2007 Annual progress report synopsis of the Center for Structures of Membrane Proteins

A synopsis of the 2007 annual progress report for the Center for Structures of Membrane Proteins, a specialized center of the Protein Structure Initiative.     

Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store

The transport of free polymannose-type oligosaccharides from the lumen of the endoplasmic reticulum into the cytosol has been recently demonstrated (Moore,S.E.H., et al., 1995, EMBO J., 14, 6034-6042), but at present little is known of the characteristics of this process. Here, it is shown that inhibition of the transport of endogenously synthesized metabolically radiolabeled free oligosaccharides out of the endoplasmic reticulum into the cytosol of permeabilized HepG2 cells occurs when assays are conducted in the presence of mannose (IC50, 4.9 mM), or its derivatives modified at the first carbon (C1) of the sugar ring; alpha-methyl mannoside (IC50, 2.0 mM), mannoheptulose (IC50, 1.6 mM), and alpha-benzyl mannoside (IC50, 0.8 mM), whereas other monosaccharides (50 mM), differing from mannose at position; C2 (glucose), C3 (altrose), C4 (talose), C5 (l-rhamnose), and C6 (mannoheptose), have little effect. N-Acetylglucosamine does not inhibit oligosaccharide transport and, furthermore, although mannobioses and a mannotriose inhibit free oligosaccharide transport, di-N-acetylchitobiose is without effect. It is also shown that if the transport assay buffer is either depleted of calcium ions, or supplemented with the Ca2+/Mg2+ATPase inhibitor, thapsigargin, or with calcium ionophores, free oligosaccharide transport out of the endoplasmic reticulum is inhibited. These results demonstrate that the terminal nonreducing mannosyl residues of free polymannose-type oligosaccharides and not their N-acetylglucosamine-containing reducing termini, play an important role in the interaction of the free oligosaccharide with the transport machinery, and that this transport process requires the presence of calcium sequestered in the lumen of the endoplasmic reticulum.      

Cloning and sequence analysis of desmosomal glycoproteins 2 and 3 (desmocollins): cadherin-like desmosomal adhesion molecules with heterogeneous cytoplasmic domains

Desmosomal glycoproteins 2 and 3 (dg2 and 3) or desmocollins have been implicated in desmosome adhesion. We have obtained a 5.0-kb-long clone for dg3 from a bovine nasal epidermal lambda gt11 cDNA library. Sequence analysis of this clone reveals an open reading frame of 2,517 bases encoding a polypeptide of 839 amino acids. The sequence consists of a signal peptide of 28 amino acids, a precursor sequence of 104 amino acids, and a mature protein of 707 amino acids. The latter has the characteristics of a transmembrane glycoprotein with an extracellular domain of 550 amino acids and a cytoplasmic domain of 122 amino acids. The sequence of a partial clone from the same library shows that dg2 has an alternative COOH terminus that is extended by 54 amino acids. Genomic DNA sequence data show that this arises by splicing out of a 46-bp exon that encodes the COOH-terminal 11 amino acids of dg3 and contains an in-frame stop codon. The extracellular domain of dg3 shows 39.4% protein sequence identity with bovine N-cadherin and 28.4% identity with the other major desmosomal glycoprotein, dg1, or desmoglein. The cytoplasmic domain of dg3 and the partial cytoplasmic domain of dg2 show 23 and 24% identity with bovine N-cadherin, respectively. The results support our previous model for the transmembrane organization of dg2 and 3 (Parrish, E.P., J.E. Marston, D.L. Mattey, H.R. Measures, R. Venning, and D.R. Garrod. 1990. J. Cell Sci. 96:239-248; Holton, J.L., T.P. Kenny, P.K. Legan, J.E. Collins, J.N. Keen, R. Sharma, and D.R. Garrod. 1990. J. Cell Sci. 97:239-246). They suggest that these glycoproteins are specialized for calcium-dependent adhesion in their extracellular domains and, cytoplasmically, for the molecular interactions involved in desmosome plaque formation. Moreover this represents the first example of alternative splicing within the cadherin family of cell adhesion molecules.     

