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The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRAS G12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRAS G12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRAS G12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Excess light causes severe photodamage to photosystem II (PSII) where the primary charge separation for electron transfer takes place. Dissection of mechanisms underlying the PSII maintenance and repair cycle in green algae promotes the usage of genetic engineering and synthetic biology to improve photosynthesis and biomass production. In this study, we systematically analyzed the high light (HL) responsive immunophilin genes in Chlamydomonas (Chlamydomonas reinhardtii) and identified one chloroplast lumen-localized immunophilin, CYN28, as an essential player in HL tolerance. Lack of CYN28 caused HL hypersensitivity, severely reduced accumulation of PSII supercomplexes and compromised PSII repair in cyn28. The thylakoid FtsH (filamentation temperature-sensitive H) is an essential AAA family metalloprotease involved in the degradation of photodamaged D1 during the PSII repair cycle and was identified as one potential target of CYN28. In the cyn28 mutant, the thylakoid FtsH undergoes inefficient turnover under HL conditions. The CYN28-FtsH1/2 interaction relies on the FtsH N-terminal proline residues and is strengthened particularly under HL. Further analyses demonstrated CYN28 displays peptidyl-prolyl isomerase (PPIase) activity, which is necessary for its physiological function. Taken together, we propose that immunophilin CYN28 participates in PSII maintenance and regulates the homeostasis of FtsH under HL stress via its PPIase activity., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is abundantly clear that the client protein influences, and is often influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 μM), and the effect of cortisol addition (0.25 μM, 0.5 μM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1., Competing Interests: Declaration of competing interest None., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi ( Tc ) and Leishmania spp. ( L spp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human ( Hs ) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca 2+ -binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits Tc CaNA1, Tc CaNA2, L sppCaNA1, L sppCaNA1_var, and L sppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of Hs CaNAα. For regulatory subunits, Hs CaNB-1 and L sppCaNB associate preferentially with the nucleus and cytoplasm, and Tc CaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.

Post-transcriptional regulation of ATP-binding cassette (ABC) proteins has been so far shown to encompass protein phosphorylation, maturation, and ubiquitination. Yet, recent accumulating evidence implicates FK506-binding proteins (FKBPs), a type of peptidylprolyl cis-trans isomerase (PPIase) proteins, in ABC transporter regulation. In this perspective article, we summarize current knowledge on ABC transporter regulation by FKBPs, which seems to be conserved over kingdoms and ABC subfamilies. We uncover striking functional similarities but also differences between regulatory FKBP-ABC modules in plants and mammals. We dissect a PPIase- and HSP90-dependent and independent impact of FKBPs on ABC biogenesis and transport activity. We propose and discuss a putative new mode of transient ABC transporter regulation by cis-trans isomerization of X-prolyl bonds., (© 2020 Federation of European Biochemical Societies.)

The FKBP39 from Drosophila melanogaster is a multifunctional regulatory immunophilin. It contains two globular domains linked by a highly charged disordered region. The N-terminal domain shows homology to the nucleoplasmin core domain, and the C-terminal domain is characteristic for the family of the FKBP immunophilin ligand binding domain. The specific partially disordered structure of the protein inspired us to investigate whether FKBP39 can drive spontaneous liquid-liquid phase separation (LLPS). Preliminary analyses using CatGranule and Pi-Pi contact predictors suggested a propensity for LLPS. Microscopy observations revealed that FKBP39 can self-concentrate to form liquid condensates. We also found that FKBP39 can lead to LLPS in the presence of RNA and peptides containing Arg-rich linear motifs derived from selected nuclear and nucleolar proteins. These heterotypic interactions have a stronger propensity for driving LLPS when compared to the interactions mediated by self-associating FKBP39 molecules. To investigate whether FKBP39 can drive LLPS in the cellular environment, we analysed it in fusion with YFP in COS-7 cells. The specific distribution and diffusion kinetics of FKBP39 examined by FRAP experiments provided evidence that immunophilin is an important driver of phase separation. The ability of FKBP39 to go into heterotypic interaction may be fundamental for ribosome subunits assembly., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Gene duplications increase organismal robustness by providing freedom for gene divergence or by increasing gene dosage. The yeast histone chaperones Fpr3 and Fpr4 are paralogs that can assemble nucleosomes in vitro ; however, the genomic locations they target and their functional relationship is poorly understood. We refined the yeast synthetic genetic array approach to enable the functional dissection of gene paralogs. Applying this method to Fpr3 and Fpr4 uncovered redundant, cooperative, and divergent functions. While Fpr3 is uniquely involved in chromosome segregation, Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. We find that the TRAMP5 RNA exosome is critical for fitness in Δ fpr3 Δ fpr4 yeast and leverage this information to identify an important role for Fpr4 at the 5' ends of protein coding genes. Additionally, Fpr4 and TRAMP5 negatively regulate RNAs from the nontranscribed spacers of ribosomal DNA. Yeast lacking Fpr3 and Fpr4 exhibit a genome instability phenotype at the ribosomal DNA, which implies that these histone chaperones regulate chromatin structure and DNA access at this location. Taken together. we provide genetic and transcriptomic evidence that Fpr3 and Fpr4 operate separately, cooperatively, and redundantly to regulate a variety of chromatin environments., (Copyright © 2019 Savic et al.)

