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Immunophilins and Their Roles in CNS Injury
Immunophilins and Spinal Cord Injury
Wise Young, Ph.D., M.D.
W. M. Keck Center for Collaborative Neuroscience
Rutgers University, Piscataway, New Jersey 08854-8082
In Construction... started 15 January 2002, last updated 17 January 2002
Several people have been asking on the CareCure Forums about FK-506 or tacrolimus and reports that this drug may be neuroprotective and neuroregenerative in spinal cord injury. Tacrolimus is a ligand for a family of proteins in neurons called immunophilins. This is one of the more complex (and beautiful) stories in biomedical research. I will not attempt to summarize the subject but rather will try to provide some perspectives on the roles and effects of immunophilin ligands, their mechanisms of action, and their potential roles in spinal cord injury.
Cyclosporin, Tacrolimus, and Rapamycin
Cyclosporin A (CyA), tacrolimus (FK-506), and rapamycin (RP) are all immunosuppressive toxins made by bacteria, presumably for the purpose of manipulating immune response of hosts that they infect. CyA was discovered by Sandoz (now Novartis) and was isolated from bacteria isolated from the Adriatic sea. FK-506 was discovered by Fujisawa in Japan and again was isolated from bacteria. These toxins are among hundreds of bacterial factors that influence the immune system.
CyA and FK-506 inhibit an enzyme called calcineurin, found mostly in neurons and lymphocytes. Calcineurin is an enzyme that is primarily found in neurons and lymphocytes. It was first discovered in neurons and is activated by calcium, hence its name. Activation of calcineurin produces several nuclear factors that turn on whole families of genes. In lymphocytes, calcineurin is responsible for activating lymphocytes to respond to create antibodies against new antigens.
CyA and FK-506 do not affect already activated lymphocytes, one of the reasons why these drugs are relatively non-toxic and do not cause life-threatening loss of immune activity when given over a short-term. Immune responses to already recognized antigens are not shut down. The discovery of the immunosuppressive effects of CyA and FK-506 transformed the field of organ transplantation, allowing heart, kidney, and liver transplants with relatively low rejection and mortality rates.
For many years, nobody understood how CyA and FK-506 inhibited calcineurin. In the late 1980's researchers at Sandoz (now Novartis) in Switzerland and Stuart Schreiber at Harvard (see below) elucidated the molecular mechanisms of CyA and FK-506 action. CyA and FK-506 block calcineurin by binding respectively to the proteins called immunophilins. These binding proteins have been a subject of much research and therapeutic speculation.
Immunophilins belong to a larger family of intracellular proteins called chaperone molecules. These molecules bind and usher messenger molecules that regulate cellular functions. These chaperone molecules are often named after their drugs that bind them. For example, cyclosporin binding proteins are called cyclophilin. CyA binds to cyclophilin A (CypA). Tacrolimus binds to proteins called FK-binding proteins (FKBP), after FK-506.
The protein complex formed by cyclosporin and its binding protein inhibits calcineurin. Likewise, tacrolimus bound to FKBP inhibits calcineurin activity. Inhibition of calcineurin prevents lymphocytic activation, accounting for the immunosuppressive effects of cyclosporin and tacrolimus. Interestingly, rapamycin also binds to FKBP but does not inhibit calcineurin. Nevertheless, rapamycin has some immunosuppressive effects, suggesting that FKBP may have targets other than calcineurin.
Chaperone and Heat Shock Proteins
Chaperone molecules not only usher molecules to their destinations inside cells but many are also rotamases or enzymes that fold proteins into the right configuration to affect specific intracellular receptors. Both FKBP and cyclophilins are rotamases. Tacrolimus binds to a specific site on FKBP called the FK binding domain responsible for its rotamase activity. Many bacterial toxins and molecules bind to the FK binding domain and block its rotamase activity, including rapamycin, but do not necessarily inhibit calcineurin activity.
Injury induces expression of many chaperone molecules in mammalian cells. Originally dubbed "heat-shock proteins" (HSP), these proteins have long been known to be massively upregulated in tissues when animals are heated artificially or through fever. Many heat shock proteins are identified by their molecular weights, i.e. HSP-60. In fact, one FKBP (FKBP52) was first identified as a heat shock protein (HSP66).
Several heat shock proteins bind the cytoplasmic steroid receptor (SR). For example, HSP90, HSP70, FKBP52-FK and Cyp40-CyA complexes bind to HSP90 and HSP70. As shown in figure 1, HSP90, FKBP52 (also called HSP66), HSP70, and p23 bind to the steroid receptor prevent . Likewise, a complex of molecules including Cyp40,HSP90, HSP70, and P23 bind to the steroid receptor. They inactivate the receptor and prevent its translocation into the nucleus.
Figure 1. Diagram of the inactivated cytoplasmic steroid receptor (SR) and various binding proteins that prevent translation of the receptor and its associated steroid (S) into the cell nucleus. These include heat shock protein 90 (hsp90), FK binding protein 52 (FKBP52 or hsp56) attached to FK506, p23, and heat shock protein 70 (hsp70). Likewise, cyclophilin 40 (Cyp40) combined with cyclosporin A (CsA), p23, hsp90, and hsp70 also bind to the steroid receptor. This complex moves into the nucleus of cells where various receptor domains interact with the DNA and other systems. The diagram also shows the different components of the steroid receptor, including a binding sites for the BuGR anti-GR antibody (BuGR), the transcription activation domain (TAD) and immune-reactive (IR) domain interacts with DNA to turn on genes, zinc finger domains (Z), DNA binding domains (DBD), nuclear localization signals (NLS), and the signal transduction domain (STD). Please see the following source for more details on the subject. (source: http://biochem1.basic-sci.georgetown.edu/nrr/srapr/ssrc.html).
A huge number of bacterial antibiotics and toxins are directed at binding proteins. Bacteria clearly know where the action is in cells because they have evolved highly specific molecules that are targetted at specific chaperone proteins in the cells, probably to prevent them for responding effectively to the infection. For example, here are two web sites that list chaperone molecules that are targetted by only two species of bacteria
Given the important role of binding proteins in the regulation of cell activity, it is not unexpected that many immunophilin ligands, including CyA and FK-506, have neuroprotective and neuroregenerative effects. It would not surprise me at all if minocycline, a member of the tetracycline family of antibiotics that was recently reported to be neuroprotective in spinal cord injury, turns out to be an immunophilin ligand. Bacteria are wily foes of the human immune system.
In my opinion, however, many studies that have reported potentially beneficial effects of such drugs on injury used these drugs like sledgehammer without considering the appropriate timing and dosing. Like methylprednisolone, immunophilin ligands are two-edged swords because they are inhibiting cellular responses that have both beneficial and deleterious effects at different stages after injury. Too much or too long can result in worse recovery.
Stuart Schrieber at Harvard is one of the leaders in the struggle to understand the profusion of chaperone and rotamase proteins. He is largely responsible for the burgeoning interest in drugs that affect binding proteins, particularly FKBP and the cyclophilins. In addition to his work, he has trained the leading scientists working in the field. He has received many awards for his work. I would not be surprised at all if he gets the Nobel prize for this work.
Guilford Pharmaceuticals (http://informagen.com/Resource_Informagen/Deprecated/2756.html) is a major player in the field. Over the past five years, they have raised hundreds of millions of dollars to develop immunophilin ligands for treating neurological disorders. Another powerhouse company that in the field is Ariad (http://www.ariad.com/about/about.html) which has licensed many of the most critical patents in the field. Almost every major pharmaceutical company has invested substantially.
©Wise Young PhD, MD