Neuroligin Trafficking Deficiencies Arising from Mutations in the α/β-Hydrolase Fold Protein Family

Despite great functional diversity, characterization of the α/β-hydrolase fold proteins that encompass a superfamily of hydrolases, heterophilic adhesion proteins, and chaperone domains reveals a common structural motif. By incorporating the R451C mutation found in neuroligin (NLGN) and associated with autism and the thyroglobulin G2320R (G221R in NLGN) mutation responsible for congenital hypothyroidism into NLGN3, we show that mutations in the α/β-hydrolase fold domain influence folding and biosynthetic processing of neuroligin3 as determined by in vitro susceptibility to proteases, glycosylation processing, turnover, and processing rates. We also show altered interactions of the mutant proteins with chaperones in the endoplasmic reticulum and arrest of transport along the secretory pathway with diversion to the proteasome. Time-controlled expression of a fluorescently tagged neuroligin in hippocampal neurons shows that these mutations compromise neuronal trafficking of the protein, with the R451C mutation reducing and the G221R mutation virtually abolishing the export of NLGN3 from the soma to the dendritic spines. Although the R451C mutation causes a local folding defect, the G221R mutation appears responsible for more global misfolding of the protein, reflecting their sequence positions in the structure of the protein. Our results suggest that disease-related mutations in the α/β-hydrolase fold domain share common trafficking deficiencies yet lead to discrete congenital disorders of differing severity in the endocrine and nervous systems.     

An endangered hemiparasitic shrub is the only host plant of the little-known Neotropical hairstreak <Emphasis Type="Italic">Strymon flavaria</Emphasis> (Lepidoptera: Lycaenidae) in the arid Andes

Larval host plants can be the main resource underlying the distribution (including altitudinal ranges) of specialized phytophagous insects such as butterflies. Strymon flavaria (Ureta) (Lepidoptera: Lycaenidae: Theclinae: Eumaeini) is a little-known hairstreak endemic to the arid belt at about 3000–3500 m elevation on the western slopes of the Andes of northern Chile. The hemiparasitic shrub Krameria lappacea (Dombey) Burdet and Simpson is here recorded as the first host plant known for S. flavaria, representing also the first record of the association of a species of Strymon Hübner with the plant family Krameriaceae. Females of S. flavaria lay eggs on flower buds and unripe fruits of K. lappacea; the larva eats mostly these plant organs. Field observations revealed that S. flavaria is a host-specialist hairstreak, since females lay eggs only on K. lappacea. These findings suggest that the altitudinal range of S. flavaria could be the result of its specialized association with K. lappacea, as this shrub is also mostly restricted to elevations above 3000 m in the Andes of northern Chile. The implications of this finding for the ecology and conservation of S. flavaria are discussed.     

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2??, which was downregulated during development similarly to XLAP2??. Embryonic isoforms XLAP2?? and XLAP2?? were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2?? were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2?? was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2?? was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains.     

Reptilian myotomal myogenesis-lessons from the sand lizard Lacerta agilis L. (Reptilia, Lacertidae)Update

Reptilian myotomal myogenesis is poorly understood. This paper reports on structural, ultrastructural and immunocytochemical studies of muscle differentiation in sand lizard (Lacerta agilis) embryos. During somitogenesis, the somites are composed of epithelial vesicles with a centrally located somitocoel. At later developmental stages the ventral portion of the somite cortex disaggregates into the sclerotome mesenchyme, while the dorsal wall of the somite differentiates into dermomyotome. At these developmental stages, mononucleated cells of the dermomyotome are Pax3-positive. The dermomyotome layer forms the dorsomedial and ventromedial lips. The myotome is first composed of mono- and then of multinucleated myotubes and small mononucleated cells that occur in the vicinity of the myotubes. These mononucleated cells exhibit low proliferative potential as revealed by the use of PCNA antibody. At subsequent stages of myogenesis the mononucleated cells express Pax7 protein, a marker of satellite cells, and assume ultrastructural features characteristic of satellite cells. Some of the mononucleated cells contribute to muscle growth, being involved in fusion with differentiating muscle fibers. This study revealed similarities of myotomal myogenesis in reptiles to that of other vertebrates.     

Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein

Restricting linear peptides to their bioactive conformation is an attractive way of improving their stability and activity. We used a cyclic peptide library with conformational diversity for selecting an active and stable peptide that mimics the structure and activity of the HIV-1 integrase (IN) binding loop from its cellular cofactor LEDGF/p75 (residues 361-370). All peptides in the library had the same primary sequence, and differed only in their conformation. Library screening revealed that the ring size and linker structure had a huge effect on the conformation, binding and activity of the peptides. One of the cyclic peptides, c(MZ 4-1), was a potent and stable inhibitor of IN activity in vitro and in cells even after 8 days. The NMR structure of c(MZ 4-1) showed that it obtains a bioactive conformation that is similar to the parent site in LEDGF/p75.     

Combinatorial docking approach for structure prediction of large proteins and multi-molecular assemblies

Protein folding and protein binding are similar processes. In both, structural units combinatorially associate with each other. In the case of folding, we mostly handle relatively small units, building blocks or domains, that are covalently linked. In the case of multi-molecular binding, the subunits are relatively large and are associated only by non-covalent bonds. Experimentally, the difficulty in the determination of the structures of such large assemblies increases with the complex size and the number of components it contains. Computationally, the prediction of the structures of multi-molecular complexes has largely not been addressed, probably owing to the magnitude of the combinatorial complexity of the problem. Current docking algorithms mostly target prediction of pairwise interactions. Here our goal is to predict the structures of multi-unit associations, whether these are chain-connected as in protein folding, or separate disjoint molecules in the assemblies. We assume that the structures of the single units are known, either through experimental determination or modeling. Our aim is to combinatorially assemble these units to predict their structure. To address this problem we have developed CombDock. CombDock is a combinatorial docking algorithm for the structural units assembly problem. Below, we briefly describe the algorithm and present examples of its various applications to folding and to multi-molecular assemblies. To test the robustness of the algorithm, we use inaccurate models of the structural units, derived either from crystal structures of unbound molecules or from modeling of the target sequences. The algorithm has been able to predict near-native arrangements of the input structural units in almost all of the cases, suggesting that a combinatorial approach can overcome the imperfect shape complementarity caused by the inaccuracy of the models. In addition, we further show that through a combinatorial docking strategy it is possible to enhance the predictions of pairwise interactions involved in a multi-molecular assembly.     

Laminopathies: The molecular background of the disease and the prospects for its treatment

Laminopathies are rare human degenerative disorders with a wide spectrum of clinical phenotypes, associated with defects in the main protein components of the nuclear envelope, mostly in the lamins. They include systemic disorders and tissue-restricted diseases. Scientists have been trying to explain the pathogenesis of laminopathies and find an efficient method for treatment for many years. In this review, we discuss the current state of knowledge about laminopathies, the molecular mechanisms behind the development of particular phenotypes, and the prospects for stem cell and/or gene therapy treatments.     

The ASPP interaction network: electrostatic differentiation between pro‐ and anti‐apoptotic proteins

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C‐terminal ankyrin repeats and SH3 domain (ANK‐SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl‐2, and NFκB. The structure of the complex between ASPP2_ANK‐SH3 and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2_ANK‐SH3 with Bcl‐2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2_ANK‐SH3 with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl‐2, and NFκB are different, yet lie on the same face of ASPP2_ANK‐SH3. The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein‐binding domains. A subset of surface‐exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl‐2, and NFκB. We also found a gain of positive charge at the non‐protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 John Wiley & Sons, Ltd.