Heterotetramer Formation Research Articles

Hsp90 chaperones hemoglobin maturation in erythroid and nonerythroid cells

Maturation of adult (α2β2) and fetal hemoglobin (α2γ2) tetramers requires that heme be incorporated into each globin. While hemoglobin alpha (Hb-α) relies on a specific erythroid chaperone (alpha Hb-stabilizing protein, AHSP), the other chaperones that may help mature the partner globins (Hb-γ or Hb-β) in erythroid cells, or may enable nonerythroid cells to express mature Hb, are unknown. We investigated a role for heat-shock protein 90 (hsp90) in Hb maturation in erythroid precursor cells that naturally express Hb-α with either Hb-γ (K562 and HiDEP-1 cells) or Hb-β (HUDEP-2) and in nonerythroid cell lines that either endogenously express Hb-αβ (RAW and A549) or that we transfected to express the globins. We found the following: (i) AHSP and hsp90 associate with distinct globin partners in their immature heme-free states (AHSP with apo-Hbα, and hsp90 with apo-Hbβ or Hb-γ) and that hsp90 does not associate with mature Hb. (ii) Hsp90 stabilizes the apo-globins and helps to drive their heme insertion reactions, as judged by pharmacologic hsp90 inhibition or by coexpression of an ATP-ase defective hsp90. (iii) In nonerythroid cells, heme insertion into all globins became hsp90-dependent, which may explain how mixed Hb tetramers can mature in cells that do not express AHSP. Together, our findings uncover a process in which hsp90 first binds to immature, heme-free Hb-γ or Hb-β, drives their heme insertion process, and then dissociates to allow their heterotetramer formation with Hb-α. Thus, in driving heme insertion, hsp90 works in concert with AHSP to generate functional Hb tetramers during erythropoiesis.

Molecular mechanism of environmental d-xylose perception by a XylFII-LytS complex in bacteria

d-xylose, the main building block of plant biomass, is a pentose sugar that can be used by bacteria as a carbon source for bio-based fuel and chemical production through fermentation. In bacteria, the first step for d-xylose metabolism is signal perception at the membrane. We previously identified a three-component system in Firmicutes bacteria comprising a membrane-associated sensor protein (XylFII), a transmembrane histidine kinase (LytS) for periplasmic d-xylose sensing, and a cytoplasmic response regulator (YesN) that activates the transcription of the target ABC transporter xylFGH genes to promote the uptake of d-xylose. The molecular mechanism underlying signal perception and integration of these processes remains elusive, however. Here we purified the N-terminal periplasmic domain of LytS (LytSN) in a complex with XylFII and determined the conformational structures of the complex in its d-xylose-free and d-xylose-bound forms. LytSN contains a four-helix bundle, and XylFII contains two Rossmann fold-like globular domains with a xylose-binding cleft between them. In the absence of d-xylose, LytSN and XylFII formed a heterodimer. Specific binding of d-xylose to the cleft of XylFII induced a large conformational change that closed the cleft and brought the globular domains closer together. This conformational change led to the formation of an active XylFII-LytSN heterotetramer. Mutations at the d-xylose binding site and the heterotetramer interface diminished heterotetramer formation and impaired the d-xylose-sensing function of XylFII-LytS. Based on these data, we propose a working model of XylFII-LytS that provides a molecular basis for d-xylose utilization and metabolic modification in bacteria.

The dominant-negative interplay between p53, p63 and p73: A family affair.

The tumor suppression activity of p53 is frequently impaired in cancers even when a wild-type copy of the gene is still present, suggesting that a dominant-negative effect is exerted by some of p53 mutants and isoforms. p63 and p73, which are related to p53, have also been reported to be subjected to a similar loss of function, suggesting that a dominant-negative interplay might happen between p53, p63 and p73. However, to which extent p53 hotspot mutants and isoforms of p53, p63 and p73 are able to interfere with the tumor suppressive activity of their siblings as well as the underlying mechanisms remain undeciphered. Using yeast, we showed that a dominant-negative effect is widely spread within the p53/p63/p73 family as all p53 loss-of-function hotspot mutants and several of the isoforms of p53 and p73 tested exhibit a dominant-negative potential. In addition, we found that this dominant-negative effect over p53 wild-type is based on tetramer poisoning through the formation of inactive hetero-tetramers and does not rely on a prion-like mechanism contrary to what has been previously suggested. We also showed that mutant p53-R175H gains the ability to inhibit p63 and p73 activity by a mechanism that is only partially based on tetramerization.

Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells.

Missense mutation of tumor suppressor p53, which exhibits oncogenic gain-of-function (GOF), not only promotes tumor progression, but also diminishes therapeutic efficacies of cancer treatments. However, it remains unclear how a p53 missense mutant contributes to induced pluripotency of cancer stem cells (CSCs) in tumors exposed to chemotherapeutic agents. More importantly, it may be possible to abrogate the GOF by restoring wild-type p53 activity, thereby overcoming the deleterious effects resulting from heterotetramer formation, which often compromises the efficacies of current approaches being used to reactivate p53 function. Herewith, we report that p53 R273H missense mutant urges cancer cells to spawn CSCs. SW48/TP53 cells, which heterozygously carry the p53 R273H hot-spot mutant (R273H/+, introduced by a CRISPR/Casp9 system), were subchronically exposed to doxorubicin in cell culture and in tumor-bearing mice. We found that p53-R273H (TP53-Dox) cells were drug-resistant and exhibited epithelial-mesenchymal transition (EMT) and increased numbers of CSCs (CD44v6+/CD133+), which resulted in enhanced wound healing and tumor formation. Inhibition of glucosylceramide synthase with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) sensitized p53-R273H cancer cells and tumor xenografts to doxorubicin treatments. Intriguingly, PDMP treatments restored wild-type p53 expression in heterozygous R273H mutant cells and in tumors, decreasing CSCs and sensitizing cells and tumors to treatments. This study demonstrated that p53-R273H promotes EMT and induced pluripotency of CSCs in cancer cells exposed to doxorubicin, mainly through Zeb1 and β-catenin transcription factors. Our results further indicate that restoration of p53 through inhibition of ceramide glycosylation might be an effective treatment approach for targeting cancers heterozygously harboring TP53 missense mutations.

Structural Insights into the Polyphyletic Origins of Glycyl tRNA Synthetases

Glycyl tRNA synthetase (GlyRS) provides a unique case among class II aminoacyl tRNA synthetases, with two clearly widespread types of enzymes: a dimeric (α2) species present in some bacteria, archaea, and eukaryotes; and a heterotetrameric form (α2β2) present in most bacteria. Although the differences between both types of GlyRS at the anticodon binding domain level are evident, the extent and implications of the variations in the catalytic domain have not been described, and it is unclear whether the mechanism of amino acid recognition is also dissimilar. Here, we show that the α-subunit of the α2β2 GlyRS from the bacterium Aquifex aeolicus is able to perform the first step of the aminoacylation reaction, which involves the activation of the amino acid with ATP. The crystal structure of the α-subunit in the complex with an analog of glycyl adenylate at 2.8 Å resolution presents a conformational arrangement that properly positions the cognate amino acid. This work shows that glycine is recognized by a subset of different residues in the two types of GlyRS. A structural and sequence analysis of class II catalytic domains shows that bacterial GlyRS is closely related to alanyl tRNA synthetase, which led us to define a new subclassification of these ancient enzymes and to propose an evolutionary path of α2β2 GlyRS, convergent with α2 GlyRS and divergent from AlaRS, thus providing a possible explanation for the puzzling existence of two proteins sharing the same fold and function but not a common ancestor.

Genetics of Phenylketonuria: Then and Now.

More than 950 phenylalanine hydroxylase (PAH) gene variants have been identified in people with phenylketonuria (PKU). These vary in their consequences for the residual level of PAH activity, from having little or no effect to abolishing PAH activity completely. Advances in genotyping technology and the availability of locus-specific and genotype databases have greatly expanded our understanding of the correlations between individual gene variant, residual PAH activity, tetrahydrobiopterin (BH4 ) responsiveness, and the clinical PKU phenotype. Most patients (∼76%) have compound heterozygous PAH gene variants and one mutated allele may markedly influence the activity of the second mutated allele, which in turn may influence either positively or negatively the activity of the biologically active heterotetrameric form of the PAH. While it is possible to predict the level of BH4 responsiveness (∼71%) and PKU severity (∼78%) from the nature of the underlying gene variants, these relationships remain complex and incompletely understood. A greater understanding of these relationships may increase the potential for individualized management of PKU in future. Inherited deficiencies in BH4 metabolism account for about 1%-2% of all hyperphenylalaninemias and are clinically more severe than PKU. Almost 90% of all patients are deficient in 6-pyruvoyl-tetrahydropterin synthase and dihydropteridine reductase.

