Tanweer Zaidi

Hyperphosphorylation induces self-assembly of τ into tangles of paired helical filaments/straight filaments

The microtubule-associated protein τ is a family of six isoforms that becomes abnormally hyperphosphorylated and accumulates in the form of paired helical filaments (PHF) in the brains of patients with Alzheimers disease (AD) and patients with several other tauopathies. Here, we show that the abnormally hyperphosphorylated τ from AD brain cytosol (AD P-τ) self-aggregates into PHF-like structures on incubation at pH 6.9 under reducing conditions at 35°C during 90 min. In vitro dephosphorylation, but not deglycosylation, of AD P-τ inhibits its self-association into PHF. Furthermore, hyperphosphorylation induces self-assembly of each of the six τ isoforms into tangles of PHF and straight filaments, and the microtubule binding domains/repeats region in the absence of the rest of the molecule can also self-assemble into PHF. Thus, it appears that τ self-assembles by association of the microtubule binding domains/repeats and that the abnormal hyperphosphorylation promotes the self-assembly of τ into tangles of PHF and straight filaments by neutralizing the inhibitory basic charges of the flanking regions.

Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.

Salmonella typhi uses CFTR to enter intestinal epithelial cells.

Homozygous mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF). In the heterozygous state, increased resistance to infectious diseases may maintain mutant CFTR alleles at high levels in selected populations. Here we investigate whether typhoid fever could be one such disease. The disease is initiated when Salmonella typhi enters gastrointestinal epithelial cells for submucosal translocation. We found that S. typhi, but not the related murine pathogen S. typhimurium, uses CFTR for entry into epithelial cells. Cells expressing wild-type CFTR internalized more S. typhi than isogenic cells expressing the most common CFTR mutation, a phenylalanine deleted at residue 508 (Δ508). Monoclonal antibodies and synthetic peptides containing a sequence corresponding to the first predicted extracellular domain of CFTR inhibited uptake of S. typhi. Heterozygous ΔF508 Cftr mice translocated 86% fewer S. typhi into the gastrointestinal submucosa than wild-type Cftr mice; no translocation occurred in ΔF508 Cftr homozygous mice. The Cftr genotype had no effect on the translocation of S. typhimurium. Immunoelectron microscopy revealed that more CFTR bound to S. typhi in the submucosa of Cftr wild-type mice than in ΔF508 heterozygous mice. We conclude that diminished levels of CFTR in heterozygotes may decrease susceptibility to typhoid fever.

Alzheimer paired helical filaments. Restoration of the biological activity by dephosphorylation.

In a normal mature neuron, microtubule associated protein tau promotes the assembly of tubulin into microtubules and maintains the structure of microtubules. In Alzheimer disease brain, tau is abnormally hyperphosphorylated and is the major protein subunit of paired helical filaments PHF). In the present study, the biological activity of tau in PHF and the effect of dephosphorylation on this activity were examined. PHF were isolated from Alzheimer disease brains and tau from the untreated or alkaline phosphatase‐treated PHF was extracted by ultrasonication in microtubule assembly buffer. Tubulin was isolated by phosphocellulose chromatography of three cycled microtubules from bovine brain. PHF‐tau did not promote assembly of bovine tubulin into microtubules whereas tau from the dephosphorylated PHF produced a robust microtubule assembly. These studies suggest (i) that in Alzheimer disease tau in PHF is functionally inactive because of abnormal phosphorylation and (ii) that the abnormally phosphorylated site(s) in PHF that inactivates PHF‐tau is accessible to enzymatic dephosphorylation in vitro.

Role of glycosylation in hyperphosphorylation of tau in Alzheimer’s disease

In Alzheimers disease (AD) brain, microtubule‐associated protein tau is abnormally modified by hyperphosphorylation and glycosylation, and is aggregated as neurofibrillary tangles of paired helical filaments. To investigate the role of tau glycosylation in neurofibrillary pathology, we isolated various pools of tau protein from AD brain which represent different stages of tau pathology. We found that the non‐hyperphosphorylated tau from AD brain but not normal brain tau was glycosylated. Monosaccharide composition analyses and specific lectin blots suggested that the tau in AD brain was glycosylated mainly through N‐linkage. In vitro phosphorylation indicated that the glycosylated tau was a better substrate for cAMP‐dependent protein kinase than the deglycosylated tau. These results suggest that the glycosylation of tau is an early abnormality that can facilitate the subsequent abnormal hyperphosphorylation of tau in AD brain.

MODULATION OF GSK-3-CATALYZED PHOSPHORYLATION OF MICROTUBULE-ASSOCIATED PROTEIN TAU BY NON-PROLINE-DEPENDENT PROTEIN KINASES

The phosphorylation of bovine tau, either by GSK‐3 alone or by a combination of GSK‐3 and several non‐proline‐dependent protein kinases (non‐PDPKs), was studied. GSK‐3 alone catalyzed the incorporation of ∼ 3 mol 32P/mot tau at a relatively slow rate. Prephosphorylation of tau by A‐kinase, C‐kinase, or CK‐2 (but not by CK‐1, CaM kinase II or Gr kinase) increased both the rate and extent of a subsequent phosphorylation catalyzed by GSK‐3 by several‐fold. These results suggest that the phosphorylation of tau by PDPKs such as GSK‐3 (and possibly MAP kinase, cdk5) may be positively modulated at the substrate level by non‐PDPK‐catalyzed phosphorylations.

