Multiple display of a protein domain on a bacterial polymeric scaffold.

The multiple display of protein domains on polymeric scaffolds is an emerging technology for many applications. BLS is a highly immunogenic protein that has an oligomeric structure formed by a 17.2 kDa subunit arranged as a dimer of pentamers. Here we describe the production as well as the structural, functional, and immunological properties of a 9 kDa double-stranded RNA-binding domain (RBD3) fused to the structure of BLS. We demonstrate that the BLS and RBD3 modules are stably and independently folded in the structure of the chimera and form a decameric structure of 255 kDa as the native BLS oligomers. The polymeric display of RBD3 in the structure of BLS increases the dsRNA binding strength of this domain both in vitro and in vivo, and also enhances its immunogenicity to the point that it breaks the tolerance of mice to the RBD3 self-antigen. Our results underscore the BLS display strategy as a powerful tool for biotechnological and therapeutic applications.

Targeting of mRNAs within the glial cell cytoplasm: how to hide the message along the journey.

The subcellular targeting of mRNAs encoding myelin proteins to the oligodendrocyte processes is an accepted fact in myelin formation. How these messengers are kept silent during their movement to the subcellular domain where they are turned on remains a mystery. This review focuses on aspects of mRNA targeting and speculates on possible molecular mechanisms for the translational control of myelin-located mRNAs.

Subcellular fractionation and association with the cytoskeleton of messengers encoding myelin proteins.

The targeting of polypeptides to restricted cytoplasmic domains by means of mRNA sorting is a widespread phenomena utilized by many cell types. In the central nervous system, in situ hybridization analysis has shown previously that the mRNAs encoding several myelin-specific proteins are specifically located within the myelinating processes of oligodendrocytes. Here, by means of biochemical and subcellular fractionation methods, we show that a myelin fraction is selectively enriched in those mRNAs. The four major myelin basic protein (MBP) mRNAs that arise by alternative splicing of exons II and VI of the MBP gene are concentrated in this subcellular fraction. Furthermore, an interaction of MBP and MOBP 81A mRNAs with the cytoskeleton was observed. This interaction might serve to mediate the anchoring of these messengers after translocation to the subcellular site of translation.

Non-coding plasmid DNA induces IFN-gamma in vivo and suppresses autoimmune encephalomyelitis.

Regulatory sequences used in plasmids for naked DNA vaccination can modulate cytokine production in vivo. We demonstrate here that injection of plasmid DNA can suppress the prototypic T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis, by inducing IFN-gamma.

Expression of autoimmune disease-related antigens by cells of the immune system.

The process of thymic selection is critical for the generation of the mature T-cell repertoire, yet the nature of the self-peptides that serve this function is not known. Several studies suggest that tissue-specific auto-antigens are expressed in the thymus. We initiated this study to examine the expression of a panel of auto-antigens related to several autoimmune diseases in the thymus, peripheral lymphoid organs, and various cell lines. We looked for the expression of these antigens by reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorter (FACS) analysis, immunoblotting, and immunoprecipitation. We found that in the thymus there is evidence for the expression of a wide variety of disease-related self-antigens including myelin antigens, insulin, cardiac myosin, and retinal S antigen. By FACS analysis, several monoclonal anti-myelin basic protein antibodies were found to bind to immune cells. In Western blotting, we could find in the thymus and other lymphoid organs the expression of myelin basic protein, proteolipid protein, and cyclic nucleotide phosphodiesterase; in contrast, the staining for myelin oligodendrocyte glycoprotein, microtubule-associated Tau protein, and insulin were negative in these organs. The results of these studies confirm that there is evidence for the expression of a variety of auto-antigens in the immune system, both at the mRNA and protein levels, potentially enabling them to participate in the process of thymic education.

Multiple sclerosis: from a myelin point of view.

Multiple sclerosis (MS) is a demyelinating disease during which an autoimmune reaction is directed against oligodendrocytes. Alterations of normal myelin structure or oligodendrocyte metabolism may be primary events that influence the susceptibility to MS. Once triggered, the immune system attacks and destroys myelin and the myelin forming cell. Evidence is presented that the oligodendrocyte responds to the attack by immune cells and their secreted products through modulation of its metabolism and gene expression. Cytokines, immunoglobulins, and complement complexes may elicit a survival response in the oligodendrocytes, involving the induction of heat shock proteins and other protective molecules. The possibility of manipulating these complex glial cell functions and controlling their pathologic interactions with immune cells will illuminate how myelin damage can be contained and how the injured tissue can be repaired.

Myelin basic protein mRNA localization and polypeptide targeting.

Myelin basic proteins (MBPs), the major peripheral membrane proteins of central nervous system (CNS) myelin, are encoded by mRNAs that are selectively segregated to the myelinating processes of oligodendrocytes. In order to test whether the intracellular mechanisms responsible for MBP mRNA translocation are oligodendrocyte-specific, or alternatively, are present in other cell types and may therefore be more general, we have studied the localization of the 14 kD MBP mRNA and its encoded polypeptide (MBP14) in transiently transfected HeLa cells (a cervical carcinoma cell line) and in the rat pheochromocytoma cell line PC12. Unlike the situation in oligodendrocytes in situ, where MBP mRNAs are translocated and become "centrifugally" distributed, in both of the non-glial cells MBP mRNA was primarily detected in the perinuclear region. The MBP14 polypeptide was found associated with intracellular membranes, and not exclusively with the plasma membrane. Our results indicate that the inability of HeLa and PC12 cells to correctly target MBP mRNAs to the cell periphery leads to a failure to incorporate MBP polypeptides directly into the plasma membrane. Further, the data lend credence to the concept that MBP mRNA segregation appears to be a specific feature of myelin-forming cells which is required for the precise delivery of the encoded polypeptides to the forming myelin membrane.

The distribution of myelin basic protein mRNAs within myelinating oligodendrocytes.

The nervous system contains oligodendrocytes with processes that are greatly extended in space. It is now clear that there are numerous complex, poorly understood mechanisms by which polypeptides are synthesized and delivered to their sites of function in these cells. One mechanism is by the active positioning of mRNAs encoding certain proteins to restricted intracellular subdomains. Perhaps the best studied example of this in the vertebrate CNS is the translocation of myelin basic protein mRNAs to the forming myelin sheath, where the newly synthesized polypeptides, which avidly associate with membranes, can be directly incorporated into the myelin membrane. Evidence for this conclusion is presented here in the context of related work on the general phenomenon of mRNA translocation that is under analysis in other systems.

In vivo biosynthesis of a stage-specific cuticle glycoprotein during early metamorphosis of the medfly Ceratitis capitata.

Cuticle proteins of an insect pest, the Medfly Ceratitis capitata, were resolved in polyacrylamide gels and partially characterized. The pupal cuticle was found to be different from cuticles of other insects since more than 80% w/w of the protein is a single mannose-containing polypeptide (PCG-100). The temporally-regulated in vivo biosynthesis and deposition of cuticle proteins was studied by microinjection of [35S]methionine followed by hand dissection of pupal cuticles. The major pupal glycoprotein, PCG-100, is cuticle- and stage-specific and was the earliest to be labeled and deposited. Its synthesis was maximal at around 46 hours after pupariation and then it decreased. The deposited PCG-100 and other minor pupal cuticle proteins become non-extractable at the end of the instar (7 days after pupariation) probably by sclerotization phenomena. These results provide insight into the temporal control of gene expression programs involved in cuticle deposition during medfly metamorphosis.