Alternative splicing leads to the secretion of multiple forms of vascular endothelial growth factor-A (VEGF-A) that differ in their activity profiles with respect to neovascularization. FSAP (factor VII activating protease) is the zymogen form of a plasma protease that is activated (FSAPa) upon tissue injury via the release of histones. The purpose of the study was to determine if FSAPa regulates VEGF-A activity in vitro and in vivo. FSAP bound to VEGF 165 , but not VEGF 121 , and VEGF 165 was cleaved in its neuropilin/proteoglycan binding domain. VEGF 165 cleavage did not alter its binding to VEGF receptors but diminished its binding to neuropilin. The stimulatory effects of VEGF 165 on endothelial cell proliferation, migration, and signal transduction were not altered by FSAP. Similarly, proliferation of VEGF receptor-expressing BAF3 cells, in response to VEGF 165 , was not modulated by FSAP. In the mouse matrigel model of angiogenesis, FSAP decreased the ability of VEGF 165 , basic fibroblast growth factor (bFGF), and their combination, to induce neovascularization. Lack of endogenous FSAP in mice did not influence neovascularization. Thus, FSAP inhibited VEGF 165 -mediated angiogenesis in the matrigel model in vivo, where VEGF's interaction with the matrix and its diffusion are important.

Destruxin A (DA), a major secondary metabolite of Metarhizium anisopliae , has anti-immunity to insects. However, the detailed mechanism and its interactions with target proteins are elusive. Previously, three immunophilins, peptidyl-prolyl cis-trans isomerase (BmPPI), FK506 binding-protein 45 (BmFKBP45) and BmFKBP59 homologue, were isolated from the silkworm, Bombyx mori Bm12 cell line following treatment with DA, which suggested that these proteins were possible DA-binding proteins. To validate the interaction between DA and the three immunophilins, we performed bio-layer interferometry (BLI) assay, and the results showed that DA has interaction with BmPPI, whose affinity constant value is 1.98 × 10 -3 M and which has no affinity with FKBP45 and FKBP59 homologue in vitro. Furthermore, we investigated the affinity between DA and human PPI protein (HsPPIA) and the affinity constant (K D ) value is 2.22 × 10 -3 M. Additionally, we compared the effects of silkworm and human PPI proteins produced by DA and immunosuppressants, cyclosporine A (CsA), and tacrolimus (FK506), by employing I2H (insect two-hybrid) in the SF-9 cell line. The results indicated that in silkworm, the effects created by DA and CsA were stronger than FK506. Furthermore, the effects created by DA in silkworm were stronger than those in humans. This study will offer new thinking to elucidate the molecular mechanism of DA in the immunity system of insects.

Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects., Competing Interests: The authors declare no conflicts of interest.

Objective- The NTs (neurotrophins), BDNF (brain-derived neurotrophic factor) and NT-3 promote vascular development and angiogenesis. This study investigated the contribution of endogenous NTs in embryonic stem cell (ESC) vascular differentiation and the potential of exogenous BDNF to improve the process of ESC differentiation to endothelial cells (ECs). Approach and Results- Mouse ESCs were differentiated into vascular cells using a 2-dimensional embryoid body (EB) model. Supplementation of either BDNF or NT-3 increased EC progenitors' abundance at day 7 and enlarged the peripheral vascular plexus with ECs and SM22α + (smooth muscle 22 alpha-positive) smooth muscle cells by day 13. Conversely, inhibition of either BDNF or NT-3 receptor signaling reduced ECs, without affecting smooth muscle cells spread. This suggests that during vascular development, endogenous NTs are especially relevant for endothelial differentiation. At mechanistic level, we have identified that BDNF-driven ESC-endothelial differentiation is mediated by a pathway encompassing the transcriptional repressor EZH2 (enhancer of zeste homolog 2), microRNA-214 (miR-214), and eNOS (endothelial nitric oxide synthase). It was known that eNOS, which is needed for endothelial differentiation, can be transcriptionally repressed by EZH2. In turn, miR-214 targets EZH2 for inhibition. We newly found that in ESC-ECs, BDNF increases miR-214 expression, reduces EZH2 occupancy of the eNOS promoter, and increases eNOS expression. Moreover, we found that NRP-1 (neuropilin 1), KDR (kinase insert domain receptor), and pCas 130 (p130 Crk-associated substrate kinase), which reportedly induce definitive endothelial differentiation of pluripotent cells, were increased in BDNF-conditioned ESC-EC. Mechanistically, miR-214 mediated the BDNF-induced expressional changes, contributing to BDNF-driven endothelial differentiation. Finally, BDNF-conditioned ESC-ECs promoted angiogenesis in vitro and in vivo. Conclusions- BDNF promotes ESC-endothelial differentiation acting via miR-214.

The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes ( AtCYP19-1/ROC3 , AtFKBP65/ROF2 , and AtCYP57 ) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for protein⁻protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; АТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3 , ROF2 , and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

Plasmodiophora brassicae is a soil-borne pathogen that belongs to Rhizaria, an almost unexplored eukaryotic organism group. This pathogen requires a living host for growth and multiplication, which makes molecular analysis further complicated. To broaden our understanding of a plasmodiophorid such as P. brassicae, we here chose to study immunophilins, a group of proteins known to have various cellular functions, including involvement in plant defense and pathogen virulence. Searches in the P. brassicae genome resulted in 20 putative immunophilins comprising of 11 cyclophilins (CYPs), 7 FK506-binding proteins (FKBPs) and 2 parvulin-like proteins. RNAseq data showed that immunophilins were differentially regulated in enriched life stages such as germinating spores, maturing spores, and plasmodia, and infected Brassica hosts (B. rapa, B. napus and B. oleracea). PbCYP3 was highly induced in all studied life stages and during infection of all three Brassica hosts, and hence was selected for further analysis. PbCYP3 was heterologously expressed in Magnaporthe oryzae gene-inactivated ΔCyp1 strain. The new strain ΔCyp1+ overexpressing PbCYP3 showed increased virulence on rice compared to the ΔCyp1 strain. These results suggest that the predicted immunophilins and particularly PbCYP3 are activated during plant infection. M. oryzae is a well-studied fungal pathogen and could be a valuable tool for future functional studies of P. brassicae genes, particularly elucidating their role during various infection phases.

