Structural and dynamic consequences of increasing repeats in a MUC1 peptide tumor antigen

MUC1 mucin is a large transmembrane glycoprotein whose extracelluler domain is composed of repeating units of a 20 amino acid sequence. In the cancer associated state, this protein expression becomes upregulated and underglycosylated. Previous studies, which show an enhanced binding of a 5-repeat over a 1-repeat MUC1 peptide to a panel of anti-MUC1 antibodies, have led us to investigate the structural and dynamic consequences of increasing repeat number. Two MUC1 peptides were studied: a 16mer corresponding to slightly less than one full repeat of the MUC1 tandem repeat sequence (GVTSAPDTRPAPGSTA) and a 40mer corresponding to two full repeats of the MUC1 sequence (VTSAPDTRPAPGSTAPPAHG)2. Isotopically labeled versions of these MUC1 peptides were cloned, expressed, purified, and evaluated structurally and dynamically using 15N- and 13C-edited NMR approaches. The data show that MUC1 structure, dynamics, and antibody binding affinity are invariant with increasing repeat number. In light of these results, we conclude that the enhanced antibody affinity of the 5-repeat over the 1-repeat MUC1 peptide is due to multivalency effects, and not due to the development of higher order structure in the longer length peptides. The implications of these results are discussed within the context of a multiple repeat MUC1 breast cancer vaccine design.     

Pathogen recognition and development of particulate vaccines: does size matter?

The use of particulate carriers holds great promise for the development of effective and affordable recombinant vaccines. Rational development requires a detailed understanding of particle up-take and processing mechanisms to target cellular pathways capable of stimulating the required immune responses safely. These mechanisms are in turn based on how the host has evolved to recognize and process pathogens. Pathogens, as well as particulate vaccines, come in a wide range of sizes and biochemical compositions. Some of these also provide 'danger signals' so that antigen 'senting cells (APC), usually dendritic cells (DC), acquire specific stimulatory activity. Herein, we provide an overview of the types of particles currently under investigation for the formulation of vaccines, discuss cellular uptake mechanisms (endocytosis, macropinocytosis, phagocytosis, clathrin-dependent and/or caveloae-mediated) for pathogens and particles of different sizes, as well as antigen possessing and presentation by APC in general, and DC in particular. Since particle size and composition can influence the immune response, inducing humoral and/or cellular immunity, activating CD8 T cells and/or CD4 T cells of T helper 1 and/or T helper 2 type, particle characteristics have a major impact on vaccine efficacy. Recently developed methods for the formulation of particulate vaccines are presented in this issue of Methods, showcasing a range of "cutting edge" particulate vaccines that employ particles ranging from nano to micro-sized. This special issue of Methods further addresses practical issues of production, affordability, reproducibility and stability of formulation, and also includes a discussion of the economic and regulatory challenges encountered in developing vaccines for veterinary use and for common Third World infectious diseases.     

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72–98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-β family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-β family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF). Received: 21 August 2000 / Accepted: 9 March 2001     

Complementation of null CF mice with a human CFTR YAC transgene.

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by approximately 5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild-type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunner's glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.     

Efficient Chromosome Biorientation and the Tension Checkpoint in Saccharomyces cerevisiae both Require Bir1

