Functionality of bone marrow mesenchymal stromal cells derived from head and neck cancer patients - A FDA-IND enabling study regarding MSC-based treatments for radiation-induced xerostomia.

PURPOSE: Salivary dysfunction is a significant side effect of radiation therapy for head and neck cancer (HNC). Preliminary data suggests that mesenchymal stromal cells (MSCs) can improve salivary function. Whether MSCs from HNC patients who have completed chemoradiation are functionally similar to those from healthy patients is unknown. We performed a pilot clinical study to determine whether bone marrow-derived MSCs [MSC(M)] from HNC patients could be used for the treatment of RT-induced salivary dysfunction. METHODS: An IRB-approved pilot clinical study was undertaken on HNC patients with xerostomia who had completed treatment two or more years prior. Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated and cultured. Culture-expanded MSC(M) were stimulated with IFNγ and cryopreserved prior to reanimation and profiling for functional markers by flow cytometry and ELISA. MSC(M) were additionally injected into mice with radiation-induced xerostomia and the changes in salivary gland histology and salivary production were examined. RESULTS: A total of six subjects were enrolled. MSC(M) from all subjects were culture expanded to > 20 million cells in a median of 15.5 days (range 8-20 days). Flow cytometry confirmed that cultured cells from HNC patients were MSC(M). Functional flow cytometry demonstrated that these IFNγ-stimulated MSC(M) acquired an immunosuppressive phenotype. IFNγ-stimulated MSC(M) from HNC patients were found to express GDNF, WNT1, and R-spondin 1 as well as pro-angiogenesis and immunomodulatory cytokines. In mice, IFNγ-stimulated MSC(M) injection after radiation decreased the loss of acinar cells, decreased the formation of fibrosis, and increased salivary production. CONCLUSIONS: MSC (M) from previously treated HNC patients can be expanded for auto-transplantation and are functionally active. Furthermore IFNγ-stimulated MSC(M) express proteins implicated in salivary gland regeneration. This study provides preliminary data supporting the feasibility of using autologous MSC(M) from HNC patients to treat RT-induced salivary dysfunction.

Ruxolitinib inhibits IFNγ-stimulated Sjögren's salivary gland MSC HLA-DR expression and chemokine-dependent T cell migration.

OBJECTIVES: Sjögren's disease (SjD) is a systemic autoimmune disease characterized by focal lymphocytic infiltrate of salivary glands (SGs) and high SG IFNγ, both of which are associated with elevated lymphoma risk. IFNγ is also biologically relevant to mesenchymal stromal cells (MSCs), a SG resident cell with unique niche regenerative and immunoregulatory capacities. In contrast to the role of IFNγ in SjD, IFNγ promotes an anti-inflammatory MSC phenotype in other diseases. The objective of this study was to define the immunobiology of IFNγ-exposed SG-MSCs with and without the JAK1 & 2 inhibitor, ruxolitinib. METHODS: SG-MSCs were isolated from SjD and controls human subjects. SG-MSCs were treated with 10 ng/ml IFNγ +/- 1000 nM ruxolitinib. Experimental methods included flow cytometry, RNA-sequencing, chemokine array, ELISA and transwell chemotaxis experiments. RESULTS: We found that IFNγ promoted expression of SG-MSC immunomodulatory markers, including HLA-DR, and this expression was inhibited by ruxolitinib. We confirmed the differential expression of CXCL9, CXCL10, CXCL11, CCL2 and CCL7, initially identified with RNA sequencing. SG-MSCs promoted CD4+ T cell chemotaxis when pre-stimulated with IFNγ. Ruxolitinib blocks chemotaxis through inhibition of SG-MSC production of CXCL9, CXCL10 and CXCL11. CONCLUSIONS: These findings establish that ruxolitinib inhibits IFNγ-induced expression of SG-MSC immunomodulatory markers and chemokines. Ruxolitinib also reverses IFNγ-induced CD4+ T cell chemotaxis, through inhibition of CXCL9, -10 and -11. Because IFNγ is higher in SjD than control SGs, we have identified SG-MSCs as a plausible pathogenic cell type in SjD. We provide proof of concept supporting further study of ruxolitinib to treat SjD.

Unique molecular characteristics and microglial origin of Kv1.3 channel-positive brain myeloid cells in Alzheimer's disease.

Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer's disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aß) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11b+CD45+ CNS-MPs acutely isolated from an Aß mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11b+Kv1.3+ CNS-MPs, CD11b+CD45int Kv1.3neg microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers (Tmem119, P2ry12) and are distinct from peripheral Ly6chigh/Ly6clow monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of CD11c, and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3+ CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aß burden, increased CD11c+ CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes (IL1b). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype.

Washed Equine Platelet Extract as an Anti-Inflammatory Biologic Pharmaceutical.

Mammalian platelets participate in the immediate tissue injury response by initiating coagulation and further promoting tissue injury mitigation and repair. The latter properties are deployed following platelet release of presynthetized morphogens, cytokines, and growth and chemotactic factors, which launch a tissue regenerative, angiogenic, and anti-inflammatory program. Several blood-derived biologic products, like platelet-rich plasma (PRP) and platelet lysate (PL), are currently on the market to allow proper healing and tissue regeneration. However, not all growth factors are released from the platelets and the final products contain plasma proteins such as albumin, fibrinogen, complement, and immunoglobulins, increasing the risks of serum sickness or allergic reaction. To address this problem, we developed a new platelet extract where equine blood platelets are concentrated, washed, and thereafter lysed by detergent Triton X-114. Distinct from PRP, this extract is devoid of albumin, fibrinogen, and immunoglobulins and is 266-fold enriched in platelet-derived growth factor content relative to PRP. Washed equine platelet extract (WEPLEX) is amenable to lyophilization without loss of biological activity. In vitro, WEPLEX significantly inhibits human and equine T cell proliferative response to phytohemagglutinin and also polarizes murine CD45+/CD11b+ peritoneal macrophages to an IL-10+ M2-like phenotype. In vivo, WEPLEX substantially improves clinical outcome of murine experimental dextran sulfate sodium colitis. We propose that equine-sourced, zoonosis-free WEPLEX may serve as an anti-inflammatory biological therapy across mammalian species.

Minor salivary gland mesenchymal stromal cells derived from patients with SjÓ§gren's syndrome deploy intact immune plasticity.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) provide minor salivary glands (MSGs) with support and niche cells for epithelial glandular tissue. Little is known about resident MSG-derived MSCs (MSG-MSCs) in primary SjÓ§gren's syndrome (PSS). The authors' objective is to define the immunobiology of endogenous PSS MSG-MSCs. METHODS: Using culture-adapted MSG-MSCs isolated from consenting PSS subjects (n = 13), the authors performed in vitro interrogation of PSS MSG-MSC immunobiology and global gene expression compared with controls. To this end, the authors performed phenotypic and immune functional analysis of indoleamine 2,3-dioxygenase (IDO), programmed death ligand 1 (PD-L1) and intercellular adhesion marker 1 (ICAM-1) before and after interferon γ (IFNγ) licensing as well as the effect of MSG-MSCs on T-cell proliferation. Considering the female predominance of PSS, the authors also addressed the influence of 17-ß-estradiol on estrogen receptor α-positive-related MSC function. RESULTS: The authors found that MSG-MSCs deployed normal immune regulatory functionality after IFNγ stimulation, as demonstrated by increased protein-level expression of IDO, PD-L1 and ICAM-1. The authors also found that MSG-MSCs suppressed T-cell proliferation in a dose-dependent manner independent of 17-ß-estradiol exposure. Gene ontology and pathway analysis highlighted extracellular matrix deposition as a possible difference between PSS and control MSG-MSCs. MSG-MSCs demonstrated increased α-smooth muscle actin expression in PSS, indicating a partial myofibroblast-like adaptation. CONCLUSIONS: These findings establish similar immune regulatory function of MSG-MSCs in both PSS and control patients, precluding intrinsic MSC immune regulatory defects in PSS. PSS MSG-MSCs show a partial imprinted myofibroblast-like phenotype that may arise in the setting of chronic inflammation, providing a plausible etiology for PSS-related glandular fibrosis.

Regulatory B Cells Normalize CNS Myeloid Cell Content in a Mouse Model of Multiple Sclerosis and Promote Oligodendrogenesis and Remyelination.

