Adaptation of the Ras-recruitment system to the analysis of interactions between membrane-associated proteins

Interactions of membrane-associated proteins play important roles in many cellular processes. The yeast two-hybrid assay is of limited utility for the analysis of such interactions, due to the need for soluble protein partners, whose interaction is assessed in the nucleus. The advent of the Ras-recruitment system (RRS) has enabled the study of membrane-associated proteins interacting with cytoplasmic proteins fused to Ras. Constitutive membrane association of the Ras fusion protein is expected to complement the growth defect of the yeast strain CDC25-2, assayed in the RRS, independent from the interaction with a membrane-bound partner. We describe the adaptation of the RRS to the analysis of interactions between two membrane-associated proteins using a model system. These results may facilitate the study of protein–protein interactions between membrane-bound proteins and further increase the utility of the RRS.     

The SBASE domain sequence library,release 10: domain architecture prediction

SBASE (http://www.icgeb.trieste.it/sbase) is an on-line collection of protein domain sequences and related computational tools designed to facilitate detection of domain homologies based on simple database search. The 10th 'jubilee release' of the SBASE library of protein domain sequences contains 1 052 904 protein sequence segments annotated by structure, function, ligand-binding or cellular topology, clustered into over 6000 domain groups. Domain identification and functional prediction are based on a comparison of BLAST search outputs with a knowledge base of biologically significant similarities extracted from known domain groups. The knowledge base is generated automatically for each domain group from the comparison of within-group ('self') and out-of-group ('non-self') similarities. This is a memory-based approach wherein group-specific similarity functions are automatically learned from the database.     

The structure of bovine lysosomal alpha-mannosidase suggests a novel mechanism for low-pH activation

Lysosomal alpha-mannosidase (LAM: EC 3.2.1.24) belongs to the sequence-based glycoside hydrolase family 38 (GH38). Two other mammalian GH38 members, Golgi alpha-mannosidase II (GIIAM) and cytosolic alpha-mannosidase, are expressed in all tissues. In humans, cattle, cat and guinea pig, lack of lysosomal alpha-mannosidase activity causes the autosomal recessive disease alpha-mannosidosis. Here, we describe the three-dimensional structure of bovine lysosomal alpha-mannosidase (bLAM) at 2.7A resolution and confirm the solution state dimer by electron microscopy. We present the first structure of a mammalian GH38 enzyme that offers indications for the signal areas for mannose phosphorylation, suggests a previously undetected mechanism of low-pH activation and provides a template for further biochemical studies of the family 38 glycoside hydrolases as well as lysosomal transport. Furthermore, it provides a basis for understanding the human form of alpha-mannosidosis at the atomic level. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession codes 1o7d and r1o7dsf).     

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.     

Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in k(cat). The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction. The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.     

Percutaneous cryoablation of colorectal liver metastases: potentiated by two consecutive freeze-thaw cycles

Cryoablation may be beneficial for selected patients with liver tumours. Two freeze-thaw cycles at the same location have been recommended during treatment as this potentiate the effect of ablation in experimental studies. However, single freeze ablations are used by some as double freeze procedures are time-consuming and have been associated with increased risk of complications. Estimation of ice-ball volume is difficult using regularly used monitoring techniques. Magnetic resonance imaging, however, allows excellent and multiplanar visualisation of the frozen region during ablation. We comment on the effect of double freeze cycles in regard to ice-ball volume as estimated from magnetic resonance imaging during percutaneous cryoablation of colorectal liver metastases. The ice-ball volume at the end of the second freeze cycle was median 42% larger than the volume at the end of the first freeze. Double freeze cycles may thus facilitate tumour destruction.     

Characterization of Stachybotrys from water-damaged buildings based on morphology, growth, and metabolite production

Stachybotrys was found to be associated with idiopathic pulmonary hemorrhage in infants in Cleveland, Ohio. Since that time, considerable effort has been put into finding the toxic components responsible for the disease. The name Stachybotrys chartarum has been applied to most of these isolates, but inconsistent toxicity results and taxonomic confusion prompted the present study. In this study, 122 Stachybotrys isolates, mainly from water-damaged buildings, were characterized and identified by combining three different approaches: morphology, colony characteristics, and metabolite production. Two different Stachybotrys taxa, S. chartarum and one undescribed species, were found in water-damaged buildings regardless of whether the buildings were in Denmark, Finland, or the USA. Furthermore, two chemotypes could be distinguished in S. chartarum. One chemotype produced atranones, whereas the other was a macrocyclic trichothecene-producer. The second undescribed taxon produced atranones and could be differentiated from S. chartarum by its growth characteristics and pigment production. Our results correlate with different inflammatory and toxicological properties reported for these same isolates and show that the three taxa/chemotypes should be treated separately. The co-occurrence of these three taxa/chemotypes in water-damaged buildings explains the inconsistent results in the literature concerning toxicity of Stachybotrys isolated from that environment.     

Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase Lck

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.