Accurate chromosome segregation requires the capture of sister kinetochores by microtubules from opposite spindle poles prior to the initiation of anaphase, a state termed chromosome biorientation. In the budding yeast Saccharomyces cerevisiae, the conserved protein kinase Ipl1 (Aurora B in metazoans) is critical for ensuring correct chromosomal alignment. Ipl1 associates with its activators Sli15 (INCENP), Nbl1 (Borealin), and Bir1 (Survivin), but while Sli15 clearly functions with Ipl1 to promote chromosome biorientation, the role of Bir1 has been uncertain. Using a temperature-sensitive bir1 mutant (bir1-17), we show that Bir1 is needed to permit efficient chromosome biorientation. However, once established, chromosome biorientation is maintained in bir1-17 cells at the restrictive temperature. Ipl1 is partially delocalized in bir1-17 cells, and its protein kinase activity is markedly reduced under nonpermissive conditions. bir1-17 cells arrest normally in response to microtubule depolymerization but fail to delay anaphase when sister kinetochore tension is reduced. Thus, Bir1 is required for the tension checkpoint. Despite their robust mitotic arrest in response to nocodazole, bir1-17 cells are hypersensitive to microtubule-depolymerizing drugs and show a more severe biorientation defect on recovery from nocodazole treatment. The role of Bir1 therefore may become more critical when spindle formation is delayed.Accurate chromosome segregation during anaphase is vital for ensuring the maintenance of genome integrity during cell division and, in turn, depends critically on the correct attachment of sister chromatids to kinetochore microtubules. For high-fidelity chromosome segregation, kinetochores must capture spindle microtubules such that sister chromatids are connected to opposite spindle poles (termed amphitelic attachment or chromosome biorientation), ensuring that they are pulled in opposite directions during the subsequent anaphase.In the budding yeast Saccharomyces cerevisiae, the majority of sister chromatids remain attached to microtubules from a single pole (mono-oriented) without the intervention of a correction mechanism to promote amphitelic attachment (36), a key element of which is the Ipl1 protein kinase. Ipl1 has been proposed to promote the detachment of incorrect microtubule-kinetochore connections so that correct attachments subsequently can form (35). In the absence of Ipl1 function, at the point of anaphase onset around two-thirds of sister chromatids remain mono-oriented, attached to microtubules originating from a single pole to which they then cosegregate (35). Kinetochore proteins such as Dam1 and Ndc80 have been proposed as key Ipl1 substrates for their role in promoting chromosome biorientation (6, 41). Ipl1 kinase also is required for cells to activate the spindle checkpoint in the absence of tension on kinetochore-microtubule attachments, and hence ipl1 mutant cells fail to delay anaphase despite their many mono-oriented chromosomes (2). Depending on the circumstances, the checkpoint role of Ipl1 involves either the generation of unattached kinetochores (26) or the phosphorylation of the checkpoint protein Mad3 (19). Ipl1 also is required in the absence of the BimC family kinesin Cin8p, probably reflecting a role in spindle assembly (9, 21), and is involved in regulating spindle disassembly following anaphase (5).Ipl1 kinase is highly conserved, and its metazoan ortholog (Aurora B) is involved in both chromosome biorientation and the spindle assembly checkpoint, forming part of the chromosomal passenger complex that also contains INCENP, Survivin, and Borealin (27, 40). The chromosomal passenger complex is so called because although these proteins colocalize throughout the cell cycle, their location changes dynamically from the chromosome arms in G₁ to centromeres in prometaphase and finally to the central spindle in anaphase. Such coordinated behavior is consistent with the recent crystal structure of the complex between INCENP, Survivin, and Borealin, in which they interact via tightly entwined helical domains (16).In budding yeast, Ipl1 interacts with Sli15, Bir1, and Nbl1, which have been proposed to be orthologs of INCENP, Survivin, and Borealin, respectively (6, 18). All three proteins are the products of essential genes. Like INCENP, Sli15 has a conserved C-terminal domain (the IN-box) that is required for Ipl1 kinase activation, and sli15 mutants have a phenotype that is very similar to that of ipl1 mutants (17, 18). Although yeast cells with reduced Bir1 function show chromosome instability, the first-described bir1 mutants failed to reveal a chromosome biorientation defect but instead conferred defects in septin dynamics during anaphase (38). Bir1 interacts with Ndc10 and is responsible for taking Ndc10 to the anaphase spindle (38, 42, 43), a role that may be linked to this septin defect (4). Yeast Bir1 is much larger than its metazoan counterpart (Survivin) and shows little sequence conservation outside the conserved BIR domain, yet this region is nonessential in yeast (42) and therefore unlikely to be involved in chromosome biorientation. Conversely, metazoan Borealin proteins are much larger than yeast Nbl1, which consists of little more than the helical region proposed to form the tight interaction with INCENP/Sli15 and Survivin/Bir1 complexes. Furthermore, a significant fraction of both Sli15 and Bir1 are present in a complex that lacks Ipl1 (29, 38) and that recent work has shown to contain Nbl1 (25), bringing into question the importance of Bir1 for chromosome biorientation. The extent to which Bir1 and Survivin function in conserved or analogous ways within the chromosomal passenger complexes of yeast and metazoans therefore was unclear at the start of our work.The Sli15-Bir1 complex has been proposed to interact both with microtubules (via the central domain of Sli15) and with kinetochores (through the Bir1-Ndc10 interaction) and through these interactions to function as a tension sensor, relaying information concerning the state of microtubule-kinetochore connections to Ipl1 kinase. Thus, when chromosomes are mono-oriented, the Bir1-Sli15-Nbl1 complex might activate Ipl1 in the absence of tension so as to promote chromosome biorientation by detaching incorrect microtubule attachments (29). This model predicts an essential role for Bir1 in promoting chromosome biorientation, but such evidence has been lacking. By generating a temperature-sensitive bir1 allele (bir1-17) and showing that it confers a profound defect in chromosome biorientation, we demonstrate that Bir1 does play a key role in the correction process needed to ensure that all yeast chromosomes become correctly aligned on the mitotic spindle. Furthermore, since the bir1-17 mutant fails to activate the spindle assembly checkpoint properly in response to reduced sister kinetochore tension, like Ipl1 it forms part of the tension checkpoint mechanism. Our data therefore are consistent with a role for Bir1 in conferring tension responsiveness on Ipl1 function.     