The unmet medical need of patients with multiple sclerosis (MS) is the inexorable loss of CNS myelin and latterly neurons leading to permanent neurologic disability. Solicitation of endogenous oligodendrocytes progenitor cells, the precursor of oligodendrocytes, to remyelinate axons may abort the onset of disability. In female mice with experimental autoimmune encephalomyelitis (EAE), a murine model of MS, adoptive transfer of IL-10+ regulatory B cells (Bregs) has been shown to reverse EAE by promoting the expansion of peripheral and CNS-infiltrating IL-10+ T cells. Here, we examined whether Bregs treatment and its bystander effect on regulatory T cells are associated with CNS repair as reflected by oligodendrogenesis and remyelination. We have found that transfusion of Bregs reverses established clinical EAE and that clinical improvement is associated with a significant increase in spinal cord remyelination as reflected by g-ratio analysis within the thoracic and lumbar spine. We further observed in the spinal cords of EAE Bregs-treated mice that CNS resident CD11b/CD45intLy6C- microglia, and infiltrating CD11b+/CD45high monocytes/macrophages content reverts to normal and polarize to a M2-like CD206+ phenotype. Concurrently, there was a substantial increase in neo-oligodendrogenesis as manifest by an increase in CD45-/low CNS cells expressing A2B5, an early marker in oligodendrocytes progenitor cell differentiation as well as GalC+/O1+ premyelinating and myelin basic protein+/myelin oligodendrocyte glycoprotein+ mature oligodendrocytes with reciprocal downregulation of paired related homeobox protein 1. These results demonstrate that the clinical benefit of Bregs is associated with normalization of CNS immune milieu and concurrent activation of oligodendrocyte progenitor cells with subsequent remyelination.SIGNIFICANCE STATEMENT In multiple sclerosis patients, demyelination progresses with aging and disease course, leading to irreversible disability. In this study, we have discovered, using a mouse model of multiple sclerosis, that the transfusion of autologous regulatory B cells (Bregs) is able to ameliorate, cure, and sustain the durable remission of the disease. We show that the adoptive transfer of Bregs dramatically decreased the frequency of myeloid-derived cells, both infiltrating monocytes/macrophages and resident microglia, and converted their phenotype to an immunosuppressive-like phenotype. Moreover, we showed that CNS oligodendrocyte progenitor cells are activated following Bregs treatment and differentiate into myelinating oligodendrocytes, which results in neo-oligodendrogenesis and remyelination of spinal cords.

A systems pharmacology-based approach to identify novel Kv1.3 channel-dependent mechanisms in microglial activation.

BACKGROUND: Kv1.3 potassium channels regulate microglial functions and are overexpressed in neuroinflammatory diseases. Kv1.3 blockade may selectively inhibit pro-inflammatory microglia in neurological diseases but the molecular and cellular mechanisms regulated by Kv1.3 channels are poorly defined. METHODS: We performed immunoblotting and flow cytometry to confirm Kv1.3 channel upregulation in lipopolysaccharide (LPS)-activated BV2 microglia and in brain mononuclear phagocytes freshly isolated from LPS-treated mice. Quantitative proteomics was performed on BV2 microglia treated with control, LPS, ShK-223 (highly selective Kv1.3 blocker), and LPS+ShK-223. Gene ontology (GO) analyses of Kv1.3-dependent LPS-regulated proteins were performed, and the most representative proteins and GO terms were validated. Effects of Kv1.3-blockade on LPS-activated BV2 microglia were studied in migration, focal adhesion formation, reactive oxygen species production, and phagocytosis assays. In vivo validation of protein changes and predicted molecular pathways were performed in a model of systemic LPS-induced neuroinflammation, employing antigen presentation and T cell proliferation assays. Informed by pathway analyses of proteomic data, additional mechanistic experiments were performed to identify early Kv1.3-dependent signaling and transcriptional events. RESULTS: LPS-upregulated cell surface Kv1.3 channels in BV2 microglia and in microglia and CNS-infiltrating macrophages isolated from LPS-treated mice. Of 144 proteins differentially regulated by LPS (of 3141 proteins), 21 proteins showed rectification by ShK-223. Enriched cellular processes included MHCI-mediated antigen presentation (TAP1, EHD1), cell motility, and focal adhesion formation. In vitro, ShK-223 decreased LPS-induced focal adhesion formation, reversed LPS-induced inhibition of migration, and inhibited LPS-induced upregulation of EHD1, a protein involved in MHCI trafficking. In vivo, intra-peritoneal ShK-223 inhibited LPS-induced MHCI expression by CD11b+CD45low microglia without affecting MHCI expression or trafficking of CD11b+CD45high macrophages. ShK-223 inhibited LPS-induced MHCI-restricted antigen presentation to ovalbumin-specific CD8+ T cells both in vitro and in vivo. Kv1.3 co-localized with the LPS receptor complex and regulated LPS-induced early serine (S727) STAT1 phosphorylation. CONCLUSIONS: We have unraveled novel molecular and functional roles for Kv1.3 channels in pro-inflammatory microglial activation, including a Kv1.3 channel-regulated pathway that facilitates MHCI expression and MHCI-dependent antigen presentation by microglia to CD8+ T cells. We also provide evidence for neuro-immunomodulation by systemically administered ShK peptides. Our results further strengthen the therapeutic candidacy of microglial Kv1.3 channels in neurologic diseases.

