The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin.

Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.

Immune responses in healthy adults elicited by a bivalent norovirus vaccine candidate composed of GI.4 and GII.4 VLPs without adjuvant.

The development of an efficacious vaccine against norovirus is of paramount importance given its potential to reduce the global burden of norovirus-associated morbidity and mortality. Here, we report a detailed immunological analysis of a phase I, double-blind, placebo-controlled clinical trial performed on 60 healthy adults, ages 18 to 40. Total serum immunoglobulin and serum IgA against vaccine strains and cross-reactive serum IgG against non-vaccine strains were measured by enzyme immunoassays, whereas cell-mediated immune responses were quantified using intracellular cytokine staining by flow cytometry. A significant increase in humoral and cellular responses, e.g., IgA and CD4+ polypositive T cells, was triggered by the GI.4 Chiba 407 (1987) and GII.4 Aomori 2 (2006) VLP-based norovirus vaccine candidate rNV-2v, which is formulated without adjuvant. No booster effect was observed after the second administration in the pre-exposed adult study population. Furthermore, a cross-reactive immune response was elicited, as shown by IgG titers against GI.3 (2002), GII.2 OC08154 (2008), GII.4 (1999), GII.4 Sydney (2012), GII.4 Washington (2018), GII.6 Maryland (2018), and GII.17 Kawasaki 308 (2015). Due to viral infection via mucosal gut tissue and the high variety of potentially relevant norovirus strains, a focus should be on IgA and cross-protective humoral and cell-mediated responses in the development of a broadly protective, multi-valent norovirus vaccine. Clinical trial registration: https://clinicaltrials.gov, identifier NCT05508178. EudraCT number: 2019-003226-25.

A randomized, double-blind, placebo-controlled, dose-escalating phase I trial to evaluate safety and immunogenicity of a plant-produced, bivalent, recombinant norovirus-like particle vaccine.

Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide and a safe and effective vaccine is needed. Here, a phase I, double-blind, placebo-controlled clinical trial was performed in 60 healthy adults, 18 to 40 years old. Safety (primary objective) and immunogenicity (secondary and exploratory objectives) of a bivalent (GI.4 and GII.4), plant-produced, virus-like particle (VLP), NoV vaccine candidate formulation were investigated at two dose levels (50 µg + 50 µg and 150 µg + 150 µg) without adjuvant. Overall, 13 subjects (65.0%) in the 50 µg group, 16 subjects (80.0%) in the 150 µg group, and 14 subjects (70.0%) in the placebo group reported at least 1 solicited local or general symptom during the 7-day post-vaccination periods following each dose. Severe solicited adverse events (AEs) were rare (2 events in the 50 µg group). A total of 8 subjects (40.0%) in each group reported at least one unsolicited AE during the 28-day post-vaccination periods. Immunogenicity was assessed on days 1, 8, 29, 57, 183 and 365. All subjects were pre-exposed to norovirus as indicated by baseline levels of the different immunological parameters examined. Vaccine-specific humoral and cellular immune responses increased after the first dose but did not rise further after the second vaccination. Increased GI.4- and GII.4-specific IgG titers persisted until day 365. The vaccine elicited cross-reactive IgG antibodies against non-vaccine NoV VLPs, which was more pronounced for NoV strains of the same genotype as the GII.4 vaccine strain than for non-vaccine genotypes. Significant blocking anti-GI.4 and anti-GII.4 VLP titers were triggered in both dose groups. Lymphoproliferation assays revealed strong cell-mediated immune responses that persisted until day 365. In conclusion, both dose levels were safe and well-tolerated, and no higher incidence of AEs was observed in the higher dose group. The data show that a single dose of the vaccine formulated at 50 µg of each VLP is sufficient to reach a peak immune response after 8 to 28 days. The results of this Phase I study warrant further evaluation of the non-adjuvanted vaccine candidate. Clinical trial registration: https://clinicaltrials.gov/ct2/show/record/NCT05508178, identifier (NCT05508178).

Safety and immunogenicity studies in animal models support clinical development of a bivalent norovirus-like particle vaccine produced in plants.

Noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis in humans worldwide. A safe and effective vaccine that prevents NoV infection or minimizes NoV disease burden is needed, especially for children and the elderly who are particularly susceptible to NoV disease. A plant-based expression system (magnICON®) was used to manufacture two different virus-like particle (VLP) immunogens derived from human NoV genogroups I and II, genotype 4 (GI.4 and GII.4), which were subsequently blended 1:1 (w/w) into a bivalent vaccine composition (rNV-2v). Here, we report on the safety and immunogenicity of rNV-2v from one pilot and two GLP-compliant toxicity studies in New Zealand White rabbits administered the vaccine subcutaneously (SC) or intramuscularly (IM). Strong genogroup-specific immune responses were induced by vaccination without adjuvant at various doses (200 to 400 µg VLP/administration) and administration schedules (Days 1 and 7; or Days 1, 15 and 29). The results showed sporadic local irritation at the injection site, which resolved over time, and was non-adverse and consistent with expected reactogenicity. There were no signs of systemic toxicity related to vaccine administration relative to vehicle-treated controls with respect to clinical chemistry, haematology, organ weights, macroscopic examinations, or histopathology. In a 3-administration regimen (n + 1 the clinical regimen), the NOAEL for rNV-2v via the SC or IM route was initially determined to be 200 µg. An improved GI.4 VLP variant mixed 1:1 (w/w) with the wild-type GII.4 VLP was subsequently evaluated via the IM route at a higher dose in the same 3-administration model, and the NOAEL was raised to 300 µg. Serology performed in samples of both toxicity studies showed significant and substantial anti-VLP-specific antibody titers for rNV-2v vaccines administered via the IM or SC route, as well as relevant NoV blocking antibody responses. These results support initiation of clinical development of the plant-made NoV vaccine.

Clinical Safety and Immunogenicity of Tumor-Targeted, Plant-Made Id-KLH Conjugate Vaccines for Follicular Lymphoma.

We report the first evaluation of plant-made conjugate vaccines for targeted treatment of B-cell follicular lymphoma (FL) in a Phase I safety and immunogenicity clinical study. Each recombinant personalized immunogen consisted of a tumor-derived, plant-produced idiotypic antibody (Ab) hybrid comprising the hypervariable regions of the tumor-associated light and heavy Ab chains, genetically grafted onto a common human IgG1 scaffold. Each immunogen was produced in Nicotiana benthamiana plants using twin magnICON vectors expressing the light and heavy chains of the idiotypic Ab. Each purified Ab was chemically linked to the carrier protein keyhole limpet hemocyanin (KLH) to form a conjugate vaccine. The vaccines were administered to FL patients over a series of ≥6 subcutaneous injections in conjunction with the adjuvant Leukine (GM-CSF). The 27 patients enrolled in the study had previously received non-anti-CD20 cytoreductive therapy followed by ≥4 months of immune recovery prior to first vaccination. Of 11 patients who became evaluable at study conclusion, 82% (9/11) displayed a vaccine-induced, idiotype-specific cellular and/or humoral immune response. No patients showed serious adverse events (SAE) related to vaccination. The fully scalable plant-based manufacturing process yields safe and immunogenic personalized FL vaccines that can be produced within weeks of obtaining patient biopsies.

Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins.

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.

New structural aspects of FKBP38 activation.

The human FK506-binding protein 38 (FKBP38) regulates Bcl-2 in neuronal apoptosis. To control Bcl-2 activity, FKBP38 requires a prior interaction with the Ca(2+)-sensor calmodulin (CaM). The resulting FKBP38/CaM complex is unique within the FKBP family. Here, we present novel insights into the structural arrangement of this complex. Chemical shift perturbation analyses of the individual protein domains revealed two separate interaction sites between FKBP38 and CaM. On the one hand, residues Glu303, Tyr307 and Leu311, belonging to the predicted CaM-binding site at the C-terminal end of FKBP38, become embedded in the hydrophobic target protein-binding cleft of the C-terminal CaM lobe. On the other hand, in a second binding interaction, the N-terminal end of the catalytic FKBP38 domain shows surface contacts to the AB and CD loops of CaM as well as the adjacent helices. Furthermore, a Glu-rich region at the non-structured FKBP38 N-terminus features additional contacts to CaM helix A. In combination with previous results, we thus conclude that the FKBP38/CaM complex is constituted by (i) a Ca(2+)-dependent interaction of the CaM-binding motif at the C-terminal end of FKBP38 with the C-terminal CaM lobe and (ii) a Ca(2+)-independent interaction between the N-terminal CaM lobe and the N-terminal region of the catalytic FKBP38 domain.

FKBP36 is an inherent multifunctional glyceraldehyde-3-phosphate dehydrogenase inhibitor.

FKBP36 has been previously shown to be a crucial factor in spermatogenesis because of its interplay with the synaptonemal complex protein SCPI. Here we show that beyond this function, FKBP36 forms complexes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) and Hsp90. Both proteins bind independently to different sites of the FKBP36 tetratricopeptide repeat domain. The interaction between FKBP36 and GAPDH directly inhibits the catalytic activity of GAPDH. In addition, FKBP36 expression causes a significant reduction of the GAPDH level and activity in COS-7 cells. Particularly in the cytosolic fraction, GAPDH was depleted by FKBP36 expression. Thus, FKBP36 diminishes GAPDH activity by direct interaction and down-regulation of GAPDH, which represents a previously unknown mechanism of GAPDH regulation and a novel function of FKBP36 in testis-specific signaling.

