Therapeutic and diagnostic agents comprising a SOCS box
The present invention relates generally to diagnostic and therapeutic agents. More particularly, the present invention provides therapeutic molecules capable of regulating signal transduction such as but not restricted to cytokine-mediated signal transduction. The molecules of the present invention are useful, therefore, in regulating cellular responsiveness to cytokines in addition to other mediators of signal transduction like endogenous or exogenous molecules, antigens, microbes and microbial products, viruses or components thereof, ions, hormones and parasites.
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Cells always monitor their environment in order to regulate biochemical and physiological processes which in turn affects future behaviour. Frequently, a mobile’s first interaction with its surroundings occurs via receptors extracted onthe plasma membrane. Activation of these receptors, whether through binding endogenous ligands (such as cytokines) or exogenous ligands (such as antigens), triggers a biochemical cascade in the membrane through the cytoplasm to the nucleus.
Of the endogenous ligands, cytokines represent a particularly important and versatile group. Cytokines are proteins which modulate the survival, proliferation, differentiation and function of many different cells within the body [Nicola, 1994].The haemopoietic cytokines have in common a four-alpha helical bundle arrangement and the huge bulk interact with a structurally related family of cell surface receptors, the type I and type II cytokine receptors [Bazan, 1990; Sprang, 1993]. In allcases, ligand-induced receptor aggregation seems to be a critical event in initiating intracellular signal transduction cascades. Some cytokines, such as growth hormone, erythropoietin (Epo) and granulocyte-colony-stimulating factor (G-CSF),trigger receptor homodimerisation, while for different cytokines, receptor heterodimersation or heterotrimerisation is crucial. In the latter circumstances, several cytokines share common receptor subunits and on this foundation can be grouped in to three subfamilieswith similar patterns of intracellular activation and comparable biological consequences [Hilton, 1994]. Interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) use the frequent .beta. -receptor subunit (.beta. C) and every cytokinestimulates the manufacturing and operational activity of granulocytes and macrophages. IL-2, IL-4, IL-7, IL-9, and IL-15 every use the common .gamma. -string (.gamma. C), whilst IL-4 and IL-13 share an alternative 7-chain (.gamma.’ C or IL-13 receptor a-chain).Each of those cytokines has an essential part in regulating acquired immunity in the lymphoid system. At length, IL-6, IL-11, leukaemia inhibitory factor (LIF), oncostatin-M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin (CT) share thereceptors subunit gp 130. Each of these cytokines seems to be very pleiotropic, having effects both within and outside of the haemopoietic system [Nicola, 1994].
In each of the above mentioned cases at least one subunit of each receptor complex includes the conserved sequence elements, termed box1 and box2, in their cytoplasmic tails [Murakami, 1991]. Box1 is a proline-rich motif that’s found more proximal tothe transmembrane domain than the acidic box two element. The box-1 region functions as the binding site for a class of cytoplasmic tyrosine kinases termed JAKs (Janus kinases). Ligand-induced receptor dimerisation functions to increase the catalytic activityof the associated JAKs through cross-phosphorylation. Activated JAKs afterward tyrosine phosphorylate many substrates, such as the receptors themselves. Specific phosphotyrosine residues on the receptor then function as docking sites for SH2-containingproteins, the best characterised of that would be the signal transducers and activators of transcription (STATs) and the adaptor protein, shc. The STATs are then phosphorylated on tyrosines, likely by JAKs, dissociate from the receptor and also shape eitherhomodimers or heterodimers through the interaction of the SH2 domain of a single STAT with the phosphotyrosine residue of the other. STAT dimers then translocate to the nucleus where they bind to particular cytokine-responsive promoters and activatetranscription [Darnell, 1994; Ihle, 1995; Ihle, 1995]. In another pathway, tyrosine phosphorylated shc interacts with another SH2 domain-containing protein, Grb-2, leading ultimately causing activation of members of the MAP kinase family and in rumtranscription factors like fos and jun (Sato, 1993; Cutler, 19931. These pathways aren’t unique to members of the cytokine receptor family because cytokines that bind receptor tyrosine kinases also having the ability to activate STATs and members of the MAPkinase household [David, 1996; Leaman, 1996; Shual, 1993; Sato, 1993; Cutler, 1993].
Four members of the JAK family of cytoplasmic tyrosine kinases are clarified, JAK1, JAK2, JAK2 and TYK2, each of which binds to a specific subset of both cytokine receptor subunits. Six STATs are clarified (STAT1 through STAT6), andthese are triggered by different cytokine/receptor complexes. For instance, STAT1 seems to be specific to the interferon system, STAT2 seems to be particular to IL-12, while STATE seems to be special for IL-4 and IL-13. Therefore,despite ordinary activation mechanics some degree of cytokine specificity might be accomplished through the usage of particular JAKs and STATs [Thierfelder, 1996; Kaplan, 1996; Takeda, 1996; Shimoda, 1996; Meraz,1996; Durbin, 1996].
Besides those described previously, there .are clearly other mechanisms of activation of the pathways. For example, the JAK/STAT pathway seems to have the ability to activate MAP kinases separate of their shc-induced pathway [David, 1995] and theSTATs themselves may be actuated with no binding to the receptor, possibly by direct interaction with JAKs [Gupta, 1996]. Conversely, total activation of STATS may require the actions of MAP kinase in addition to that of JAKs [David, 1995; Wen, 1995].
While the activation of the signalling pathways is becoming better known, little is known of the regulation of the pathways, including employment of negative or positive feedback loops. That is essential since when a cell has begunto respond to a stimulus, it’s critical that the strength and duration of the reaction is controlled and that signal transduction is switched off. It is likewise desirable to increase the strength of a reaction systemically or even locally as thesituation needs.
In work leading to the current invention, the historians sought to isolate negative regulators of signal transduction. The inventors have now identified a new family of proteins that are capable of behaving as regulators of signalling. Thenew family of proteins is traditionally described as the suppressor of cytokine signalling (SOCS) family depending on the ability of the initially identified SOCS molecules to curb cytokine-mediated signalling. It should be said, however, that not all members of theSOCS family need always share suppressor role nor target entirely cytokine mediated signalling. The SOCS family contains at least three types of protein molecules based on amino acid sequence motifs situated N-terminal of a C-terminal motifcalled that the SOCS box. The identification of the new family of regulatory molecules allows the creation of a range of effector or modulator molecules capable of regulating signal transduction and, hence, cellular responsiveness to a range of moleculesincluding cytokines. The present invention, therefore, provides diagnostic and therapeutic agents based on SOCS proteins, derivatives, homologues, analogues and mimetics thereof as well as agonists and antagonists of SOCS proteins.
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