Influence of Heparin Mimetics on Assembly of the FGF·FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM₆), octasaccharide (HM₈), and decasaccharide (HM₁₀), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM₈ and HM₁₀ are significantly more potent than HM₆ in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM₈ relative to HM₆ in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM₈ to bind and dimerize FGF2. Notably FGF2·HM₈ exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.     

p38 MAP Kinase and MAPKAP Kinases MK2/3 Cooperatively Phosphorylate Epithelial Keratins

The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser⁷³ is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser⁷³ phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser⁵² and K20-Ser¹³ but not of K8-Ser⁴³¹ or K18-Ser³³. Interestingly, K18-Ser⁵² and K20-Ser¹³ are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser¹³ and K18-Ser⁵² are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser¹³ phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser¹³ phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.     

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond—the flavinylation reaction—has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl-flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.     

Widespread Infection with Homologues of Human Parvoviruses B19, PARV4, and Human Bocavirus of Chimpanzees and Gorillas in the Wild

Infections with human parvoviruses B19 and recently discovered human bocaviruses (HBoVs) are widespread, while PARV4 infections are transmitted parenterally and prevalent specifically in injecting drug users and hemophiliacs. To investigate the exposure and circulation of parvoviruses related to B19 virus, PARV4, and HBoV in nonhuman primates, plasma samples collected from 73 Cameroonian wild-caught chimpanzees and gorillas and 91 Old World monkey (OWM) species were screened for antibodies to recombinant B19 virus, PARV4, and HBoV VP2 antigens by enzyme-linked immunosorbent assay (ELISA). Moderate to high frequencies of seroreactivity to PARV4 (63% and 18% in chimpanzees and gorillas, respectively), HBoV (73% and 36%), and B19 virus (8% and 27%) were recorded for apes, while OWMs were uniformly negative (for PARV4 and B19 virus) or infrequently reactive (3% for HBoV). For genetic characterization, plasma samples and 54 fecal samples from chimpanzees and gorillas collected from Cameroonian forest floors were screened by PCR with primers conserved within Erythrovirus, Bocavirus, and PARV4 genera. Two plasma samples (chimpanzee and baboon) were positive for PARV4, while four fecal samples were positive for HBoV-like viruses. The chimpanzee PARV4 variant showed 18% and 15% nucleotide sequence divergence in NS and VP1/2, respectively, from human variants (9% and 7% amino acid, respectively), while the baboon variant was substantially more divergent, mirroring host phylogeny. Ape HBoV variants showed complex sequence relationships with human viruses, comprising separate divergent homologues of HBoV1 and the recombinant HBoV3 species in chimpanzees and a novel recombinant species in gorillas. This study provides the first evidence for widespread circulation of parvoviruses in primates and enables future investigations of their epidemiology, host specificity, and (co)evolutionary histories.Autonomous parvoviruses known to infect humans comprise parvovirus B19 (18) and the recently discovered PARV4 (22) and human bocavirus (HBoV) (3). Members of the family Parvoviridae are genetically and biologically diverse and are classified into several genera or groups, showing marked differences in host range, pathology, and tissue/cellular tropisms (18). Human parvovirus B19, a member of the Erythrovirus genus, is transmitted primarily by the respiratory route but causes systemic infections. Erythroid progenitor cells are specifically targeted through expression of globoside P antigen, which acts as the B19 virus receptor for entry (5). In common with infections by most parvoviruses, B19 virus infections are acute; a period of intense viremia is followed by seroconversion for antibody to B19 virus and lifelong immunity from reinfection (29). Despite the clearance of viremia and seroconversion for antibody, lifelong persistence of viral DNA in tissues has been shown to occur (12, 20, 26, 28, 43, 58). Three genotypes of B19 virus have been described, differing in nucleotide sequence by approximately 13 to 14% (7, 21, 41, 53); genotypes 1 and 2 have been found in Europe, the United States, and other Western countries, while genotype 3 is restricted to sub-Saharan Africa and South America (7, 47, 49). B19 virus widely circulates in human populations worldwide; in Western countries, several studies have documented increasing frequencies of B19 virus seropositivity with age, rising to approximately 60 to 70% by adulthood (15, 39, 48, 61).Another human parvovirus, PARV4, shows markedly different epidemiology and transmission routes. It was originally detected in plasma from an individual with an “acute infection syndrome” resembling that of primary human immunodeficiency virus (HIV) infection (22). While this clinical presentation has not been observed again, infection with PARV4 is known to be widespread specifically in individuals with a history of parenteral exposure (injecting drug users [IDUs], hemophiliacs, polytransfused individuals), with a strikingly higher incidence in those infected with HIV-1 (13, 14, 30, 35, 