Solution structure and backbone dynamics of Calsensin, an invertebrate neuronal calcium-binding protein

Calsensin is an EF-hand calcium-binding protein expressed by a subset of peripheral sensory neurons that fasciculate into a single tract in the leech central nervous system. Calsensin is a 9-kD protein with two EF-hand calcium-binding motifs. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone dynamics of calcium-bound Calsensin. Calsensin consists of four helices forming a unicornate-type four-helix bundle. The residues in the third helix undergo slow conformational exchange indicating that the motion of this helix is associated with calciumbinding. The backbone dynamics of the protein as measured by (15)N relaxation rates and heteronuclear NOEs correlate well with the three-dimensional structure. Furthermore, comparison of the structure of Calsensin with other members of the EF-hand calcium-binding protein family provides insight into plausible mechanisms of calcium and target protein binding.     

The JIL-1 kinase regulates the structure of Drosophila polytene chromosomes

The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. Here we have performed a detailed ultrastructural and cytological analysis of the defects in JIL-1 mutant chromosomes. We show that all autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes we propose a model in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions. Electronic Supplementary Material Supplementary material is available for this article at and accessible for authorised users     

JIL-1 kinase, a member of the male-specific lethal (MSL) complex, is necessary for proper dosage compensation of eye pigmentation in Drosophila

The upregulation of the JIL-1 kinase on the male X chromosome and its association with the male-specific lethal (MSL) complex suggest that JIL-1 may play a role in regulating dosage compensation. To directly test this hypothesis we measured eye pigment levels of mutants in the X-linked white gene in an allelic series of JIL-1 hypomorphic mutants. We show that dosage compensation of w(a) alleles that normally do exhibit dosage compensation was severely impaired in the JIL-1 mutant backgrounds. As a control we also examined a hypomorphic white allele w(e) that fails to dosage compensate in males due to a pogo element insertion. In this case the relative pigment level measured in males as compared to females remained approximately the same even in the most severe JIL-1 hypomorphic background. These results indicate that proper dosage compensation of eye pigment levels in males controlled by X-linked white alleles requires normal JIL-1 function.     

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

Chiral separation and quantification of R/S-amphetamine, R/S-methamphetamine, R/S-MDA, R/S-MDMA, and R/S-MDEA in whole blood by GC-EI-MS

The enantioselective composition of the amphetamines is of interest, as the enantiomers show differences in their pharmacological effects and several methods for chiral separation of amphetamines have been described. Only a few methods have used whole blood as matrix and none of these separates both classic amphetamines (amphetamine and methamphetamine) and designer amphetamines (MDA, MDMA and MDEA). The aim of this study was, therefore, to develop a method for enantioselective analysis of AM, MA, MDA, MDMA, and MDEA in whole blood. The amphetamines were extracted from 0.5 g of whole blood by liquid-liquid extraction. After derivatization with R-MTPCl, the resulting diastereomers were separated by GC on a HP-5MS column and detected by SIM-MS. R-MTPCl was used as derivatization reagent because of the stability of this reagent and good separation of these analytes. Through the method, development time and temperature of the derivatization were optimized, and by admixture of 0.02% triethylamine it became possible to detect the amphetamines in adequately low concentrations as more analytes were derivatized. The method was validated and it was linear from 0.004 to 3 microg/g per enantiomer. The accuracy was within 91-115%, while the repeatability and reproducibility were < or =15% R.S.D. A method suitable for enantioselective separation and analysis of the amphetamines has been achieved, and the method was applied to analysis of whole blood samples originating from traffic and criminal cases and post mortem cases.     

FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport

Autophagy is the main eukaryotic degradation pathway for long-lived proteins, protein aggregates, and cytosolic organelles. Although the protein machinery involved in the biogenesis of autophagic vesicles is well described, very little is known about the mechanism of cytosolic transport of autophagosomes. In this study, we have identified an adaptor protein complex, formed by the two autophagic membrane-associated proteins LC3 and Rab7 and the novel FYVE and coiled-coil (CC) domain–containing protein FYCO1, that promotes microtubule (MT) plus end–directed transport of autophagic vesicles. We have characterized the LC3-, Rab7-, and phosphatidylinositol-3-phosphate–binding domains in FYCO1 and mapped part of the CC region essential for MT plus end–directed transport. We also propose a mechanism for selective autophagosomal membrane recruitment of FYCO1.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

Identification of a polymeric Ig receptor binding phage-displayed peptide that exploits epithelial transcytosis without dimeric IgA competition

The polymeric Ig receptor (pIgR), also called membrane secretory component (SC), mediates epithelial transcytosis of polymeric immunoglobulins (pIgs). J Chain-containing polymeric IgA (pIgA) and pentameric IgM bind pIgR at the basolateral epithelial surface. After transcytosis, the extracellular portion of the pIgR is cleaved at the apical side, either complexed with pIgs as bound SC or unoccupied as free SC. This transport pathway may be exploited to target bioactive molecules to the mucosal surface. To identify small peptide motifs with specific affinity to human pIgR, we used purified free SC and selection from randomized, cysteine-flanked 6- and 9-mer phage-display libraries. One of the selected phages, called C9A, displaying the peptide CVVWMGFQQVC, showed binding both to human free SC and SC complexed with pIgs. However, the pneumococcal surface protein SpsA (Streptococcus pneumoniae secretory IgA-binding protein), which binds human SC at a site distinct from the pIg binding site, competed with the C9A phage for binding to SC. The C9A phage showed greatly increased transport through polarized Madin-Darby canine kidney cells transfected with human pIgR. This transport was not affected by pIgA nor did it inhibit pIgR-mediated pIgA transcytosis. A free peptide of identical amino acid sequence as that displayed by the C9A phage inhibited phage interaction with SC. This implied that the C9A peptide sequence may be exploited for pIgR-mediated epithelial transport without interfering with secretory immunity.