Intranasal immunization with heterologously expressed polysaccharide protects against multiple Pseudomonas aeruginosa infections

Surface-expressed bacterial polysaccharides are often immunodominant, protective antigens. However, these antigens are chemically and serologically highly heterogeneous, and conjugation to protein carriers is often necessary to enhance their immunogenicity. Here we show the efficacy of intranasal immunization of mice with attenuated Salmonella enterica serovar Typhimurium expressing the O antigen portion of Pseudomonas aeruginosa lipopolysaccharide. P. aeruginosa is an ideal model system because it can cause a myriad of localized and systemic infections. In particular, this bacterium is a leading cause of hospital-acquired pneumonia and is responsible for infections after burns and after eye injury. In addition, there are mouse models of infection that mimic the clinical manifestations of P. aeruginosa infections. Immunized mice were highly protected against infection, with long-lasting immunity to acute P. aeruginosa pneumonia, whereas mice immunized with Salmonella containing only the cloning vector or PBS were not. Prophylactic and therapeutic administration of sera from vaccinated animals protected naive mice. Intranasal vaccination also provided complete protection from infections after burns and reduced pathology after corneal abrasions. These results indicate that intranasal delivery of heterologously expressed polysaccharide antigens provides protection at distinct sites of infection. This approach for the expression and delivery of polysaccharide antigens as recombinant immunogens could be easily adapted to develop vaccines for many infectious agents, without the need for complicated purification and conjugation procedures.

Inhibitors of protein phosphatase-2A from human brain structures, immunocytological localization and activities towards dephosphorylation of the Alzheimer type hyperphosphorylated tau

Protein phosphatase (PP)‐2A, which regulates the phosphorylation of tau, is regulated by two endogenous inhibitor proteins, I 1 PP 2 A and I 2 PP 2 A , in mammalian tissues. Here, we report the cloning of I 1 PP 2 A and I 2 PP 2 A from human brain, and show that in PC12 cells and in I 1 PP 2 A ‐ GFP or I 2 PP 2 A ‐ GFP transfected NIH3T3 and human neural progenitor cells, I 1 PP 2 A is localized mostly in the cell cytoplasm and I 2 PP 2 A mostly in the nucleus. The recombinant I 1 PP ‐ 2 A and I 2 PP ‐ 2 A inhibit PP‐2A activity towards hyperphosphorylated tau in vitro; the dephosphorylation of the hyperphosphorylated tau at specific sites is selectively inhibited. Overexpression of I 1 PP 2 A as well as I 2 PP 2 A results in tau hyperphosphorylation and degeneration of PC 12 cells.

The galU Gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung.

ABSTRACT Acute pneumonias and corneal infections due to Pseudomonas aeruginosa are typically caused by lipopolysaccharide (LPS)-smooth strains. In cystic fibrosis patients, however, LPS-rough strains of P. aeruginosa, which lack O antigen, can survive in the lung and cause chronic infection. It is not clear whether an LPS-rough phenotype affects cytotoxicity related to the type III secretion system (TTSS). We previously reported that interruption of the galU gene in P. aeruginosa results in production of a rough LPS and truncated LPS core. Here we evaluated the role of the galU gene in the pathogenesis of murine lung and eye infections and in cytotoxicity due to the TTSS effector ExoU. We studied galU mutants of strain PAO1, of its cytotoxic variant expressing ExoU from a plasmid, and of the inherently cytotoxic strain PA103. The galU mutants were more serum sensitive than the parental strains but remained cytotoxic in vitro. In a corneal infection model, the galU mutants were significantly attenuated. In an acute pneumonia model, the 50% lethal doses of the galU mutants were higher than those of the corresponding wild-type strains, yet these mutants could cause mortality and severe pneumonia, as judged by histology, even with minimal systemic spread. These findings suggest that the galU gene is required for corneal infection and for efficient systemic spread following lung infection but is not required for infection confined to the lung. Host defenses in the lung appear to be insufficient to control infection with LPS-rough P. aeruginosa when local bacterial levels are high.

Non-proline-dependent protein kinases phosphorylate several sites found in tau from Alzheimer disease brain

Of 21 phosphorylation sites identified in PHF-tau 11 are on ser/thr-X motifs and are probably phosphorylated by non-proline-dependent protein kinases (non-PDPKs). The identities of the non-PDPKs and how they interact to hyperphosphorylate PHF-tau are still unclear. In a previous study we have shown that the rate of phosphorylation of human tau 39 by a PDPK (GSK-3) was increased several fold if tau were first prephosphorylated by non-PDPKs (Singh et al., FEBS Lett 358: 267-272, 1995). In this study we have examined how the specificity of a non-PDPK for different sites on human tau 39 is modulated when tau is prephosphorylated by other non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) as well as a PDPK (GSK-3). We found that the rate of phosphorylation of tau 39 by a non-PDPK can be stimulated if tau were first prephosphorylated by other non-PDPKs. Of the four non-PDPKs only CK-1 can phosphorylate sites (thr 231, ser 396, ser 404) known to be present in PHF-tau. Further, these sites were phosphorylated more rapidly and to a greater extent by CK-1 if tau 39 were first prephosphorylated by A-kinase, CaM kinase II or GSK-3. These results suggest that the site specificities of the non-PDPKs that participate in PHF-tau hyperphosphorylation can be modulated at the substrate level by the phosphorylation state of tau.