Licensing of Centromeric Chromatin Assembly through the Mis18α-Mis18β Heterotetramer

Centromeres are specialized chromatin domains specified by the centromere-specific CENP-A nucleosome. The stable inheritance of vertebrate centromeres is an epigenetic process requiring deposition of new CENP-A nucleosomes by HJURP. We show HJURP is recruited to centromeres through a direct interaction between the HJURP centromere targeting domain and the Mis18α-β C-terminal coiled-coil domains. We demonstrate Mis18α and Mis18β form a heterotetramer through their C-terminal coiled-coil domains. Mis18α-β heterotetramer formation is required for Mis18BP1 binding and centromere recognition. S. pombe contains a single Mis18 isoform that forms a homotetramer, showing tetrameric Mis18 is conserved from fission yeast to humans. HJURP binding disrupts the Mis18α-β heterotetramer and removes Mis18α from centromeres. We propose stable binding of Mis18 to centromeres in telophase licenses them for CENP-A deposition. Binding of HJURP deposits CENP-A at centromeres and facilitates the removal of Mis18, restricting CENP-A deposition to a single event per cell cycle.

Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

The ability of inositol 1,4,5-trisphosphate receptors (IP3R) to precisely initiate and generate a diverse variety of intracellular Ca(2+) signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP3, Ca(2+), and ATP). However, the contribution of IP3R heterotetramerization to Ca(2+) signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca(2+) signals. Under conditions where key regulators of IP3R function are optimal for Ca(2+) release, we demonstrate that individual monomers within heteromeric IP3Rs contributed equally toward generating a distinct 'blended' sensitivity to IP3 that is likely dictated by the unique IP3 binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP3R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP3R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP3-induced Ca(2+) signaling in cells expressing multiple IP3R isoforms.

Emerging Associations of the ALDH2*2 Polymorphism with Disease Susceptibility

Ethanol is metabolized by Alcohol Dehydrogenase (ADH) to acetaldehyde and then irreversibly oxidized by Aldehyde Dehydrogenase (ALDH) to nontoxic acetate. In individuals expressing the ALDH2*2 variant enzyme, the rate of conversion from acetaldehyde to acetate is reduced and leads to flushing, nausea, and tachycardia due to increased blood levels of acetaldehyde. The ALDH2*2 variant has a lowered NAD+ coenzyme binding affinity, which results in a lowered clearance capacity toward acetaldehyde. This polymorphism is caused by the substitution of glutamate for lysine at position 487 within the catalytic active site of ALDH2, resulting in effects on subunit and quaternary complex activity. ALDH2*2 alleles are dominant over ALDH2*1 and therefore are expected to contribute to the formation of inactive heterotetramers decreased enzymatic activity in both homozygous and heterozygous individuals. Consequently, a higher susceptibility to various diseases such as Alzheimer’s, osteoporosis, and acute coronary syndrome has been associated with ALDH2*2 carriers. Additionally, the polymorphism seems to affect the efficacy of Glyceryl Trinitrate (GTN), a drug intended to treat coronary heart disease, in carriers of ALDH2*2 alleles. However, the polymorphism is believed to afford a protective effect against alcoholism as the side effects of acetaldehyde build-up are undesirable. Disulfiram, a drug historically used to treat alcohol dependency, induces the same undesirable physiological effects as the variant enzyme in non-carriers by inhibiting the normal functioning of ALDH2 enzyme.

