FKBP-type peptidyl prolyl cis/trans isomerases (PPIases) are folding helper enzymes involved in the control of functional regrowth of damaged sciatic, cortical cholinergic, dopaminergic and 5-HT neurones. Here, we show that the constitutively inactive human FK506-binding protein 38 (FKBP38) is capable of responding directly to intracellular Ca 2 þ rise through formation of a heterodimeric Ca 2 þ /calmodulin/FKBP38 complex. Only complex formation creates an enzymatically active FKBP, displaying affinity for Bcl-2 mediated through the PPIase site. Association between Bcl-2 and the active site of Ca 2 þ / calmodulin/FKBP38 regulates Bcl-2 function and thereby participates in the promotion of apoptosis in neuronal tissues. FKBP38 proapoptotic function mediated by this interaction is abolished by either potent inhibitors of the PPIase activity of the Ca 2 þ /calmodulin/FKBP38 complex or RNA interference-mediated depletion of FKBP38, promoting neuronal cell survival.

Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant-and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.antimicrobials | colicin | EHEC | food safety | plant-made recombinant proteins

FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerasesinhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N,N-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 g/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases. Interestingly, FK506 and its open chain derivatives were shown to display neuroprotective and neuroregenerative effects in a wide range of animal models mimicking Parkinson disease, dementia, stroke, and nerve damage (3-11). For example, FK506 administration resulted in protection against ischemic brain injury (12), prevention of long term depression in the rat hippocampus (13), modulation of long term potentiation (14), prevention of N-methyl-D-aspartate receptor desensitization (15), alteration in neurotransmitter release (16), and attenuation of glutamate neurotoxicity ex vivo (17). FK506 increased neurite outgrowth in SH-SY5Y and PC12 cell cultures, but also in primary cultures of chicken dorsal root ganglion and of hippocampal neurons, as well (8,18,19). However, the molecular mechanism of the FK506-mediated neuroprotection and neuroregeneration remained elusive. Members of the enzyme class of peptidyl prolyl cis/trans-isomerasesIn general, the interpretation of effects caused by FK506 in cells is difficult, because FK506 inhibits not only the enzymatic activity of FKBPs, but also the protein phosphatase activity of calcineurin (CaN, PP2B). CaN inhibition is mediated by complex formation with FK506⅐FKBP complexes and is thought to be the initial process leading to immunosuppression (20 -22). CaN inhibition by immunophilin-immunosuppressant complexes is used to prevent allograft rejection in transplantation medicine, to treat autoimmune diseases and to circumvent graft-versus-host diseases. Additionally, inhibition of the protein phosphatase was the proposed basis of FK506-mediated neuroprotection, because the FKBP ligand rapamycin, which has no effects on CaN activity, did not exhibit neuroprotective properties (12,17,23).In contrast, monofunctional inhibitors of FKBPs, such as GPI1046, GPI1048, GPI1485 (Guilford Pharmaceuticals and Amgen), and V10,367 (Vertex Pharmaceuticals) have been developed, that have no influence on CaN activity, while neuroprotective and neuroregenerative effects of FK506 remain conserved. In the central nervous system, GPI1046 promotes protection and sprouting o...

Multiple intracellular receptors of the FK506 binding protein (FKBP) family of peptidylprolyl cis/trans-isomerases are potential targets for the immunosuppressive drug FK506. Inhibition of the protein phosphatase calcineurin (CaN), which has been implicated in the FK506-mediated blockade of T cell proliferation, was shown to involve a gain of function in the FKBP12/FK506 complex. We studied the potential of six human FKBPs to contribute to CaN inhibition by comparative examination of inhibition constants of the respective FK506/FKBP complexes. Interestingly, these FKBPs form tight complexes with FK506, exhibiting comparable dissociation constants, but the resulting FK506/FKBP complexes differ greatly in their affinity for CaN, with IC50 values in the range of 0.047-17 microM. The different capacities of FK506/FKBP complexes to affect CaN activity are partially caused by substitutions corresponding to the amino acid side chains K34 and I90 of FKBP12. Only the FK506 complexes of FKBP12, FKBP12.6, and FKBP51 showed high affinity to CaN; small interfering RNA against these FKBP allowed defining the contribution of individual FKBP in an NFAT reporter gene assay. Our results allow quantitative correlation between FK506-mediated CaN effects and the abundance of the different FKBPs in the cell.

FKBP38 is a negative effector of the anti-apoptotic Bcl-2 protein in neuroblastoma cells. The interaction with Bcl-2 and the enzyme activity of FKBP38 depend on prior binding of calmodulin-Ca 2؉ (CaM-Ca 2؉ ) at high Ca 2؉ concentrations. The FKBP38 protein structure contains three tetratricopeptide repeat (TPR) motifs corresponding to the Hsp90 interaction sites of other immunophilins. In this study we show that the TPR domain of FKBP38 interacts with the C-terminal domain of Hsp90, but only if the FKBP38-CaM-Ca 2؉ complex is preformed. Hence, FKBP38 is the first example of a TPR-containing immunophilin that interacts cofactor-dependently with Hsp90. In the ternary Hsp90-FKBP38-CaM-Ca 2؉ complex the active site of FKBP38 is blocked, thus preventing interactions with Bcl-2. The dual control of the active site cleft of FKBP38 by CaM-Ca 2؉ and Hsp90 highlights the importance of the enzyme activity of the FKBP38-CaM-Ca 2؉ complex in the regulation of programmed cell death.The human FKBP38 (FK506-binding protein 38), the product of the FKBP8 gene, belongs to the enzyme class of peptidylprolyl cis/trans isomerases (PPIases, 2 EC 5.2.1.8) that assist protein folding by catalyzing the slow interconversion of cis and trans isomers of the peptide bond preceding proline in polypeptide chains. This isomerization reaction is involved in both de novo protein folding and regulation of biological activity of native proteins. The accelerated isomerization catalyzed by PPIases is essential in signal transduction pathways (1, 2).FKBP38 was found to play an important role in the apoptosis regulation of neuroblastoma cells by inhibiting interactions of Bcl-2 with its cellular targets, such as calcineurin and Bad (3, 4). The negative regulation of Bcl-2 by FKBP38 depends on functional integrity of the FKBP domain, which is controlled by the cellular Ca 2ϩ concentration and the interaction with the Ca 2ϩ sensor CaM, characterizing FKBP38 as the first cofactor-regulated PPIase (3). Potent neuroprotective and neuroregenerative effects of specific low molecular weight FKBP38 inhibitors and FKBP38 short interfering RNA constructs in neuroblastoma cells outline the importance of FKBP38 activity in the control of neuronal apoptosis. Furthermore, the administration of N-(NЈ,NЈ-dimethylcarboxamidomethyl)-cycloheximide, a low molecular weight FKBP38 inhibitor, to an endothelin-1-induced middle cerebral artery occlusion stroke model in the rat brain demonstrated the potency of FKBP38 inhibition for neuroregeneration and neuroprotection in rat brain tissue (5). The crucial role of FKBP38 in the control of neuronal apoptosis is indicated as well by a pronounced FKBP38 expression in neuronal cells (5, 6). Additionally, FKBP38 is also involved in the apoptosis control of other cell types, as recently shown by the anti-apoptotic effect resulting from the interaction of FKBP38 and the NS5A protein of the hepatitis C virus in hepatoma cells (7,8). The FKBP38 ortholog in mice was described to be involved in the regulation of neuronal development by aff...