Therapeutic implications of GIPC1 silencing in cancer

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.     

Structural basis for recruitment of Ubc12 by an E2 binding domain in NEDD8's E1

E2 conjugating enzymes play a central role in ubiquitin and ubiquitin-like protein (ublp) transfer cascades: the E2 accepts the ublp from the E1 enzyme and then the E2 often interacts with an E3 enzyme to promote ublp transfer to the target. We report here the crystal structure of a complex between the C-terminal domain from NEDD8's heterodimeric E1 (APPBP1-UBA3) and the catalytic core domain of NEDD8's E2 (Ubc12). The structure and associated mutational analyses reveal molecular details of Ubc12 recruitment by NEDD8's E1. Interestingly, the E1's Ubc12 binding domain resembles ubiquitin and recruits Ubc12 in a manner mimicking ubiquitin's interactions with ubiquitin binding domains. Structural comparison with E2-E3 complexes indicates that the E1 and E3 binding sites on Ubc12 may overlap and raises the possibility that crosstalk between E1 and E3 interacting with an E2 could influence the specificity and processivity of ublp transfer.     

Development of Trypanosoma (M.) theileri in Tabanids

Thus far the life cycle of Trypanosoma (Megatrypanum) theileri has not been studied. We collected tabanids during the mass hatching, when only few tabanids are infected with trypanosomes. Tabanids were caught immediately after attacking a bait cow to serve as controls or after they had been allowed to engorge on the Trypanosoma (M.) theileri-infected cow. Tabanids were kept in the laboratory and used to study the developmental cycle of T. (M.) theileri in the tabanid gut. From day 1 to day 10 the presumably unfed controls and the engorged tabanids were dissected and cytological smears made from the mid- and hindgut. In total 2.6% (1/38) of the controls and 39% (23/59) of the engorged tabanids were positive for trypanosomes in the 1991 season. From day 1 to day 4 after engorgement trypanosomes were found in the midgut. Epimastigotes with a length of 29 μm on day 1 after infection multiplied by inequal division to form smaller epimastigotes of 26 μm on day 3. On day 4 morphologically indistinguishable trypanosomes of 21 μm total length were found in both mid- and hindgut. From day 5 to day 10 trypanosomes were found only in the hindgut in which the transformation to metacyclics was demonstrated, i.e. epimastigotes transformed to amastigote stages of 5 μm in total length.     

Comparison of image analysis software packages in the assessment of adhesion of microorganisms to mucosal epithelium using confocal laser scanning microscopy

We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+, and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms, the dynamic range that could be detected by the software packages was 1x10(6)-1x10(9) cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our 'Region of Interest' method, with the software's 'Standard Area Method' of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By pre-incubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250 mug/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells.     

Localization of polyadenylated RNAs during teloplasm formation and cleavage in leech embryos

In the embryos of glossiphoniid leeches, as in many annelids, cytoplasmic reorganization prior to first cleavage generates domains of yolk-deficient cytoplasm (called teloplasm) that are sequestered during the first three cell divisions to the D' macromere. Subsequently, the D' macromere generates a set of embryonic stem cells (teloblasts) that are the progenitors of the definitive segmental tissues. The hypothesis that fate-determining substances are localized within the teloplasm and segregated to the D macromere during cleavage is supported by experiments in which a redistribution of yolk-defcient cytoplasm changes the fate of blastomeres that inherit it (Astrow et al. 1987; Devries 1973; Nelson and Weisblat 1992). As a step toward identifying fate-determining factors in teloplasm, we describe the distribution of polyadenylated RNAs (polyA+ RNA) in the early embryo of the leech, Helobdella triserialis, as inferred from in situ hybridization using tritiated polyuridylic acid (3H-polyU). Our results indicate that polyA+ RNA colocalizes with teloplasm during cytoplasmic rearrangements resulting in teloplasm formation, and that it remains concentrated in the teloplasm during the cell divisions and a second cytoplasmic rearrangement during early embryogenesis. Lesser amounts of polyA+ RNA appear to be localized in cortical cytoplasm at most stages.