Novartis Foundation Symposium最新文献

What are molecular chaperones and how should we think about them? We propose that it is better to think in terms of a chaperone function rather than in terms of chaperone molecules. We define the chaperone function as the prevention or reversal of incorrect interactions that may occur when reactive macromolecular surfaces are transiently exposed to the intracellular environment. We suggest that this function is a distinct and essential cellular function, mediated by many different proteins. Chaperones have evolved to reduce, by a variety of mechanisms, the aggregation of proteins into non-functional, and sometimes cytotoxic, structures. Chaperones may also have evolved to have additional roles. A cellular or extracellular event mediated by a chaperone protein is not necessarily a consequence of that protein's chaperone function. The aim of this article is to provide a brief summary of the origin and concepts used in the intracellular chaperone field, to provide a backdrop for discussion of their possible roles outside the cell.

Thioredoxin (TRX) is a small multifunctional protein with a redox-active dithiol/disulfide in the conserved active site. Human TRX was originally identified as a cytokine-like factor in virus-transformed cells. The TRX family of proteins share the active site sequence: -Cys-Xxx-Xxx-Cys-. Intracellularly TRX scavenges reactive oxygen species and regulates various signal transduction pathways in cellular activation and apoptosis. TRX is induced and released from cells in response to oxidative stress. TRX levels in plasma or serum are good markers for oxidative stress. Extracellularly, TRX shows anti-inflammatory effects. Circulating TRX in plasma inhibits neutrophil extravasation into the inflammatory sites. More recently we found that TRX inhibits the expression and release of macrophage migration inhibitory factor (MIF), which is a classical proinflammatory cytokine and a member of TRX family. Administration of recombinant TRX protein may become a novel therapeutic strategy for acute inflammatory disorders.

A number of molecular chaperones in mammalian cells are localized in mitochondria and they are presumed to function mainly within this organelle. However, there is now compelling evidence that these chaperones are also localized at a variety of other sites/compartments in cells where they perform important functions. These proteins include: (i) the major chaperonin Hsp60 (or P1), which was identified in mammalian cells as a protein altered in mutants resistant to microtubule inhibitors and is involved in numerous functions at the cell surface and in other compartments; (ii) the Hspl0 or Cpn10 protein, which is a co-chaperone for Hsp60 in protein folding but also serves as an early pregnancy factor in maternal serum; and (iii) the mHsp70 protein, which plays a central role in mitochondrial protein import but is also important for cellular senescence (mortalin) and antigen presentation processes. The presence of these mitochondrial chaperones at specific extramitochondrial locations greatly broadens the range of functions that they can carry out in cells. However, these observations also raise important questions regarding the mechanisms by which these proteins reach these extramitochondrial locations. My paper will review some work in this area and discuss the significance of these results.

For many years the perception has been that mammalian stress proteins are intracellular molecules that are only present in the extracellular environment as a consequence of pathological situations such as necrotic cell death. However, many investigators have now shown that these proteins can be released from a variety of viable (non-necrotic) cell types in vitro, by a mechanism which has yet to be fully established. Moreover, we and a number of others have reported Hsp60 and/or Hsp70 to be present in the peripheral circulation of normal individuals. These observations have profound implications for the perceived role of these proteins as universal pro-inflammatory intercellular 'danger' signalling molecules, and the functional significance and role(s) of these ubiquitously expressed and highly conserved families of molecules must therefore be critically re-evaluated. This paper reviews the evolving evidence which indicates that stress proteins such as Hsp60 and Hsp70 are present in, and can be released into the extracellular compartment under normal physiological conditions, and puts into context their pro- and anti-inflammatory potential.

Heat shock proteins (HSP) were initially identified as a family of stress-induced proteins characterized by their chaperone activity. HSP, however, are also important players in the control of the immune response: HSP are targeted by HSP-specific T cells and antibodies in healthy subjects and also during the course of autoimmune disorders and, conversely, HSP influence the activity of several immune cell types via innate receptor signalling pathways. In addition, the immune response to HSP can be mined for information on the state of the immune system. Newborns carry autoantibodies to HSP. However, this natural autoreactivity to HSP is modified by inflammation, and these changes can be monitored as biomarkers during postnatal life. Using antigen microarrays, we have shown that autoantibody patterns identify individuals prone to develop autoimmune diabetes before disease onset. Moreover, changes in the inflammatory process in multiple sclerosis are also reflected in the antibody response to self-HSP. Herein, we discuss the relevance of HSP and their immune activities for the regulation and monitoring of inflammation and autoimmune disease.

Nerve growth factor (NGF) binds to TrkA receptors (neurotrophic) and P75(NTR) (apoptosis or other pathways depending on the coupled adaptor proteins). Brain derived growth factor (BDNF) can bind to TrkB (neurotrophic) and P75(NTR) receptors. BDNF is the main, activity-dependent, neurotrophin and sculpts neuronal organisation dependent on activity, thereby coupling and balancing effects on excitatory (glutamate) and inhibitory (GABA) transmission--in a synapse-specific manner. Some drugs can interact in a specific way. Positive modulators of AMPA receptors induce BDNF and favour long term potentiation (LTP) and memory processes. Some antidepressants such as tianeptine reverse stress-induced inhibition of LTP and restore neuronal plasticity in brain areas at risk. Inflammatory cytokines are produced in sickness behaviour mimicking depression. Interleukin (IL)1beta can exacerbate the immediate effects of stressors, and enhance and prolong the overall effects, which may be protective in preventing overuse or by increasing conservation-withdrawal: in some synapses IL1beta induces long term depression (LTD) or blocks LTP. The interactions with neurotrophins are complex and frequently reciprocal. However, NGF also contributes to inflammatory situations and mediates pain responses. This interplay is poorly understood but may be critical in cerebral palsy, neurodegenerative disorders such as amyotrophic lateral sclerosis and multiple sclerosis, and even Alzheimer's disease.

The endoplasmic reticulum chaperone and stress protein BiP has hitherto been considered as having only crucial intracellular cell protective functions. However, we have shown that BiP can be present in the extracellular environment and that it binds to a putative but as yet uncloned cell surface receptor. It will stimulate human monocytes via this receptor to express a gene profile that is anti-inflammatory. It will generate T cells with a regulatory function from human peripheral blood most likely by altering dendritic cell development. Intravenous BiP will both prevent and treat ongoing collagen induced arthritis in the DBA/1 mouse. Part of the suppression of arthritis is linked to interleukin (IL)4 as BiP-specific lymph node and spleen cells from these mice secrete IL4, and BiP has no suppressive effect on collagen induced arthritis in IL4 knockout mice. Lymph node and spleen cells isolated from mice administered intravenous BiP will suppress arthritis when transferred intravenously into recipient arthritic mice without any further BiP having to be given. Thus, both in vitro work with human peripheral blood mononuclear cells and in vivo work in the collagen arthritis model lead to the conclusion that BiP induces regulatory cells. Finally, intravenous BiP will ablate the inflammatory cell infiltrate and inflammatory cytokine expression in rheumatoid synovial membrane tissue transplanted subcutaneously into SCID mice. The conclusion from all this experimental work is that BiP may be a novel therapy for the treatment of patients with rheumatoid arthritis.

Long-lasting changes in the basis of memory storage require delivery of newly synthesized proteins to the affected synapses. While most of these proteins are generated in the cell body, several key molecules for plasticity can be delivered in the form of silent mRNAs at synapses in extra-somatic compartments where they are locally translated in response to specific stimuli. One such mRNA encodes brain derived neurotrophic factor (BDNF), a key molecule in neuronal development that plays a critical role in learning and memory, and which displays abnormal levels in several neuropsychiatric disorders. BDNF mRNA accumulates in distal dendrites in response to stimuli that trigger activation of NMDAR and TrkB receptors. A single BDNF protein is produced from several splice variants having different 5'UTRs. We have shown that these mRNA variants have a different subcellular localization (soma, proximal or distal dendritic compartment) and that the protein is co-localized with the transcript from which it originated. As these splice variants are also differentially expressed in response to various stimuli and antidepressants, we propose that they represent a spatial and temporal code to regulate BDNF protein expression locally.

A common single-nucleotide polymorphism in the human brain-derived neurotrophic factor (BDNF) gene, a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met), is associated with alterations in brain anatomy and memory, but its relevance to clinical disorders is unclear. We generated a variant BDNF mouse (BDNF(MET/Met)) that reproduces the phenotypic hallmarks in humans with the variant allele. Variant BDNF(Met) was expressed in brain at normal levels, but its secretion from neurons was defective. In this context, the BDNF(Met/Met) mouse represents a unique model that directly links altered activity-dependent release of BDNF to a defined set of in vivo consequences. Our subsequent analyses of these mice elucidated a phenotype that had not been established in human carriers: increased anxiety. When placed in conflict settings, BDNF(Met/Met) mice display increased anxiety-related behaviours that were not normalized by the antidepressant, fluoxetine. A genetic variant BDNF may thus play a key role in genetic predispositions to anxiety and depressive disorders.