Crystal structures of the Tie2 receptor ectodomain and the angiopoietin-2-Tie2 complex

The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.     

Neuroprotective roles of the P2Y2 receptor

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

Exostosin-like 2 regulates FGF2 signaling by controlling the endocytosis of FGF2

Background

Heparan sulfate proteoglycans are ubiquitously expressed on cell surfaces and in extracellular matrices, and are engaged in heparin-binding growth factor-related signal transduction. Thus, changes in the amounts, structures, and chain lengths of heparan sulfate have profound effects on aspects of cell growth controlled by heparin-binding growth factors such as FGF2. Exostosin glycosyltransferases (EXT1, EXT2, EXTL1, EXTL2, and EXTL3) control heparan sulfate biosynthesis, and the expression levels of their genes regulate the amounts, chain lengths, and sulfation patterns of heparan sulfate. Unlike EXT1, EXT2, and EXTL3, EXTL2 functions chain termination of heparan sulfate. Here, we examined the importance of EXTL2 in FGF2-dependent signaling.

Tight interplay between the Ca2+ affinity of the cardiac SERCA2 Ca2+ pump and the SERCA2 expression level

A reduced activity of the sarcoplasmic reticulum Ca2+ pump SERCA2a is a hallmark of cardiac dysfunction in heart failure. In SERCA2b/b mice, the normal SERCA2a isoform is replaced by SERCA2b, displaying a higher Ca2+ affinity. This elicited decreased cardiac SERCA2 expression and cardiac hypertrophy. Here, the interplay was studied between the increased Ca2+ affinity and a reduced expression of the pump and its role in the cardiac remodeling was investigated. First, SERCA2b/b mice were crossed with SERCA2b transgenes to boost cardiac SERCA2b expression. However, the enforced expression of SERCA2b was spontaneously countered by an increased inhibition by phospholamban (PLB), reducing the pump's Ca2+ affinity. Moreover, the higher SERCA2 content did not prevent hypertrophy. Second, we studied heterozygous SERCA2b/WT mice, which also express lower SERCA2 levels compared to wild-type. Hypertrophy was not observed. In heterozygotes, SERCA2b expression was specifically suppressed, explaining the reduced SERCA2 content. The SERCA2b/WT model strikingly differs from the homozygote models because SERCA2a (not SERCA2b) is the major isoform and because the inhibition of the pump by PLB is decreased instead of being increased. Thus, a tight correlation exists between the SERCA2 levels and Ca2+ affinity (controlled by PLB). This compensatory response may be important to prevent cardiac remodeling.     

Casein kinase-2 phosphorylates serine-2 in the beta-subunit of initiation factor-2

We have previously presented evidence which suggests that casein kinase-2 phosphorylates a serine residue near the N-terminus of the beta-subunit of the initiation factor eIF-2 (Clark, S.J. et al. Biochim. Biophys. Acta 968, 211-219). We now report further data which confirm that it is serine-2 which is phosphorylated by casein kinase-2. This data includes (1) the electrophoretic mobilities of the phosphopeptides produced by different cleavage techniques, (2) the amino acid composition of the principal phosphopeptide generated by treatment with cyanogen bromide and (3) the resistance of this phosphopeptide to Edman degradation.     

Nek2A specifies the centrosomal localization of Erk2

Nek2A is a cell-cycle-regulated protein kinase that localizes to the centrosome and kinetochore. Our recent studies provide a link between Nek2A and spindle checkpoint signaling [J. Biol. Chem. 279 (2004) 20049]. Extracellular signal-regulated kinase 2 (Erk2) is an important kinase, which belongs to mitogen activating protein (MAP) kinase family. Here we demonstrated that Nek2A binds specifically to Erk2. Erk2 interacts with Nek2A via a conserved Erk2 docking site located to the C-terminus of Nek2A. Our studies indicate this docking site is essential and sufficient for a direct Nek2A-Erk2 interaction. In addition, our immunocytochemical studies show that Nek2A and Erk2 are co-localized to centrosome. Significantly, elimination of Nek2A by RNA interference delocalized Erk2 from its centrosomal location, while inhibition of Erk2 kinase activity did not affect the localization of Nek2A in centrosome. We propose that Erk2 links extracellular signaling to centrosome dynamics by Nek2A.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Both thioredoxin 2 and glutaredoxin 2 contribute to the reduction of the mitochondrial 2-Cys peroxiredoxin Prx3