54). These observations suggest that PARV4 is primarily transmitted though parenteral routes in Western countries (54, 56). In common with infection with the better-characterized human parvovirus B19, infection with PARV4 is associated with a period of acute viremia, followed by seroconversion for antibody and long-term persistence of viral DNA sequences in lymphoid and other tissue (33, 37, 52). Circulating variants of PARV4 have been classified into three distinct genotypes exhibiting approximately 8% nucleotide sequence divergence from each other. Genotypes 1 and 2 circulate in Western countries, while genotype 3 has to date been recorded only in sub-Saharan Africa (45, 55).The third human parvovirus, HBoV (3), shows a number of epidemiological and clinical attributes different from those of both B19 virus and PARV4. HBoV was originally found in the respiratory tract of young children and has been the subject of intense investigation as a potential cause of human respiratory disease (reviewed in references 1, 51, and 62). Although it is frequently detected by PCR in the nasopharynx of viremic individuals with primary infections with lower respiratory tract disease, other coinfecting respiratory viruses are frequently detected (19). HBoV additionally shows long-term, low-level carriage in the respiratory tract after primary infection, which further complicates PCR-based etiological studies (2, 38) and warrants the use of other diagnostic strategies, such as serology (30, 32, 59). In contrast to the rather minimal genetic diversity of B19 virus and PARV4 genotypes, bocaviruses infecting humans are now known to comprise three to four major genetic variants (termed types or species 1 to 4) (23, 24). HBoV1 and HBoV2 show 22%, 33%, and 20% amino acid sequence divergence from each other in the encoded viral nonstructural (NS), NP-1, and structural VP1/VP2 proteins, respectively, the latter potentially leading to antigenic diversity and some loss of antigenic cross-reactivity. A third type/species of HBoV is a chimeric form with a nonstructural gene region (NS, NP1) most similar to HBoV1, a recombination breakpoint in the intergenic region between NP1 and VP1, and structural genes related to those of HBoV2 (4, 23). Current data suggest that only HBoV1 is capable of infecting the respiratory tract; most published large-scale screening studies have failed to detect HBoV2 (or HBoV3) in respiratory samples (10, 11, 60), while all three types/species are detectable in fecal samples, indicating the existence of an alternative or additional site of virus replication (23). Despite extensive inquiry, the exact role of HBoV1 in respiratory disease remains unclear, as is the proposed etiological role of HBoV2 (and possibly HBoV3) in gastroenteritis (4, 11, 23, 50). Very recently, a fourth species/type, HBoV4, has been detected in fecal samples; genetically it also shows evidence for past recombination, with NS and NP1 region sequences grouping with HBoV2, while VP1/VP2 is more closely related to HBoV3 (23).We have little understanding of the past epidemiology, evolution, and origins of human parvoviruses. For both B19 virus and PARV4, evidence has been obtained for a temporal succession of genotypes over time (37, 43); in Europe, B19 virus genotype 1 largely replaced type 2 in the 1960 and 1970s (43), while current data indicate that a similar replacement of PARV4 genotypes occurred within the last 20 years (37). The highly restricted sequence diversity of currently circulating variants of PARV4 and B19 virus and of HBoV1 variants supports the hypothesis of a relatively recent emergence and spread of these viruses in human populations (36, 42, 64).The existence and evolution of parvoviruses on a much longer time scale is suggested by the observations that members of the Erythrovirus and Parvovirus genera both contain viruses that are highly host species specific and that the molecular phylogenies of both genera are largely congruent with those of their hosts (34). This has led to the hypothesis of long-term coevolution of parvoviruses with their host over the 90 million years of mammalian evolution and perhaps beyond. Among erythroviruses, simian homologues of B19 virus have been found in cynomolgus monkeys (44) and rhesus and pig-tailed macaques (16) and more genetically distant viruses have been characterized in chipmunks and cows (9, 63). Divergent homologues of PARV4 in pigs and cows have been described (31), while the bovine and canine parvoviruses distantly related to HBoV are the originally described members of the Bocavirus genus. However, the process of virus-host codivergence is known to be punctuated by occasional cross-species transmissions, including the well-documented spread of feline parvovirus to dogs (46). Based on serological evidence, the possible transmission of simian erythroviruses to animal handlers has been proposed (6).To gain further insights into the origins and evolution of human parvoviruses, we have performed large-scale serological and PCR-based screening of nonhuman primates (chimpanzees and gorillas) and of several species of Old World monkeys (OWMs) for evidence of infection with parvoviruses that are antigenically related to the human B19, PARV4, and HBoV viruses. By PCR, we have sought to genetically characterize homologues of the three autonomous human parvoviruses in apes and Old World monkey species and to analyze their evolutionary relationship to human and other mammalian homologues of these viruses.     

