The primary structure of sperm whale hemoglobin (Physeter catodon, cetacea)

The complete primary structure of the two major hemoglobin components of sperm whale (Physeter catodon) is presented. The major components A and B account for 55% and 40% respectively whereas the minor component constitutes for 5% of the total hemoglobin. The globin chains were separated on CM-Cellulose in 8M urea buffer. The sequence was determined by automatic Edman degradation of tryptic and hydrolytic peptides in a liquid phase sequencer. Alignment of the sequence with human hemoglobin shows 22 exchanges each for the alpha I and alpha II and 21 exchanges for the beta I and beta II chains. Within the two beta-chains three differences have been located, beta NA2 His/Gln, beta A2 Gly/Ala and beta A8 Leu/Val. The two alpha-chains are characterized by heterogeneities at position alpha A8 Val/Ile or Ala/Ile (ratio of the phenylthiohydantoin derivatives of the amino acids 1:1) and alpha AB1 Asn/Ser (ratio of the phenylthiohydantoin derivatives of the amino acids 6:4). The role of these exchanges in modulating oxygen affinity is discussed.     

A split luciferase-based reporter for detection of a cellular macromolecular complex

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear RNPs (snRNPs). To process large numbers of introns with a limited number of snRNPs, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage. However, the mechanism that maintains cellular snRNP levels is unknown. Molecules that modulate cellular snRNP levels may help to define this mechanism but are not available. Therefore, the goal of the current study was to develop a reporter for snRNP levels using split luciferase based on proteomic analysis of snRNPs. We constructed an expression library of a luciferase fragment fused to core components of U5 snRNP and used it to isolate pre-mRNA processing factor 6 (PRPF6) and small nuclear ribonucleoprotein 40 kDa (U5-40K) that specifically reconstitute luciferase activity in the U5 snRNP complex. Here we show that this reporter detects the effects of small molecules on the levels of the U5 snRNP reporter protein complex. Our approach provides an alternative assay to discover small molecules targeting a macromolecular complex when the structure of the complex is not precisely identified.     

Fluorescence Lifetime Imaging of Interactions between Golgi Tethering Factors and Small GTPases in Plants

Peripheral tethering factors bind to small GTPases in order to obtain their correct location within the Golgi apparatus. Using fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) we visualized interactions between Arabidopsis homologues of tethering factors and small GTPases at the Golgi stacks in planta . Co-expression of the coiled-coil proteins AtGRIP and golgin candidate 5 (GC5) [TATA element modulatory factor (TMF)] and the putative post-Golgi tethering factor AtVPS52 fused to green fluorescent protein (GFP) with mRFP (monomeric red fluorescent protein) fusions to the small GTPases AtRab-H1^b, AtRab-H1^c and AtARL1 resulted in reduced GFP lifetimes compared to the control proteins. Interestingly, we observed differences in GFP quenching between the different protein combinations as well as selective quenching of GFP-AtVPS52-labelled structures. The data presented here indicate that the FRET-FLIM technique should prove invaluable in assessing protein interactions in living plant cells at the organelle level.     

Colonization of segmented filamentous bacteria and its interaction with the luminal IgA level in conventional mice

Segmented filamentous bacteria (SFB) colonize in the ileum. They promote the development of intraepithelial lymphocytes and immunoglobulin A (IgA)-producing cells in the small intestine. In SFB-monoassociated mice, changes in SFB colonization of the small intestine were related to the level of IgA derived from maternal milk during the suckling period and self-produced in the small intestine after weaning. In this study, we investigated whether or not maternal and neonatal IgA influence the colonization of SFB in conventional mice from 18 to 105 days old. The pups were forcedly weaned at 20 days old. SFB could be detected in the distal small intestine after day 22, and their number rapidly reached a maximum on day 28. Thereafter, they gradually declined to one-fourth of the maximum level. The lowest concentrations of IgA in the small intestinal and cecal contents were detected on day 22. Thereafter, they increased as the age of the mice increased. The expression of the polymeric immunoglobulin receptor gene in the distal small intestine increased after weaning. These results suggested that the colonization of SFB in the pre-weaning and post-weaning periods might be prevented with IgA derived from maternal milk and self-produced IgA, respectively.     

Autoinhibition regulates cellular localization and actin assembly activity of the diaphanous-related formins FRLalpha and mDia1

Diaphanous-related formins (DRFs) are key regulators of actin cytoskeletal dynamics whose in vitro actin assembly activities are thought to be regulated by autoinhibition. However, the in vivo consequences of autoinhibition and the involvement of DRFs in specific biological processes are not well understood. In this study, we show that in the DRFs FRLalpha (formin-related gene in leukocytes alpha) and mouse diaphanous 1, autoinhibition regulates a novel membrane localization activity in vivo as well as actin assembly activity in vitro. In FRLalpha, the Rho family guanosine triphosphatase Cdc42 relieves the autoinhibition of both membrane localization and biochemical actin assembly activities. FRLalpha is required for efficient Fc-gamma receptor-mediated phagocytosis and is recruited to the phagocytic cup by Cdc42. These results suggest that mutual autoinhibition of biochemical activity and cellular localization may be a general regulatory principle for DRFs and demonstrate a novel role for formins in immune function.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

Crystal Structure of the Formin mDia1 in Autoinhibited Conformation

Background

Formin proteins utilize a conserved formin homology 2 (FH2) domain to nucleate new actin filaments. In mammalian diaphanous-related formins (DRFs) the FH2 domain is inhibited through an unknown mechanism by intramolecular binding of the diaphanous autoinhibitory domain (DAD) and the diaphanous inhibitory domain (DID).

Methodology/Principal Findings

Here we report the crystal structure of a complex between DID and FH2-DAD fragments of the mammalian DRF, mDia1 (mammalian diaphanous 1 also called Drf1 or p140mDia). The structure shows a tetrameric configuration (4 FH2 + 4 DID) in which the actin-binding sites on the FH2 domain are sterically occluded. However biochemical data suggest the full-length mDia1 is a dimer in solution (2 FH2 + 2 DID). Based on the crystal structure, we have generated possible dimer models and found that architectures of all of these models are incompatible with binding to actin filament but not to actin monomer. Furthermore, we show that the minimal functional monomeric unit in the FH2 domain, termed the bridge element, can be inhibited by isolated monomeric DID. NMR data on the bridge-DID system revealed that at least one of the two actin-binding sites on the bridge element is accessible to actin monomer in the inhibited state.

Conclusions/Significance

Our findings suggest that autoinhibition in the native DRF dimer involves steric hindrance with the actin filament. Although the structure of a full-length DRF would be required for clarification of the presented models, our work here provides the first structural insights into the mechanism of the DRF autoinhibition.     

Clinically Relevant Characterization of Lung Adenocarcinoma Subtypes Based on Cellular Pathways: An International Validation Study

Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient''s prognosis. Additional results suggest the ability to use a similar approach to determine a patient''s sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.