Aim: To increase in vivo DNA transfection efficiency of a nonviral delivery vehicle, its tumor targeting and nuclear delivery ability was improved., Materials & Methods: A novel bifunctional peptide tLyP-1-NLS (named K12) was prepared by coupling a tumor-targeting peptide (tLyP-1) with a nuclear localization signal (NLS), and then was used to modify a degradable polyethyleneimine (PEI) derivative called "N-octyl-N-quaternary chitosan (OTMCS)-PEI". The carrier OTMCS-PEI-K12 was characterized in terms of the physicochemical properties, in vitro gene transfection and antitumor effect in vivo., Results: OTMCS-PEI-K12 showed good suitability, stability and transfection capacity in vitro on the premise of noncytotoxicity. OTMCS-PEI-K12/pING4 complexes induced extensive apoptosis of tumor tissues and shrunk the tumor volume of mice noticeably in vivo., Conclusion: This study offers a way to enhance in vivo transfection of a nonviral carrier.

Cyclophilins (EC 5.2.1.8) belong to a subgroup of proteins known as immunophilins, which also include FK506-binding proteins and parvulins. Members of the immunophilins have two main characteristic properties: (i) peptidyl-prolyl cis-trans isomerase activity and (ii) the ability to bind immunosuppressant molecules of fungal origin. Cyclophilins are some of the most conserved proteins present in eukaryotes and prokaryotes, and they have been implicated in diverse cellular processes and responses to multiple biotic and abiotic stresses. Cyclophilins have been exploited in humans and plants extensively, but they have only recently received attention in regard to phytopathogens. In Phellinus sulphurascens and species of the genus Leptosphaeria and Phytophthora, high expression of cyclophilins was found to be related to infection. Moreover, recent studies of cyclophilins in certain phytopathogens, such as Magnaporthe oryzae, Botrytis cinerea, Cryphonectria parasitica, and Puccinia triticina, demonstrated their roles as a pathogenicity factors. In addition to pathogenicity, cyclophilins have high affinity for the immunosuppressive drug cyclosporin A, which is a potent antifungal agent. Although cyclophilins are highly conserved in phytopathogens, because they have been less studied, their role remains largely unknown. In this review, we provide detailed information on the cyclophilins in several phytopathogens, including fungi and oomycetes, as well as their role in suppressing plant immunity.

The novel class of dual-family immunophilins (henceforth abbreviated as DFI) represents naturally occurring chimera of classical FK506-binding protein (FKBP) and cyclophilin (CYN), connected by a flexible linker that may include a three-unit tetratricopeptide (TPR) repeat. Here, I report a comprehensive analysis of all current DFI sequences and their host organisms. DFIs are of two kinds: CFBP (cyclosporin- and FK506-binding protein) and FCBP (FK506- and cyclosporin-binding protein), found in eukaryotes. The CFBP type occurs in select bacteria that are mostly extremophiles, such as psychrophilic, thermophilic, halophilic, and sulfur-reducing. Essentially all DFI organisms are unicellular. I suggest that DFIs are specialized bifunctional chaperones that use their flexible interdomain linker to associate with large polypeptides or multisubunit megacomplexes to promote simultaneous folding or renaturation of two clients in proximity, essential in stressful and denaturing environments. Analysis of sequence homology and predicted 3D structures of the FKBP and CYN domains as well as the TPR linkers upheld the modular nature of the DFIs and revealed the uniqueness of their TPR domain. The CFBP and FCBP genes appear to have evolved in parallel pathways with no obvious single common ancestor. The occurrence of both types of DFI in multiple unrelated phylogenetic clades supported their selection in metabolic and environmental niche roles rather than a traditional taxonomic relationship. Nonetheless, organisms with these rare immunophilins may define an operational taxonomic unit (OTU) bound by the commonality of chaperone function.

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation., (© 2017 Melani, Valko, Romero, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Aberrant restoration of AR activity is linked with prostate tumor growth, therapeutic failures and development of castrate-resistant prostate cancer. Understanding the processes leading to AR-reactivation should provide the foundation for novel avenues of drug discovery. A differential gene expression study was conducted using biopsies from CaP and BPH patients to identify the components putatively responsible for reinstating AR activity in CaP. From the set of genes upregulated in CaP, FKBP52, an AR co-chaperone, was selected for further analysis. Expression of FKBP52 was positively correlated with that of c-Myc. The functional cross-talk between c-Myc and FKBP52 was established using c-Myc specific-siRNA to LNCaP cells that resulted in reduction of FKBP52. A non-canonical E-box sequence housing a putative c-Myc binding site was detected on the FKBP4 promoter using in silico search. LNCaP cells transfected with the FKBP52 promoter cloned in pGL3 basic showed increased luciferase activity which declined considerably when the promoter-construct was co-transfected with c-Myc specific-siRNA. ChIP-PCR confirmed the binding of c-Myc with the conserved E-box located in the FKBP52 promoter. c-Myc downregulation concomitantly affected expression of FGF8. Since expression of FGF8 is controlled by AR, our study unveiled a novel functional axis between c-Myc, AR and FGF8 operating through FKBP52.

Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ionomycin Ca 2+ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca 2+ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca 2+ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca 2+ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The K D obtained for Cyp D- autumnalamide was 1.51±1.399. Moreover, the K D for Cyp A- autumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08±1.23 and 6.85±1.1μM respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca 2+ channels., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In meiotic prophase I, homologous chromosome pairing is promoted through chromosome movement mediated by nuclear envelope proteins, microtubules, and dynein. After proper homologue pairing has been established, the synaptonemal complex (SC) assembles along the paired homologues, stabilizing their interaction and allowing for crossing over to occur. Previous studies have shown that perturbing chromosome movement leads to pairing defects and SC polycomplex formation. We show that FKB-6 plays a role in SC assembly and is required for timely pairing and proper double-strand break repair kinetics. FKB-6 localizes outside the nucleus, and in its absence, the microtubule network is altered. FKB-6 is required for proper movement of dynein, increasing resting time between movements. Attenuating chromosomal movement in fkb-6 mutants partially restores the defects in synapsis, in agreement with FKB-6 acting by decreasing chromosomal movement. Therefore, we suggest that FKB-6 plays a role in regulating dynein movement by preventing excess chromosome movement, which is essential for proper SC assembly and homologous chromosome pairing., (© 2017 Alleva et al.)

Understanding the mechanisms that lead to autoimmunity is critical for defining potential therapeutic pathways. In this regard there have been considerable efforts in investigating the interacting roles of TGF-β and IL-2 on the function regulatory T cells. We have taken advantage of dnTGF-βRII Il2ra -/- (abbreviated as Il2ra -/- Tg) mouse model, which allows a direct mechanistic approach to define the relative roles of TGF-β and IL-2 on Treg development. Il2ra -/- Tg mice spontaneously developed multi-organ autoimmune diseases with expansion of pathogenic T cells and enhanced germinal center response at 3-4 weeks of age. Importantly, peripheral Treg cells from Il2ra -/- Tg mice demonstrated an activated Th1-like stable phenotype and normal in vitro suppressive function, while thymus Treg increased but manifested decreased suppressive function. Interestingly, neither thymus nor peripheral Treg cells of Il2ra -/- Tg mice contained Neuropilin-1 + or PD-1 hi phenotype, resulting in defective follicular regulatory T (Tfr) cell development. Such defective Tfr development led to elevated follicular T helper cells, enhanced germinal center responses and increased plasma cell infiltration. These data demonstrate an important synergetic role of TGF-β and IL-2 in the generation, activation and stability of Treg cells, as well as their subsequent development into Tfr cells.

The nuclear immunophilin FKBP25 interacts with chromatin-related proteins and transcription factors and is suggested to interact with nucleic acids. Currently the structural basis of nucleic acid binding by FKBP25 is unknown. Here we determined the nuclear magnetic resonance (NMR) solution structure of full-length human FKBP25 and studied its interaction with DNA. The FKBP25 structure revealed that the N-terminal helix-loop-helix (HLH) domain and C-terminal FK506-binding domain (FKBD) interact with each other and that both of the domains are involved in DNA binding. The HLH domain forms major-groove interactions and the basic FKBD loop cooperates to form interactions with an adjacent minor-groove of DNA. The FKBP25-DNA complex model, supported by NMR and mutational studies, provides structural and mechanistic insights into the nuclear immunophilin-mediated nucleic acid recognition., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Immunophilins are a family of intracellular receptors for immunosuppressive drugs. Those immunophilins that are related to immunosuppression are the smallest proteins of the family, i.e., FKBP12 and CyPA, whereas the other members of the family have higher molecular weight because the show additional domains to the drug-binding site. Among these extra domains, the TPR-domain is perhaps the most relevant because it permits the interaction of high molecular weight immunophilins with the 90-kDa heat-shock protein, Hsp90. This essential molecular chaperone regulates the biological function of several protein-kinases, oncogenes, protein phosphatases, transcription factors and cofactors . Hsp90-binding immunophilins where first characterized due to their association with steroid receptors. They regulate the cytoplasmic transport and the subcellular localization of these and other Hsp90 client proteins, as well as transcriptional activity, cell proliferation, cell differentiation and apoptosis. Hsp90-binding immunophilins are frequently overexpressed in several types of cancers and play a key role in cell survival. In this article we analyze the most important biological actions of the best characterized Hsp90-binding immunophilins in both steroid receptor function and cancer development and discuss the potential use of these immunophilins for therapeutic purposes as potential targets of specific small molecules., (© 2015 UICC.)

Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼ 5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼ 5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly(219)-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms., (Copyright © 2016 Elsevier Inc. All rights reserved.)

VEGF is widely investigated for therapeutic angiogenesis, but while short-term delivery is desirable for safety, it is insufficient for new vessel persistence, jeopardizing efficacy. Here, we investigated whether and how VEGF dose regulates nascent vessel stabilization, to identify novel therapeutic targets. Monoclonal populations of transduced myoblasts were used to homogeneously express specific VEGF doses in SCID mouse muscles. VEGF was abrogated after 10 and 17 days by Aflibercept treatment. Vascular stabilization was fastest with low VEGF, but delayed or prevented by higher doses, without affecting pericyte coverage. Rather, VEGF dose-dependently inhibited endothelial Semaphorin3A expression, thereby impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-β1 production and endothelial SMAD2/3 activation. TGF-β1 further initiated a feedback loop stimulating endothelial Semaphorin3A expression, thereby amplifying the stabilizing signals. Blocking experiments showed that NEM recruitment required endogenous Semaphorin3A and that TGF-β1 was necessary to start the Semaphorin3A/NEM axis. Conversely, Semaphorin3A treatment promoted NEM recruitment and vessel stabilization despite high VEGF doses or transient adenoviral delivery. Therefore, VEGF inhibits the endothelial Semaphorin3A/NEM/TGF-β1 paracrine axis and Semaphorin3A treatment accelerates stabilization of VEGF-induced angiogenesis., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Immunophilins comprise two protein families, cyclophilins (CYPs) and FK506-binding proteins (FKBPs), and are the major receptors for the immunosuppressive drugs cyclosporin A (CsA) and FK506 (tacrolimus), respectively. Most eukaryotic species have at least one immunophilin and some of them have been associated with pathogenesis of infectious or parasitic diseases or the action of antiparasitic drugs. The human malarial parasite Plasmodium falciparum has 13 immunophilin or immunophilin-like genes but the functions of their products are unknown. We set out to identify the parasite proteins that interact with the major CYPs, PfCYP19A and PfCYP19B, and the FKBP, PfFKBP35, using a combination of co-immunoprecipitation and yeast two-hybrid screening. We identified a cohort of putative interacting partners and further investigation of some of these revealed potentially novel roles in parasite biology. We demonstrated that (i) P. falciparum CYPs interacted with the heat shock protein 70, (ii) treatment of parasites with CYP ligands disrupted transport of the rhoptry-associated protein 1, and (iii) PfFKBP35 interacted with parasite histones in a way that might modulate gene expression. These findings begin to elucidate the functions of immunophilins in malaria. Furthermore, the known antimalarial effects of CsA, FK506 and non-immunosuppressive derivatives of these immunophilin ligands could be mediated through these partner proteins.

The 12- and 12.6-kDa FK506-binding proteins, FKBP12 (12-kDa FK506-binding protein) and FKBP12.6 (12.6-kDa FK506-binding protein), have been implicated in the binding to and the regulation of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs), both tetrameric intracellular Ca2+-release channels. Whereas the amino acid sequences responsible for FKBP12 binding to RyRs are conserved in IP3Rs, FKBP12 binding to IP3Rs has been questioned and could not be observed in various experimental models. Nevertheless, conservation of these residues in the different IP3R isoforms and during evolution suggested that they could harbour an important regulatory site critical for IP3R-channel function. Recently, it has become clear that in IP3Rs, this site was targeted by B-cell lymphoma 2 (Bcl-2) via its Bcl-2 homology (BH)4 domain, thereby dampening IP3R-mediated Ca2+ flux and preventing pro-apoptotic Ca2+ signalling. Furthermore, vice versa, the presence of the corresponding site in RyRs implied that Bcl-2 proteins could associate with and regulate RyR channels. Recently, the existence of endogenous RyR-Bcl-2 complexes has been identified in primary hippocampal neurons. Like for IP3Rs, binding of Bcl-2 to RyRs also involved its BH4 domain and suppressed RyR-mediated Ca2+ release. We therefore propose that the originally identified FKBP12-binding site in IP3Rs is a region critical for controlling IP3R-mediated Ca2+ flux by recruiting Bcl-2 rather than FKBP12. Although we hypothesize that anti-apoptotic Bcl-2 proteins, but not FKBP12, are the main physiological inhibitors of IP3Rs, we cannot exclude that Bcl-2 could help engaging FKBP12 (or other FKBP isoforms) to the IP3R, potentially via calcineurin.

FK506-binding protein-like (FKBPL) has established roles as an anti-tumor protein, with a therapeutic peptide based on this protein, ALM201, shortly entering phase I/II clinical trials. Here, we evaluated FKBPL's prognostic ability in primary breast cancer tissue, represented on tissue microarrays (TMA) from 3277 women recruited into five independent retrospective studies, using immunohistochemistry (IHC). In a meta-analysis, FKBPL levels were a significant predictor of BCSS; low FKBPL levels indicated poorer breast cancer specific survival (BCSS) (hazard ratio (HR) = 1.30, 95% confidence interval (CI) 1.14-1.49, p < 0.001). The prognostic impact of FKBPL remained significant after adjusting for other known prognostic factors (HR = 1.25, 95% CI 1.07-1.45, p = 0.004). For the sub-groups of 2365 estrogen receptor (ER) positive patients and 1649 tamoxifen treated patients, FKBPL was significantly associated with BCSS (HR = 1.34, 95% CI 1.13-1.58, p < 0.001, and HR = 1.25, 95% CI 1.04-1.49, p = 0.02, respectively). A univariate analysis revealed that FKBPL was also a significant predictor of relapse free interval (RFI) within the ER positive patient group, but it was only borderline significant within the smaller tamoxifen treated patient group (HR = 1.32 95% CI 1.05-1.65, p = 0.02 and HR = 1.23 95% CI 0.99-1.54, p = 0.06, respectively). The data suggests a role for FKBPL as a prognostic factor for BCSS, with the potential to be routinely evaluated within the clinic.

In stallions, impaired acrosome reaction (IAR) may often cause subfertility. Single nucleotide polymorphisms (SNPs) within FK506-binding protein (FKBP6) seem to be associated with IAR in stallions. However, their effect on stallion fertility has not yet been quantified. Using whole-genome sequence data of seven stallions, we searched FKBP6 for mutations to perform an association study in Hanoverian stallions with estimated breeding values for the paternal component of the pregnancy rate per oestrus cycle (EBV-PAT) as target trait. Genotyping five exonic mutations within FKBP6 revealed a significant association of the SNP g.11040379C>A (p.167H>N) with EBV-PAT in 216 Hanoverian stallions. The difference among the two homozygous genotypes was 7.62% in EBV-PAT, corresponding to one standard deviation of EBV-PAT. In conclusion, in Hanoverian stallions, the FKBP6-associated SNP g.11040379C>A confers higher conception rates in A/A homozygous and lower conception rates in C/C homozygous Hanoverian stallions. Thus, an FKBP6-associated missense mutation is significantly associated with stallion fertility., (© 2014 Blackwell Verlag GmbH.)

Objective: The antitumor effects of FK506-binding protein like (FKBPL) and its extracellular role in angiogenesis are well characterized; however, its role in physiological/developmental angiogenesis and the effect of FKBPL ablation has not been evaluated. This is important as effects of some angiogenic proteins are dosage dependent. Here we evaluate the regulation of FKBPL secretion under angiogenic stimuli, as well as the effect of FKBPL ablation in angiogenesis using mouse and zebrafish models., Approach and Results: FKBPL is secreted maximally by human microvascular endothelial cells and fibroblasts, and this was specifically downregulated by proangiogenic hypoxic signals, but not by the angiogenic cytokines, VEGF or IL8. FKBPL's critical role in angiogenesis was supported by our inability to generate an Fkbpl knockout mouse, with embryonic lethality occurring before E8.5. However, whilst Fkbpl heterozygotic embryos showed some vasculature irregularities, the mice developed normally. In murine angiogenesis models, including the ex vivo aortic ring assay, in vivo sponge assay, and tumor growth assay, Fkbpl(+/-) mice exhibited increased sprouting, enhanced vessel recruitment, and faster tumor growth, respectively, supporting the antiangiogenic function of FKBPL. In zebrafish, knockdown of zFkbpl using morpholinos disrupted the vasculature, and the phenotype was rescued with hFKBPL. Interestingly, this vessel disruption was ineffective when zcd44 was knocked-down, supporting the dependency of zFkbpl on zCd44 in zebrafish., Conclusions: FKBPL is an important regulator of angiogenesis, having an essential role in murine and zebrafish blood vessel development. Mouse models of angiogenesis demonstrated a proangiogenic phenotype in Fkbpl heterozygotes., (© 2015 The Authors.)

We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge., (© 2015 Howell et al.)

The steroid receptor-associated TPR cochaperones FKBP51, FKBP52, CyP40 and PP5 have non-redundant roles in steroid receptor function that impact steroid hormone-binding affinity, nucleocyoplasmic shuttling and transcriptional activation of target genes in a tissue-specific manner. Aberrant expression of these TPR immunophilins has the potential to cause steroid-based diseases, including breast and prostate cancer, diabetes and metabolic disorders, male and female infertility and major depressive and neurodegenerative disorders. This review summaries the function of these proteins as cochaperones in steroid receptor-Hsp90 complexes and elaborates on their role in alternative, Hsp90-dependent and -independent signalling pathways not involving steroid receptors. The review also extensively covers current knowledge of the link between the steroid receptor-associated immunophilins and human disease. An improved understanding of their mechanisms of action has revealed opportunities for molecular therapies to enhance or inhibit cellular processes under their control that contribute both to human health and disease.

Immunophilins consist of a family of highly conserved proteins which possess binding abilities to immunosuppressive drugs. Cyclophilins (Cyps) and FK506-binding proteins (FKBP) are family proteins collectively referred as immunophilins. Most Cyps and FKBP family members catalyse peptidyl-prolyl cis/trans isomerase (PPIase) mediated reactions and form binary complexes with their ligands cyclosporine A and FK506. Immunophilins are also involved in key biochemical processes including protein folding, receptor signalling, protein trafficking, and transcription and exhibit versatile biological functions, when complexed with their ligands. Therapeutic implications of immunophilins and effects of their ligands in neurodegenerative disorders, cancer, and infectious diseases have been accumulating in recent years. This review focuses on molecular characteristics of the canonical and non-canonical immunophilin family members from human and Plasmodium falciparum and P. vivax, recent progress on immunophilin inhibitor development, and future perspectives of structure-based design of non-immunosuppressive immunophilin ligands with potential pharmacological activities against infectious diseases.

The centromeric histone H3 variant Cse4 in Saccharomyces cerevisiae is polyubiquitylated and degraded in a proteasome-dependent manner. We report here that the proline isomerase Fpr3 regulates Cse4 proteolysis. Structural change in Cse4 by Fpr3 might be important for the interaction between Cse4 and the E3 ubiquitin ligase Psh1.

Arabidopsis contains 16 putative chloroplast lumen-targeted immunophilins (IMMs). Proteomic analysis has enabled the subcellular localization of IMMs experimentally, but the exact biological and physiological roles of most luminal IMMs remain to be discovered. FK506-binding protein (FKBP) 16-1, one of the lumenal IMMs containing poorly conserved amino acid residues for peptidyl-prolyl isomerase (PPIase) activity, was shown to play a possible role in chloroplast biogenesis in Arabidopsis, and was also found to interact with PsaL in wheat. In this study, further evidence is provided for the notion that Arabidopsis FKBP16-1 (AtFKBP16-1) is transcriptionally and post-transcriptionally regulated by environmental stresses including high light (HL) intensity, and that overexpression of AtFKBP16-1 plants exhibited increased photosynthetic stress tolerance. A blue native-polyacrylamide gel electrophoresis/two-dimensional (BN-PAGE/2-D) analysis revealed that the increase of AtFKBP16-1 affected the levels of photosystem I (PSI)-light harvesting complex I (LHCI) and PSI-LHCI-light harvesting complex II (LHCII) supercomplex, and consequently enhanced tolerance under conditions of HL stress. In addition, plants overexpressing AtFKBP16-1 showed increased accumulation of PsaL protein and enhanced drought tolerance. Using a protease protection assay, AtFKBP16-1 protein was found to have a role in PsaL stability. The AtPsaL levels also responded to abiotic stresses derived from drought, and from methyl viologen stresses in wild-type plants. Taken together, these results suggest that AtFKBP16-1 plays a role in the acclimation of plants under photosynthetic stress conditions, probably by regulating PsaL stability., (© 2013 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Semaphorins were originally identified as axon-guidance molecules that function during neuronal development. However, cumulative evidence indicates that semaphorins also participate in immune responses, both physiological and pathological, and they are now considered to be potential diagnostic and/or therapeutic targets for a range of diseases. The primary receptors for semaphorins are neuropilins and plexins, which have cell type-specific patterns of expression and are involved in multiple signalling responses. In this Review, we focus on the roles of neuropilin 1 (NRP1) and plexins in the regulation of the immune system, and we summarize recent advances in our understanding of their pathological implications.