FKBP36 forms complexes with clathrin and Hsp72 in spermatocytes.

The testes-specific peptidyl-prolyl cis/ trans isomerase FKBP36 plays a crucial role in male meiosis. Here we show that the catalytic domain of FKBP36 binds to clathrin heavy chain (CHC) of clathrin. Despite wild-type FKBP36 not displaying PPIase activity, the introduction of the R81L substitution resulted in catalysis of prolyl isomerization, which is comparable to the regulated activity of FKBP38. Furthermore, the TPR domain of FKBP36 interacts with Hsp72. In fact, FKBP36 preferentially binds to Hsp72 among the members of the Hsp70 family and is thus the first TPR-containing protein which discriminates between Hsp70 proteins. The clathrin-FKBP36-Hsp72 complexes resulting from both identified interactions are bound to the matrices of clathrin-coated vesicles in spermatocytes, which indicates a possible role of FKBP36 and Hsp72 in the disassembly of clathrin coats.

Hsp90-mediated inhibition of FKBP38 regulates apoptosis in neuroblastoma cells.

The FK506-binding protein 38 (FKBP38) is a pro-apoptotic regulator of Bcl-2 in neuroblastoma cells. Hsp90 inhibits the pro-apoptotic FKBP38/CaM/Ca(2+) complex and thus prevents interactions between FKBP38 and Bcl-2. Here we show that Hsp90 increases cell survival rates of neuroblastoma cells after apoptosis induction. Depletion of FKBP38 by short interference RNA significantly decreased the anti-apoptotic effect of Hsp90 expression. In addition, the influence of high cellular Hsp90 levels was only observed in post-stimulation apoptosis that is sensitive to selective FKBP38 active site inhibition. Similar anti-apoptotic effects in neuroblastoma cells were observed after stimulation of endogenous Hsp90 expression. Hence, the inhibition of FKBP38 by Hsp90 participates in programmed cell death control of neuroblastoma cells.

A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38.

The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.

The Bcl-2 regulator FKBP38-calmodulin-Ca2+ is inhibited by Hsp90.

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.

Comparative analysis of calcineurin inhibition by complexes of immunosuppressive drugs with human FK506 binding proteins.

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.

The specific FKBP38 inhibitor N-(N',N'-dimethylcarboxamidomethyl)cycloheximide has potent neuroprotective and neurotrophic properties in brain ischemia.

FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remains unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca(2+) (CaM/Ca(2+))-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38.CaM/Ca(2+) 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38.CaM/Ca(2+) inhibition 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 mug/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.

Bcl-2 regulator FKBP38 is activated by Ca2+/calmodulin.

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 Ca2+ rise through formation of a heterodimeric Ca2+/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 Ca2+/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 Ca2+/calmodulin/FKBP38 complex or RNA interference-mediated depletion of FKBP38, promoting neuronal cell survival.

A reassessment of the inhibitory capacity of human FKBP38 on calcineurin.

The microbial peptidomacrolide FK506 affects many eukaryotic developmental and cell signaling programs via calcineurin inhibition. Prior formation of a complex between FK506 and intracellular FK506-binding proteins (FKBPs) is the precondition for the interaction with calcineurin. A puzzling difference has emerged between the mammalian multidomain protein hFKBP38 and other FKBPs. It was shown that hFKBP38 not only binds to calcineurin but also inhibits the protein phosphatase activity of calcineurin on its own [Shirane, M. and Nakayama, K.I. (2003) Nature Cell Biol. 5, 28-37]. Inherent calcineurin inhibition by hFKBP38 would completely eliminate the need for FK506 in controlling many signal transduction pathways. To address this issue, we have characterized the functional and physical interactions between calcineurin and hFKBP38. A recombinant hFKBP38 variant and endogenous hFKBP38 were tested both in vitro and in vivo. The proteins neither directly inhibited calcineurin activity nor affected NFAT reporter gene activity in SH-SY5Y and Jurkat cells. In addition, a direct physical interaction between calcineurin and hFKBP38 was not detected in co-immunoprecipitation experiments. However, hFKBP38 indirectly affected the subcellular distribution of calcineurin by interaction with typical calcineurin ligands, as exemplified by the anti-apoptotic protein Bcl-2. Our data suggest that hFKBP38 cannot substitute for the FKBP/FK506 complex in signaling pathways controlled by the protein phosphatase activity of calcineurin.