubunit protein complex that serves as a platform for caspase activation. In this review we summarize the mechanisms of apoptosome assembly and activation, and our current understanding of the regulation of these processes. We detail the evidence that loss-of-function of the apoptosome pathway may contribute to the development of specific cancers. Finally we discuss recent results showing enhanced sensitivity of some tumor cells to cytochrome c-induced apoptosis, suggesting that agents able to directly or indirectly trigger apoptosome-catalyzed caspase activation in tumor cells could provide new approaches to cancer treatment.

The orf virus inhibitor of apoptosis functions in a Bcl-2-like manner, binding and neutralizing a set of BH3-only proteins and active Bax.

We have previously shown that the Orf virus protein, ORFV125, is a potent inhibitor of the mitochondrial pathway of apoptosis and displays rudimentary sequence similarities to cellular anti-apoptotic Bcl-2 proteins. Here we investigate the proposal that ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. We show that the viral protein interacted with a range of BH3-only proteins (Bik, Puma, DP5, Noxa and all 3 isoforms of Bim) and neutralized their pro-apoptotic activity. In addition, ORFV125 bound to the active, but not the inactive, form of Bax, and reduced the formation of Bax dimers. Mutation of specific amino acids in ORFV125 that are conserved and functionally important in mammalian Bcl-2 family proteins led to loss of both binding and inhibitory functions. We conclude that ORFV125's mechanism of action is Bcl-2-like and propose that the viral protein's combined ability to bind to a range of BH3-only proteins as well as the active form of Bax provides significant protection against apoptosis. Furthermore, we demonstrate that the binding profile of ORFV125 is distinct to that of other poxviral Bcl-2-like proteins.

Oxidation of mitochondrial peroxiredoxin 3 during the initiation of receptor-mediated apoptosis.

It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-alpha. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis.