Evolution of major milk proteins in Mus musculus and Mus spretus mouse species: a genoproteomic analysis

<p>Abstract</p> <p>Background</p> <p>Due to their high level of genotypic and phenotypic variability, <it>Mus spretus </it>strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. <it>Mus spretus </it>diverged from <it>Mus musculus </it>around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP) in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of <it>Mus spretus </it>and <it>Mus musculus </it>species, respectively.</p> <p>Results</p> <p>The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L <it>vs</it>. 125 ± 12 g/L, respectively). Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (β and α_s1) and the whey acidic protein (WAP), showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas <it>Csn1s1 </it>(11), <it>Csn2 </it>(7) and <it>Wap </it>(8) genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas <it>Wap </it>gene.</p> <p>Conclusion</p> <p>SNP frequencies found in three milk protein-encoding genes between <it>Mus spretus </it>and <it>Mus musculus </it>is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence), already identified in casein genes from other species, likely explain the existence of multiple α_s1-casein isoforms both in SEG/Pas and C57BL/6J strains. Finally, we propose a possible mechanism by which the hallmark tandem duplication of a 18-nt exon (14 copies) may have occurred in the mouse genome.</p

Propofol Directly Increases Tau Phosphorylation

In Alzheimer's disease (AD) and other tauopathies, the microtubule-associated protein tau can undergo aberrant hyperphosphorylation potentially leading to the development of neurofibrillary pathology. Anesthetics have been previously shown to induce tau hyperphosphorylation through a mechanism involving hypothermia-induced inhibition of protein phosphatase 2A (PP2A) activity. However, the effects of propofol, a common clinically used intravenous anesthetic, on tau phosphorylation under normothermic conditions are unknown. We investigated the effects of a general anesthetic dose of propofol on levels of phosphorylated tau in the mouse hippocampus and cortex under normothermic conditions. Thirty min following the administration of propofol 250 mg/kg i.p., significant increases in tau phosphorylation were observed at the AT8, CP13, and PHF-1 phosphoepitopes in the hippocampus, as well as at AT8, PHF-1, MC6, pS262, and pS422 epitopes in the cortex. However, we did not detect somatodendritic relocalization of tau. In both brain regions, tau hyperphosphorylation persisted at the AT8 epitope 2 h following propofol, although the sedative effects of the drug were no longer evident at this time point. By 6 h following propofol, levels of phosphorylated tau at AT8 returned to control levels. An initial decrease in the activity and expression of PP2A were observed, suggesting that PP2A inhibition is at least partly responsible for the hyperphosphorylation of tau at multiple sites following 30 min of propofol exposure. We also examined tau phosphorylation in SH-SY5Y cells transfected to overexpress human tau. A 1 h exposure to a clinically relevant concentration of propofol in vitro was also associated with tau hyperphosphorylation. These findings suggest that propofol increases tau phosphorylation both in vivo and in vitro under normothermic conditions, and further studies are warranted to determine the impact of this anesthetic on the acceleration of neurofibrillary pathology

Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation

Degradation of Microtubule‐Associated Protein 2 and Brain Spectrin by Calpain: A Comparative Study

: The in vitro degradation of microtubule‐associated protein 2 (MAP‐2) and spectrin by the calcium‐dependent neutral protease calpain was studied. Five major results are reported. First, MAP‐2 isolated from twice‐cycled microtubules (2XMT MAP‐2) was extremely sensitive to calpain‐induced hydrolysis. Even at an enzyme‐to‐substrate ratio (wt/wt) of 1:200, 2XMT MAP‐2 was significantly degraded by calpain. Second, MAP‐2 purified from the total brain heat‐stable fraction (total MAP‐2) was significantly more resistant to calpain‐induced hydrolysis compared with 2XMT MAP‐2. Third, MAP‐2a and MAP‐2b were proteolyzed similarly by calpain, although some relative resistance of MAP‐2b was observed. Fourth, the presence of calmodulin significantly increased the extent of calpain‐induced hydrolysis of the α‐subunit of spectrin. Fifth, the two neuronal isoforms of brain spectrin (240/235 and 240/235E, referred to as α/βN and α/βE, respectively) showed different sensitivities to calpain. αN‐spectrin was significantly more sensitive to calpain‐induced degradation compared to αE‐spectrin. Among other things, these results suggest a role for the calpain‐induced degradation of MAP‐2, as well as spectrin, in such physiological processes as alterations in synaptic efficacy, dendritic remodeling, and in pathological processes associated with neurodegeneration

The interrelationship between selective tau phosphorylation and microtubule association

The purpose of this study was to examine the modulation of tau phosphorylation mediated by protein kinase A, a kinase with low intrinsic activity, and by the constitutively active glycogen synthase kinase, as well as to examine the subsequent effects on tau-microtubule association in differentiated human SH-SY5Y neuroblastoma cells. Activation of protein kinase A with forskolin and rolipram significantly increased tau phosphorylation at Ser262/356 only in the presence of okadaic acid, indicating that phosphates at these sites are normally turned over rapidly. In contrast, glycogen synthase kinase appears to maintain tau phosphorylation at Thr181 and Ser396/404 since inhibition of glycogen synthase kinase with lithium reduced phosphorylation at these sites. Lithium treatment also significantly decreased tau and tyrosinated α-tubulin levels. Perturbation of microtubules with nocodazole or taxol induced tau dephosphorylation at Tau-1 sites, Thr181 and Ser396/404, indicating that both constitutive kinase activity and microtubule state modulate tau phosphorylation at these sites. Nocodazole- or taxol-induced tau dephosphorylation was blocked by the protein phosphatase 2A/1 inhibitor okadaic acid, but not by the protein phosphatase 2B inhibitor cyclosporin A. In addition, osmotic stress, such as treatment with 20 mM NaCl, selectively increased tau phosphorylation at the Tau-1 epitope. To investigate the effect of phosphorylation on tau association with microtubules and microtubule stability in situ, a Triton X-100 extraction assay was utilized to separate the detergent-soluble cytosolic components from the detergent-insoluble cytoskeletal components. In control cells or cells treated with lithium very little tau was detected in the cytosolic fraction. Activation of protein kinase A in the presence of okadaic acid elevated tau levels in the detergent-soluble fraction, which contained all the tau phosphorylated at Ser262/356, and also decreased microtubule stability, as indicated by decreased acetylated α-tubulin levels. In conclusion, the phosphorylation state of tau in differentiated SH-SY5Y cells is regulated by glycogen synthase kinase, microtubule dynamics and osmotic stress at overlapping sites which apparently have little influence on tau-microtubule association. In contrast, phosphorylation of tau at Ser262/356 within the microtubule-binding, which was mediated in part by protein kinase A, prevented the association of tau with microtubules in situ