Direct Demonstration of Half-of-the-sites Reactivity in the Dimeric Cytochrome bc1 Complex: ENZYME WITH ONE INACTIVE MONOMER IS FULLY ACTIVE BUT UNABLE TO ACTIVATE THE SECOND UBIQUINOL OXIDATION SITE IN RESPONSE TO LIGAND BINDING AT THE UBIQUINONE REDUCTION SITE*

We previously proposed that the dimeric cytochrome bc₁ complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc₁ monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289–22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc₁ complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10–16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c₁ by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wild-type dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.     

Pannexin1 and Pannexin2 Channels Show Quaternary Similarities to Connexons and Different Oligomerization Numbers from Each Other

Pannexins are homologous to innexins, the invertebrate gap junction family. However, mammalian pannexin1 does not form canonical gap junctions, instead forming hexameric oligomers in single plasma membranes and intracellularly. Pannexin1 acts as an ATP release channel, whereas less is known about the function of Pannexin2. We purified cellular membranes isolated from MDCK cells stably expressing rat Pannexin1 or Pannexin2 and identified pannexin channels (pannexons) in single membranes by negative stain and immunogold labeling. Protein gel and Western blot analysis confirmed Pannexin1 (Panx1) or Pannexin2 (Panx2) as the channel-forming proteins. We expressed and purified Panx1 and Panx2 using a baculovirus Sf9 expression system and obtained doughnut-like structures similar to those seen previously in purified connexin hemichannels (connexons) and mammalian membranes. Purified pannexons were comparable in size and overall appearance to Connexin46 and Connexin50 connexons. Pannexons and connexons were further analyzed by single-particle averaging for oligomer and pore diameters. The oligomer diameter increased with increasing monomer molecular mass, and we found that the measured oligomeric pore diameter for Panxs was larger than for Connexin26. Panx1 and Panx2 formed active homomeric channels in Xenopus oocytes and in vitro vesicle assays. Cross-linking and native gels of purified homomeric full-length and a C-terminal Panx2 truncation mutant showed a banding pattern more consistent with an octamer. We purified Panx1/Panx2 heteromeric channels and found that they were unstable over time, possibly because Panx1 and Panx2 homomeric pannexons have different monomer sizes and oligomeric symmetry from each other.     

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis

N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.     

Isoform-specific O-Glycosylation of Osteopontin and Bone Sialoprotein by Polypeptide N-Acetylgalactosaminyltransferase-1

Mucin-type O-glycan biosynthesis is regulated by the family of UDP-GalNAc polypeptide:N-acetylgalactosaminlytransfersases (ppGalNAcTs) that catalyzes the first step in the pathway by transferring GalNAc to Ser or Thr residues in a protein from the sugar donor UDP-GalNAc. Because not all Ser/Thr residues are glycosylated, rules must exist that signal which hydroyxamino acids acquire sugar. To date, no universal consensus signal has emerged. Therefore, strategies to deduce the subset of proteins that will be glycosylated by distinct ppGalNAcTs must be developed. Mucin-type O-glycoproteins are present abundantly in bone, where we found multiple ppGalNAcT isoforms, including ppGalNAcT-1, to be highly expressed. Thus, we compared glycoproteins expressed in wild-type and Galnt1-null mice to identify bone-associated proteins that were glycosylated in a ppGalNAcT-1-dependent manner. A reduction in the apparent molecular masses of two SIBLINGs (small integrin binding ligand N-linked glycoproteins), osteopontin (OPN) and bone sialoprotein (BSP) in the Galnt1-null mice relative to those of the wild-type was observed. Several synthetic peptides derived from OPN and BSP sequences were designed to include either known or predicted (in silico) glycosylation sites. In vitro glycosylation assays of these peptides with recombinant ppGalNAcT-1, ppGalNAcT-2, or ppGalNAcT-3 demonstrated that both SIBLINGs contained Thr/Ser residues that were preferentially glycosylated by ppGalNAcT-1. In addition, lysates prepared from wild-type, but not those from Galnt1-null derived osteoblasts, could glycosylate these peptides efficiently, suggesting that OPN and BSP contain sites that are specific for ppGalNAcT-1. Our study presents a novel and systematic approach for identification of isoform-specific substrates of the ppGalNAcT family and suggests ppGalNAcT-1 to be indispensable for O-glycosylation at specific sites of the bone glycoproteins OPN and BSP.