The proteins from the thioredoxin family are crucial actors in redox signaling and the cellular response to oxidative stress. The major intracellular source for oxygen radicals are the components of the respiratory chain in mitochondria. Here, we show that the mitochondrial 2-Cys peroxiredoxin (Prx3) is not only substrate for thioredoxin 2 (Trx2), but can also be reduced by glutaredoxin 2 (Grx2) via the dithiol reaction mechanism. Grx2 reduces Prx3 exhibiting catalytic constants (K(m), 23.8 μmol·liter(-1); V(max), 1.2 μmol·(mg·min)(-1)) similar to Trx2 (K(m), 11.2 μmol·liter(-1); V(max), 1.1 μmol·(mg·min)(-1)). The reduction of the catalytic disulfide of the atypical 2-Cys Prx5 is limited to the Trx system. Silencing the expression of either Trx2 or Grx2 in HeLa cells using specific siRNAs did not change the monomer:dimer ratio of Prx3 detected by a specific 2-Cys Prx redox blot. Only combined silencing of the expression of both proteins led to an accumulation of oxidized protein. We further demonstrate that the distribution of Prx3 in different mouse tissues is either linked to the distribution of Trx2 or Grx2. These results introduce Grx2 as a novel electron donor for Prx3, providing further insights into pivotal cellular redox signaling mechanisms.     

On the Mechanism of Action of H2O2 in the Cellular Stress

We propose a hypothesis according to which the reactive and reduced species of oxygen could be the intracellular inducers of the stress (or “heat-shock”) response. This hypothesis is based on the following observations on Drosphila cells: -a) the return to normoxia after 24 h anaerobiosis is suficient to induce the synthesis of the ‘heat shock’ proteins without elevation of temperature together with a rapid increase of O₂ consumption; -b) hydrogen peroxide introduced in the culture medium induces the early transcrip-tional activation of the ‘heat shock’ genes (maximal after 5 minutes); -c) hydrogen peroxide added to cellular extracts in vitro (thus acting as an intracellular metabolite) activates instantaneously the binding capacity of a ‘heat shock’ factor to a DNA ‘heat shock’ regulatory element. Thus, hydrogen peroxide, and possibly other reactive reduced species ofoxygen, could trigger the onset of the stress (or ‘heat shock’) response.     

Predicting the angiotensin converting enzyme 2 (ACE2) utilizing capability as the receptor of SARS-CoV-2

SARS-CoV-2, the newly identified human coronavirus causing severe pneumonia pandemic, was probably originated from Chinese horseshoe bats. However, direct transmission of the virus from bats to humans is unlikely due to lack of direct contact, implying the existence of unknown intermediate hosts. Angiotensin converting enzyme 2 (ACE2) is the receptor of SARS-CoV-2, but only ACE2s of certain species can be utilized by SARS-CoV-2. Here, we evaluated and ranked the receptor-utilizing capability of ACE2s from various species by phylogenetic clustering and sequence alignment with the currently known ACE2s utilized by SARS-CoV-2. As a result, we predicted that SARS-CoV-2 tends to utilize ACE2s of various mammals, except murines, and some birds, such as pigeon. This prediction may help to screen the intermediate hosts of SARS-CoV-2.     

Lipid interaction of alpha-synuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2

Alpha-synuclein co-exists with lipids in the Lewy bodies, a pathological hallmark of Parkinson's disease. Molecular interaction between alpha-synuclein and lipids has been examined by observing lipid-induced protein self-oligomerization in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Lipids such as phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and even arachidonic acid induced the self-oligomerization whereas phosphatidylcholine did not affect the protein. Because the oligomerizations occurred from critical micelle concentrations of the lipids, the self interaction of alpha-synuclein was shown to be a lipid-surface dependent phenomenon with head group specificity. By employing beta-synuclein and a C-terminally truncated alpha-synuclein (alpha-syn97), the head-group dependent self-oligomerization was demonstrated to occur preferentially at the N-terminal region while the fatty acid interaction leading to the protein self-association required the presence of the acidic C-terminus of alpha-synuclein. In the presence of Cu2+ and H2O2, phosphatidylinositol (PI), along with other acidic lipids, actually enhanced the metal-catalyzed oxidative self-oligomerization of alpha-synuclein. The dityrosine crosslink formation responsible for the PI-enhanced covalent self-oligomerization was more sensitive to variation of copper concentrations than that of H2O2 during the metal-catalyzed oxidation. The enhancement by PI was shown to be due to facilitation of copper localization to the protein because actual binding affinity between copper and alpha-synuclein increased from Kd of 44.7 microm to 5.9 microm in the presence of the lipid. Taken together, PI not only affects alpha-synuclein to be more self-interactive by providing the lipid surface, but also enhances the metal-catalyzed oxidative protein self-oligomerization by facilitating copper localization to the protein when the metal and H2O2 are provided. This observation therefore could be implicated in the formation of Lewy bodies as lipids and metal-catalyzed oxidative stress have been considered to be a part of pathological causes leading to the neurodegeneration.