Structural Basis of Neurohormone Perception by the Receptor Tyrosine Kinase Torso

In insects, brain-derived Prothoracicotropic hormone (PTTH) activates the receptor tyrosine kinase (RTK) Torso to initiate metamorphosis through the release of ecdysone. We have determined the crystal structure of silkworm PTTH in complex with the ligand-binding region of Torso. Here we show that ligand-induced Torso dimerization results from the sequential and negatively cooperative formation of asymmetric heterotetramers. Mathematical modeling of receptor activation based upon our biophysical studies shows that ligand pulses are "buffered" at low receptor levels, leading to a sustained signal. By contrast, high levels of Torso develop the signal intensity and duration of a noncooperative system. We propose that this may allow Torso to coordinate widely different functions from a single ligand by tuning receptor levels. Phylogenic analysis indicates that Torso is found outside arthropods, including human parasitic roundworms. Together, our findings provide mechanistic insight into how this receptor system, with roles in embryonic and adult development, is regulated.

The interaction between delayed rectifier channel alpha-subunits does not involve hetero-tetramer formation.

We have previously reported a physiologically relevant interaction between KCNQ1 (Q1) and KCNH2 (H2). While the H2 C-terminus has been suggested to play a role, so far, no more detailed information regarding the interaction site is available. The methods used in the study are cell culture, PCR for mutagenesis, patch clamp for ion current recordings, co-immunoprecipitation for determination of protein interaction. Co-expression of Q1 and H2 resulted in an increase of I H2 (tails after +50mV; Q1 + H2, 36 ± 6pA/pF; H2, 14 ± 2pA/pF; n = 10; 12; P < 0.05). Upon expressing a non-conductive (dominant-negative) Q1-pore mutation (dnQ1), there was still an increase in I H2 (tails after +50mV; H2 + dnQ1, 24 ± 4pA/pF; n = 10; P < 0.05) making the pore region unlikely as an interaction site. Experiments using the KCNH2-pore blocking agent quinidine supported these findings. If Q1 and H2 formed hetero-tetramers, steric changes within the pore should change the quinidine half-inhibitory concentrations (IC50). However, I H2 sensitivity did not significantly change in the presence or absence of Q1 (IC50 341 ± 63 vs. 611 ± 293nmol/L, respectively, P = n.s.), providing further evidence that the pore is not a likely H2-Q1 interaction site. To obtain further insights into the role of intra-cytoplasmic structures, we used both C- and N-terminally truncated mutant H2 proteins. Both H2 mutants co-immunoprecipitated with Q1, suggesting no specific role of C- or N-termini. Accordingly, rather than these, the transmembrane domains of the α-subunits appear relevant for the interaction. Our results largely exclude the formation of hetero-tetramers between H2 and Q1 comprising the pore region or H2 C- or N-termini.

Remodeling of the AAA+ ATPase p97 by the UBX Adaptor Protein ASPL

The hexameric mammalian AAA+ ATPase p97, also known as VCP (valosin-containing protein; CDC48 in yeast), is a very abundant cytosolic protein and serves a wide variety of cellular functions. p97 is a central component in endoplasmic reticulum-associated degradation (ERAD) of proteins where it delivers ubiquitinated ERAD substrates to the proteasome. In addition, cellular roles of p97 in organelle membrane fusion, mitosis, DNA repair and suppression of apoptosis have been described. These different functions are linked to the binding of adaptor proteins to p97. Many of these adaptors contain ubiquitin regulatory X (UBX) domains. ASPL (alveolar soft part sarcoma locus, also known as TUG) was recenty identified as a p97 adaptor protein. As shown by crystal structure analysis, ASPL uses a substantially extended UBX domain for binding to the N domain of p97 where a lariat-like, mostly α-helical extension wraps around one subunit of p97. By this binding ASPL triggers the dissociation of functional p97 hexamers and the formation of p97:ASPL heterotetramers with 2:2 stoichiometry, leading to inactivation of the AAA+ ATPase. The p97-ASPL interaction in the heterotetramer is very tight, but p97 hexamer dissociation and heterotetramer formation may be suppressed by single-site mutations at p97-ASPL interfaces. p97 hexamer dissociation and p97-ASPL heterotetramer formation are linked to reduced ATPase activity of p97, cellular accumulation of ERAD substrates and apoptosis induction. To the best of our knowledge, this is the first time that the structural basis for adaptor protein-induced inactivation by hexamer dissociation of p97 and, indeed, any AAA+ ATPase has been demonstrated. This observation has far reaching implications for AAA+ ATPase-regulated processes.

Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1) Co-Expressing Cells.

Transient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1) receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1.

Heteromerization of PIP aquaporins affects their intrinsic permeability.

The plant aquaporin plasma membrane intrinsic proteins (PIP) subfamily represents one of the main gateways for water exchange at the plasma membrane (PM). A fraction of this subfamily, known as PIP1, does not reach the PM unless they are coexpressed with a PIP2 aquaporin. Although ubiquitous and abundantly expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here, we unravel how FaPIP1;1, a fruit-specific PIP1 aquaporin from Fragaria x ananassa, contributes to the modulation of membrane water permeability (Pf) and pH aquaporin regulation. Our approach was to combine an experimental and mathematical model design to test its activity without affecting its trafficking dynamics. We demonstrate that FaPIP1;1 has a high water channel activity when coexpressed as well as how PIP1-PIP2 affects gating sensitivity in terms of cytosolic acidification. PIP1-PIP2 random heterotetramerization not only allows FaPIP1;1 to arrive at the PM but also produces an enhancement of FaPIP2;1 activity. In this context, we propose that FaPIP1;1 is a key participant in the regulation of water movement across the membranes of cells expressing both aquaporins.

Allosteric Activation of Trypanosomatid Deoxyhypusine Synthase by a Catalytically Dead Paralog

Polyamine biosynthesis is a key drug target in African trypanosomes. The "resurrection drug" eflornithine (difluoromethylornithine), which is used clinically to treat human African trypanosomiasis, inhibits the first step in polyamine (spermidine) biosynthesis, a highly regulated pathway in most eukaryotic cells. Previously, we showed that activity of a key trypanosomatid spermidine biosynthetic enzyme, S-adenosylmethionine decarboxylase, is regulated by heterodimer formation with a catalytically dead paralog (a prozyme). Here, we describe an expansion of this prozyme paradigm to the enzyme deoxyhypusine synthase, which is required for spermidine-dependent hypusine modification of a lysine residue in the essential translation factor eIF5A. Trypanosoma brucei encodes two deoxyhypusine synthase paralogs, one that is catalytically functional but grossly impaired, and the other is inactive. Co-expression in Escherichia coli results in heterotetramer formation with a 3000-fold increase in enzyme activity. This functional complex is also present in T. brucei, and conditional knock-out studies indicate that both DHS genes are essential for in vitro growth and infectivity in mice. The recurrent evolution of paralogous, catalytically dead enzyme-based activating mechanisms may be a consequence of the unusual gene expression in the parasites, which lack transcriptional regulation. Our results suggest that this mechanism may be more widely used by trypanosomatids to control enzyme activity and ultimately influence pathogenesis than currently appreciated.

Inefficient TLR4/MD-2 Heterotetramerization by Monophosphoryl Lipid A

Synthetic forms of E. coli monophosphoryl lipid A (sMLA) weakly activate the MyD88 (myeloid differentiation primary response protein) branch of the bifurcated TLR4 (Toll-like receptor 4) signaling pathway, in contrast to diphosphoryl lipid A (sDLA), which is a strong activator of both branches of TLR4. sMLA’s weak MyD88 signaling activity is apparent downstream of TLR4/MyD88 signaling as we show that sMLA, unlike sDLA, is unable to efficiently recruit the TNF receptor-associated factor 6 (TRAF6) to the Interleukin-1 receptor-associated kinase 1 (IRAK1). This reduced recruitment of TRAF6 explains MLA’s lower MAPK (Mitogen Activated Protein Kinase) and NF-κB activity. As further tests of sMLA’s ability to activate TLR4/Myeloid differentiation factor 2 (MD-2), we used the antibody MTS510 as an indicator for TLR4/MD-2 heterotetramer formation. Staining patterns with this antibody indicated that sMLA does not effectively drive heterotetramerization of TLR4/MD-2 when compared to sDLA. However, a F126A mutant of MD-2, which allows lipid A binding but interferes with TLR4/MD-2 heterotetramerization, revealed that while sMLA is unable to efficiently form TLR4/MD-2 heterotetramers, it still needs heterotetramer formation for the full extent of signaling it is able to achieve. Monophosphoryl lipid A’s weak ability to form TLR4/MD-2 heterotetramers was not restricted to synthetic E. coli type because cells exposed to a biological preparation of S. minnesota monophosphoryl lipid A (MPLA) also showed reduced TLR4/MD-2 heterotetramer formation. The low potency with which sMLA and MPLA drive heterotetramerization of TLR4/MD-2 contributes to their weak MyD88 signaling activities.