Wheat scab, caused by the fungal pathogen Fusarium graminearum is a devastating disease worldwide. Despite an extensive and coordinated effort to investigate this pathosystem, little progress has been made to understand the molecular basis of host-pathogen interactions, for example how the pathogen causes disease in plant. Recently, a secreted lipase (FGL1) has been identified from the fungus and shown to be an important virulence factor; however, the intrinsic function of FGL1 in plant is unknown. Here, we report the identification of the molecular components that may possibly be involved in the FGL virulence pathway using yeast two hybrid system. FGL gene was amplified from a local virulent strain (F15) and shown to be 99.5% identical to the original published FGL at the amino acid level. We showed that transient expression of this FGL gene by Agroinfiltration in tobacco leaves causes cell death further implicating the role of FGL in virulence. To identify FGL initial physical target in plant, we screened two wheat cDNA libraries using the FGL protein as the bait. From both libraries, a small FKBP-type immunophilin protein, designated wFKBP12, was found to physically interact with FGL. The direct interaction of FGL with wFKBP12 was confirmed in living onion epidermal cells by biomolecular fluorescence complementation (BiFC) assay. To investigate further, we then used wFKBP12 protein as bait and identified an elicitor-responsive protein that contains a potential Ca(2+) binding domain. Semi-quantitative PCR showed that this elicitor-responsive gene is down-regulated during the F. graminearum infection suggesting that this protein may be an important component in FGL virulence pathway. This work serves as an initial step to reveal how fungal lipases act as a general virulence factor., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Protein phosphatase-1 (PP1) controls many processes in eukaryotic cells. Modulation of mitosis by reversing phosphorylation of proteins phosphorylated by aurora protein kinase is a critical function for PP1. Overexpression of the sole PP1, Glc7, in budding yeast, Saccharomyces cerevisiae, is lethal. This work shows that lethality requires the function of Glc7 regulatory proteins Sds22, Reg2, and phosphorylated Glc8. This finding shows that Glc7 overexpression induced cell death requires a specific subset of the many Glc7-interacting proteins and therefore is likely caused by promiscuous dephosphorylation of a variety of substrates. Additionally, suppression can occur by reducing Glc7 protein levels by high-copy Fpr3 without use of its proline isomerase domain. This divulges a novel function of Fpr3. Most suppressors of GLC7 overexpression also suppress aurora protein kinase, ipl1, temperature-sensitive mutations. However, high-copy mutant SDS22 genes show reciprocal suppression of GLC7 overexpression induced cell death or ipl1 temperature sensitivity. Sds22 binds to many proteins besides Glc7. The N-terminal 25 residues of Sds22 are sufficient to bind, directly or indirectly, to seven proteins studied here including the spindle assembly checkpoint protein, Bub3. These data demonstrate that Sds22 organizes several proteins in addition to Glc7 to perform functions that counteract Ipl1 activity or lead to hyper Glc7 induced cell death. These data also emphasize that Sds22 targets Glc7 to nuclear locations distinct from Ipl1 substrates.

Purpose: FK506-binding protein like (FKBPL) and its peptide derivative, AD-01, have already shown tumor growth inhibition and CD44-dependent antiangiogenic activity. Here, we explore the ability of AD-01 to target CD44-positive breast cancer stem cells (BCSC)., Experimental Design: Mammosphere assays and flow cytometry were used to analyze the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anticancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples, and xenografts. Delays in tumor initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, quantitative PCR (qPCR), and immunofluorescence., Results: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere-forming efficiency and ESA(+)/CD44(+)/CD24(-) or aldehyde dehydrogenase (ALDH)(+) cell subpopulations in vitro and tumor initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism seems to be due to AD-01-mediated BCSC differentiation shown by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4, and Sox2, were also significantly reduced. Furthermore, we showed additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy-induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signaling., Conclusions: AD-01 has dual antiangiogenic and anti-BCSC activity, which will be advantageous as this agent enters clinical trial.

Immunophilins are proteins that contain a PPIase domain as a family signature. Low-molecular-weight immunophilins were first described associated to immunosuppressive action and protein folding. Recent studies of other members of the family have led to the identification of their participation in basic processes such as protein-protein interactions, signal transduction cascades, cell differentiation, cell cycle progression, metabolic activity, apoptosis mechanisms, microorganisms infection, cancer, neurotrophism and neuroprotection, among several other physiological and pathophysiological processes. Due to all these emerging features, the development of specific ligands for immunophilins appears to have promising perspectives, in particular in the fields of cancer biology and neuroregeneration fields. We review the emerging role of immunophilins in protein transport, transcription regulation, malignancies development and neurotrophic action, in addition to a number of biological properties that transform these proteins in potential targets for novel therapeutic strategies.