Integrins of Anopheles gambiae and a putative role of a new beta integrin, BINT2, in phagocytosis of E. coli

Mosquitoes use effective immune responses, including phagocytosis, to fight microbial infection. Here we show that in an Anopheles gambiae immune responsive cell line, RGD recognizing receptors play an important role in the phagocytic response, suggesting overlap between molecular components implicated in adhesion and phagocytosis. Integrins are a major class of adhesive receptors that recognize ligands containing an RGD motif. We have cloned a gene encoding a new beta integrin, BINT2, and demonstrated its involvement in Escherichia coli engulfment. Based on molecular modeling, we propose a structural reason for the role of BINT2, but not BINT1, on phagocytosis of Gram-negative bacteria. Using bioinformatic tools, we have identified and compared the complete A. gambiae integrin repertoire as a prelude to a future systematic functional study.     

Pax6 is misexpressed in Sox1 null lens fiber cells

Sox1 null lens fiber cells fail to elongate and have disrupted expression of gamma-crystallin. We have evaluated the expression of Sox1 and Pax6 proteins during critical stages of lens morphogenesis, with particular focus on fiber cell differentiation. While Pax6 and Sox1 are co-expressed during early stages of fiber cell differentiation, Sox1 up-regulation coincides temporally with the down-regulation of Pax6, and these proteins therefore display a striking inverse expression pattern in the lens fiber cell compartment. Furthermore, Pax6 is inappropriately expressed in the fiber cells of Sox1 null mice and the Pax6 target, alpha5 integrin, is simultaneously misexpressed. Finally, we demonstrate a genetic interaction between Sox1 and Pax6, as Sox1 heterozygosity partially rescues the diameter of Pax6(Sey) lenses by increasing the number of cells in the fiber cell compartment.     

Changes in muscle proteomics in the course of the Caudwell Research Expedition to Mt. Everest

This study employed differential proteomic and immunoassay techniques to elucidate the biochemical mechanisms utilized by human muscle (vastus lateralis) in response to high altitude hypoxia exposure. Two groups of subjects, participating in a medical research expedition (A, n = 5, 19d at 5300 m altitude; B, n = 6, 66d up to 8848 m) underwent a ≈ 30% drop of muscular creatine kinase and of glycolytic enzymes abundance. Protein abundance of most enzymes of the tricarboxylic acid cycle and oxidative phosphorylation was reduced both in A and, particularly, in B. Restriction of α‐ketoglutarate toward succinyl‐CoA resulted in increased prolyl hydroxylase 2 and glutamine synthetase. Both A and B were characterized by a reduction of elongation factor 2alpha, controlling protein translation, and by an increase of heat shock cognate 71 kDa protein involved in chaperone‐mediated autophagy. Increased protein levels of catalase and biliverdin reductase occurred in A alongside a decrement of voltage‐dependent anion channels 1 and 2 and of myosin‐binding protein C, suggesting damage to the sarcomeric structures. This study suggests that during acclimatization to hypobaric hypoxia the muscle behaves as a producer of substrates activating a metabolic reprogramming able to support anaplerotically the tricarboxylic acid cycle, to control protein translation, to prevent energy expenditure and to activate chaperone‐mediated autophagy.