Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/p32/gC1qR

Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg2+ concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function

Structural flexibility of multifunctional HABP1 may be important for regulating its binding to different ligands

Hyaluronan-binding protein 1 (HABP1)/p32/gC1qR was characterized as a highly acidic and oligomeric protein, which binds to different ligands like hyaluronan, C1q, and mannosylated albumin. It exists as trimer in high ionic and reducing conditions as shown by crystal structure. In the present study, we have examined the structural changes of HABP1 under a wide range of ionic environments. HABP1 exhibits structural plasticity, which is influenced by the ionic environment under in vitro conditions near physiological pH. At low ionic strength HABP1 exists in a highly expanded and loosely held trimeric structure, similar to that of the molten globule-like state, whereas the presence of salt stabilizes the trimeric structure in a more compact fashion. It is likely that the combination of the high net charge asymmetrically distributed along the faces of the molecule and the relatively low intrinsic hydrophobicity of HABP1 result in its expanded structure at neutral pH. Thus, the addition of counter ions in the molecular environment minimizes the intramolecular electrostatic repulsion in HABP1 leading to its stable and compact conformations, which reflect in its differential binding toward different ligands. Whereas the binding of HABP1 toward HA is enhanced on increasing the ionic strength, no significant effect was observed with the two other ligands, C1q and mannosylated albumin. Thus, although HA interacts only with compact HABP1, C1q and mannosylated albumin can bind to loosely held oligomeric HABP1 as well. In other words, structural changes in HABP1 mediated by changes in the ionic environment are responsible for recognizing different ligands

A novel mechanism of RNase L inhibition: Theiler\u27s virus L* protein prevents 2-5A from binding to RNase L

<div><p>The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L <i>in vivo</i>. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A.</p></div

Evidence for inhibitory interaction of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) with Streptococcus pneumoniae hyaluronidase

Bacterial hyaluronan lyase enzymes are the major virulence factors that enable greater microbial ingress by cleaving hyaluronan (HA) polymers present predominantly in extracellular space of vertebrates. Based on the premise that effective inhibitors may bind to and stabilize HA thereby protecting it from degradation, here we investigated inhibitory activity of human hyaluronan-binding protein 1 (HABP1) on bacterial hyaluronidase because it is highly specific to HA and localized on the cell surface. Biochemical characterization revealed that HABP1 is a competitive inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of 22 &#956;m. This is thus the first report of an endogenous protein inhibitor that may be used during natural antibacterial defense. Our findings also support a novel multipronged mechanism for the high efficacy of HABP1-mediated inhibition based on structural modeling of enzyme, substrate, and inhibitor. Evidence from docking simulations and contact interface interactions showed that the inherent charge asymmetry of HABP1 plays a key role in the inhibitory activity. This novel role of HABP1 may pave the way for peptide inhibitors as alternatives to synthetic chemicals in antibacterial research

Schwarzwald, Eugenie (Genia); geb. Nußbaum

(1872 - 1940), Pädagogin, Schulreformerin und Germanisti

Demethylation of Non-CpG Sites in DNA Is Initiated by TET2 5-Methylcytosine Dioxygenase

In the mammalian genome, cytosine methylation predominantly occurs at CpG sites. In addition, a number of recent studies have uncovered extensive C5 cytosine methylation (5mC) at non-CpG (5mCpH, where H = A/C/T) sites. Little is known about the enzyme responsible for active demethylation of 5mCpH sites. Using a very sensitive and quantitative LC–MS/MS method, we demonstrate that the human TET2, an iron (II)- and 2OG-dependent dioxygenase, which is a frequently mutated gene in several myeloid malignancies, as well as in a number of other types of cancers, can oxidize 5mCpH sites in double-stranded DNA in vitro. Similar to oxidation of 5mCpG, oxidation of 5mC at CpH sites produces 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC) bases in DNA. After 5mCpG, which is the most preferred substrate, TET2 prefers 5mCpC as a substrate, followed by 5mCpA and then 5mCpT. Since the TDG/BER pathway and deformylation or decarboxylation of 5fC or 5caC, respectively, can convert 5fCpH and 5caCpH to an unmodified cytosine base in DNA, our results suggest a novel demethylation pathway of 5mCpH sites initiated by TET2 dioxygenase

L* can act on non-macrophage cell lines and in absence of other viral components.

<p>A. L* inhibition of the 2–5A/RNase L pathway is not restricted to macrophages: L929 cells were incubated with 5 U/ml of murine IFN-β for 24 h or left untreated prior to infection with 2 PFU per cell of indicated viruses. Histograms show the mean +/− SD of viral genome copies detected by quantitative RT-PCR in RNA samples isolated 16 h after infection. Values for mock samples were lower than the detection limit (10 cDNA copies). B. L* inhibits the 2–5A/RNase L pathway in the absence of other viral components: L929 cells stably expressing L* (L929-L*) or the empty vector (L929-NEO) were primed with 5 U/ml of murine IFN-β and transfected with poly(I:C) for 7 hours. Alternatively, the cells were directly transfected without priming, with crude 2–5A at a concentration of 5 uM. Total cell RNA was then isolated and analyzed on RNA chips.</p

Expression plasmids used in this study.

1<p>Neo: G418/Geneticin, puro: puromycin, hygro: hygromycin.</p

Theiler's virus L* protein inhibits the 2–5A/RNase L pathway in infected peritoneal macrophages.

<p>A. Peritoneal macrophages prepared from RNase L ^−/− (central panel) or RNase L^+/+ (left) C57BL/6 mice and, as an additional control, from RNase L^+/+ 129/Sv mice (right), were mock-infected or infected at a MOI of 20 with VV18 (L* WT), TM770 (L* 1–92) or FS58 (L* 1–12). Nine hours post infection, total cell RNA was extracted and analyzed on RNA chips. For control purposes, peritoneal macrophages were also transfected with 2.5 µg/ml poly(I:C) for 7 h before total cell RNA extraction. Prominent rRNA cleavage products are indicated. B. Replication levels of wild-type or L*-mutant viruses in peritoneal macrophages. Peritoneal macrophages isolated from indicated mouse strains were plated for four days and infected as in (A). Viral RNA was quantified by quantitative RT-PCR. Histograms show the mean and SD of viral cDNA copies detected in samples from a representative triplicate infection experiment. Values for mock samples were lower than the detection limit (10 cDNA copies). The experiment was repeated twice, using two independent productions of viruses and macrophages.</p