In the course of our screening program for a new inhibitor of the Ca(2+) signalling pathway using mutant yeast [Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ)], a mouse PP2Cα activator, pisiferdiol, isolated from Chamaecyparis pisifera, was found to alleviate the Ca(2+) signal-mediated growth inhibition. Pisiferdiol showed growth inhibition activity against the mpk1Δ strain compared with the cnb1Δ strain and induced Li(+) sensitivity to the wild-type strain, indicating that it suppresses the calcineurin pathway in the yeast. However, the Li(+) sensitivity to ptc1Δ strain by pisiferdiol was diminished. Pisiferdiol showed growth restored activity in the zds1Δ strain without immunophilins Fkb1p or Cph1p, and in the pmc1Δ strain. It inhibited calcineurin-induced expression in the reporter gene assay and decreased the protein expression (Western blots) of calcineurin (Cnb1p) in addition to a decrease of Swe1p and phosphorylation of Cdc28p in the mutant yeast. These results showed that pisiferdiol could suppress indirectly the action of calcineurin and restored the growth inhibition of the mutant yeast through Ptc1p activation., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Unlabelled: Platelet hyperaggregability might contribute to vascular complications associated with type 2 diabetes mellitus (DM2).Experimental evidence supports a direct link between altered Ca(2+) entry and hyperaggregability in DM2 patients., Objectives: We aimed to investigate whether altered immunophilin expression and function are involved in the abnormal Ca(2+) entry observed in platelets from DM2 patients., Results: Inhibition of immunophilins by tacrolimus (FK506) and sirolimus (rapamycin) reduced Ca(2+) entry in platelets from healthy donors and DM2 patients. Similarly, immunophilin inhibitors reduced platelet degranulation in both healthy and DM2 subjects. Nevertheless, α-granule secretion reduction was greater than that observed for dense granules in platelets from DM2 patients. However, no difference was observed in the inhibition of secretion in platelets from healthy subjects. Additionally, altered expression of FK506 binding protein-52 (FKBP52) and coupling to Ca(2+) channels were found in platelets from DM2 patients compared to healthy subjects. Finally, reduction in platelet function from healthy subjects and DM2 patients in the presence of immunophilin antagonists was observed, being this dysfunction more evident in platelets from DM2 patients., Conclusions: We suggest that, among others, FKBP52 expression and function are altered in platelets from DM2 patients, contributing to the altered Ca(2+) entry and hyperaggregability in these cells.

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

Fungi rely on regulatory networks to coordinate sensing of environmental stress with initiation of responses crucial for survival. Antifungal drugs are a specific type of environmental stress with broad clinical relevance. Small molecules with antifungal activity are ubiquitous in the environment, and are produced by a myriad of microbes in competitive natural communities. The echinocandins are fungal fermentation products and the most recently developed class of antifungals, with those in clinical use being semisynthetic derivatives that target the fungal cell wall by inhibiting 1,3-β-D-glucan synthase. Recent studies implicate the protein phosphatase calcineurin as a key regulator of cellular stress responses required for fungal survival of echinocandin-induced cell wall stress. Pharmacological inhibition of calcineurin can be achieved using the natural product and immunosuppressive drug cyclosporin A, which inhibits calcineurin by binding to the immunophilin Cpr1. This drug-protein complex inhibits the interaction between the regulatory and catalytic subunits of calcineurin, an interaction necessary for calcineurin function. Here, we report on potent activity of cyclosporin A when combined with the echinocandin micafungin against the model yeast Saccharomyces cerevisiae that is independent of its known mechanism of action of calcineurin inhibition. This calcineurin-independent synergy does not involve any of the 12 immunophilins known in yeast, individually or in combination, and is not mediated by any of the multidrug transporters encoded or controlled by YOR1, SNQ2, PDR5, PDR10, PDR11, YCF1, PDR15, ADP1, VMR1, NFT1, BPT1, YBT1, YNR070w, YOL075c, AUS1, PDR12, PDR1 and/or PDR3. Genome-wide haploinsufficiency profiling (HIP) and homozygous deletion profiling (HOP) strongly implicate the cell wall biosynthesis and integrity pathways as being central to the calcineurin-independent activity of cyclosporin A. Thus, systems level chemical genomic approaches implicate key cellular pathways in a novel mechanism of antifungal drug synergy.

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.

Objectives: FK1706, a non-immunosuppressive immunophilin ligand, potentiated nerve growth factor-induced neurite outgrowth, putatively mediated via FKBP-52 and the Ras/Raf/MAPK signaling pathway. It also improved mechanical allodynia accompanied by the recovery of intraepidermal nerve fiber density in a painful diabetic neuropathy in rats. The aim of this study was to demonstrate the gene expression profiling in dorsal root ganglion in streptozotocin-induced diabetic rats related to pain and anti-allodynia effects of FK1706 administration to elucidate the putative mechanisms of its neurotrophic activity in vivo. Here, we analyzed gene expression of the dorsal root ganglia using microarray together with behavioral measurement of mechanical allodynia in diabetic rats to try to capture the global fingerprint of changes in gene expression associated with FK1706 administration., Methods: The withdrawal threshold of streptozotocin-induced diabetic rats was measured by an electronic von Frey system. The gene expression of the ganglia from L4 to L6 obtained from streptozotocin-treated rats with or without chronic administration of FK1706 was analyzed using an Affymetrix GeneChip to extract interesting genes in the development of mechanical allodynia in diabetes and anti-allodynia effect of FK1706., Results: Daily oral administration of FK1706 improved mechanical allodynia without decreasing plasma glucose levels. From gene expression analysis, the expression of thioredoxin interacting protein gene was sustained to increased change, whereas those of collagen I alpha1, II alpha1 and IX alpha1 genes were decreased from 2 to 4 weeks after streptozotocin injection. While no changes occurred after 1 week of commencing of FK1706 administration (2 weeks after streptozotocin injection), changes in expression more than 1.5-fold were observed for genes such as Ckm, Actn3, Atp2a1, Bglap, Acta1, Myl1, Tnnc2, and Mylpf at 2 weeks of FK1706 administration (3 weeks after streptozotocin injection). The genes RGD1564519, Hbb, LOC689064, Arpc4 and S100a9 were upregulated in comparison with streptozotocin-injected control group at 3 weeks of FK1706 administration; on the other hand, those of Actn3, Atp2a1 were downregulated by FK1706., Discussion: FK1706 ameliorates mechanical allodynia with accompanying increases in gene expressions possibly related to neurite outgrowth, development, differentiation, and nociceptive sensitivity.