Probing the histone binding requirements of the Hif1 histone chaperone, a component of the Hat1 chromatin assembly complex

Hif1 (Hat1‐interacting factor 1) is a component of the Hat1 chromatin assembly complex. This complex contains the Hat1 histone acetyltransferase enzyme, that specifically acetylates histone H4, and acts in a pathway that deposits newly synthesized histone H3 and histone H4 onto DNA during chromatin assembly. The Hat1 complex is highly conserved, found in organisms ranging from yeast to humans, and plays a role in gene regulation and DNA repair. Hif1 binds histones and has histone chaperone activity. Evidence suggests that the Hat1 complex acts in histone deposition upstream of the Asf1 histone chaperone. Asf1 interacts with a heterodimer of histones H3 and H4 and blocks formation of the heterotetrameric form of histones H3 and H4 found in chromatin. This raises the question of whether Hif1 binds to a similar H3–H4 heterodimer or to a heterotetrameric complex. This is especially intriguing as Hif1 forms oligomers. To test this question, we have created site‐directed mutants of histone H3 and produced mutant H3–H4 complexes in bacteria via co‐expression methods. Mutations were introduced at the H3–H3 interaction surface that destabilize heterotetramer formation and produce obligate heterodimers. We are currently analyzing the ability of Hif1 to interact with native and mutant histone complexes to assess whether it prefers to bind heterodimeric or heterotetrameric histone complexes.

Domains I and IV of Annexin A2 Affect the Formation and Integrity of In Vitro Capillary-Like Networks

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

Voltage- and Camp-Dependent Gating in Heterotetrameric HCN2/4-Pacemaker Channels

HCN pacemaker channels play an important role in generating and regulating rhythmicity of special neurons and cardiac cells. They are activated by hyperpolarizing voltages and modulated by the binding of cyclic nucleotides. Four isoforms, HCN1-HCN4, have been identified. HCN2 and HCN4, expressed in cardiac sinoatrial node and ventricular cells, build functional homotetrameric and heterotetrameric channels in various heterologous cell systems. Heterotetrameric HCN2/4 channels in Xenopus oocytes show two changes compared to each of the homotetramers: the voltage of half maximum activation is shifted to more depolarized voltages and activation kinetics are faster (Zhang et al., Biochim Biophys Acta, 2009). However, little is known about the ligand dependence of these channels. Herein, we studied the differences in activation gating of HCN2/4 channels in comparison to the respective homotetramers, thereby focusing on the effect of cAMP. We monitored activation under both steady-state and non steady-state conditions in the presence and absence of cAMP, as well as the ligand-dependent activation kinetics after ligand jumps. We found (1) that in HCN2/4 the apparent affinity for cAMP was between that of the two homotetramers, whereas the Hill coefficient was lowest, (2) that cAMP accelerates voltage-induced activation in HCN2/4 only slightly (factor ∼2), resembling HCN4, whereas in HCN2 it accelerates activation in a voltage-dependent manner by a factor of up to ∼12, (3) that the activation kinetics following a cAMP jump to channels pre-activated by voltage was fastest in HCN2/4, whereas the increase of current amplitude by a concentration jump was similar to homotetramers. Our results confirm that coinjection of the HCN2 and HCN4 isoforms in Xenopus oocytes leads to heterotetrameric channels. They suggest that in native heart cells the formation of heterotetramers leads to pacemaker channels with specific characteristics, thereby fine-tuning the process of pacemaking.

A Bridging [4Fe-4S] Cluster and Nucleotide Binding Are Essential for Function of the Cfd1-Nbp35 Complex as a Scaffold in Iron-Sulfur Protein Maturation

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.