GM-CSF and IL-4 Fusion Cytokine Induces B Cell-Dependent Hematopoietic Regeneration.

Hematopoietic stem cells (HSCs) have the capacity to self-renew and differentiate into hematopoietic cells and have been utilized to replace diseased bone marrow for patients with cancers and blood disorders. Although remarkable progress has been made in developing new tools to manipulate HSCs for clinic use, there is still no effective method to expand HSCs in vivo for quick repopulation of hematopoietic cells following sublethal irradiation. We have recently described a novel synthetic cytokine that is derived from the fusion of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4; named as GIFT4), and we have now discovered that GIFT4 fusokine promotes long-term hematopoietic regeneration in a B cell-dependent manner. We found that GIFT4 treatment triggered a robust expansion of endogenous bone marrow HSCs and multipotent progenitors in vivo. Delivery of GIFT4 protein together with B cells rescued lethally irradiated mice. Moreover, adoptive transfer of autologous or allogeneic GIFT4-treated B cells (GIFT4-B cells) enhanced long-term hematopoietic recovery in radiated mice and prevented the mice from irradiation-induced death. Our data suggest that GIFT4 as well as GIFT4-B cells could serve as means to augment HSC engraftment in the setting of bone marrow transplantation for patients with hematological malignancy.

Regulatory B Cells Induce Formation of IL-10-Expressing T Cells in Mice with Autoimmune Neuroinflammation.

Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How these regulatory B cells (Bregs) suppress the immune response remains to be worked out in detail. In this article, we show that Bregs can induce the formation of conventional FoxP3+ regulatory T cells (Tregs), as well as a more recently described CD49b+CD223+ regulatory T-cell subset, known as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, they home to the spleen and mesenteric lymph nodes, leading to an expansion of Tregs and Tr1 in vivo Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms. The discovery that Bregs induce the formation of regulatory T-cell subsets in vivo may herald their use as immunosuppressive agents in adoptive cellular therapies for autoimmune pathologies. SIGNIFICANCE STATEMENT: Although B cells are traditionally known for their role in propagating proinflammatory immune responses, their immunosuppressive effects have only recently begun to be appreciated. How regulatory B cells (Bregs) suppress the immune response remains to be fully understood. In this article, we show that Bregs can induce the formation of conventional regulatory T cells (Tregs) as well as type 1 regulatory T cells (Tr1s). When Bregs are transferred into mice with experimental autoimmune encephalomyelitis (EAE), they home to secondary lymphoid organs, leading to an expansion of Tregs and Tr1s in vivo Tregs and Tr1s are also found in greater proportions in the CNS of mice with EAE treated with Bregs and are correlated with the remission of symptoms.

Stimulation of Natural Killer Cell-Mediated Tumor Immunity by an IL15/TGFß-Neutralizing Fusion Protein.

The clinical efficacy of immune cytokines used for cancer therapy is hampered by elements of the immunosuppressive tumor microenvironment such as TGFß. Here we demonstrate that FIST15, a recombinant chimeric protein composed of the T-cell-stimulatory cytokine IL15, the sushi domain of IL15Rα and a TGFß ligand trap, can overcome immunosuppressive TGFß to effectively stimulate the proliferation and activation of natural killer (NK) and CD8+ T cells with potent antitumor properties. FIST15-treated NK and CD8+ T cells produced more IFNγ and TNFα compared with treatment with IL15 and a commercially available TGFß receptor-Fc fusion protein (sTßRII) in the presence of TGFß. Murine B16 melanoma cells, which overproduce TGFß, were lysed by FIST15-treated NK cells in vitro at doses approximately 10-fold lower than NK cells treated with IL15 and sTßRII. Melanoma cells transduced to express FIST15 failed to establish tumors in vivo in immunocompetent murine hosts and could only form tumors in beige mice lacking NK cells. Mice injected with the same cells were also protected from subsequent challenge by unmodified B16 melanoma cells. Finally, mice with pre-established B16 melanoma tumors responded to FIST15 treatment more strongly compared with tumors treated with control cytokines. Taken together, our results offer a preclinical proof of concept for the use of FIST15 as a new class of biological therapeutics that can coordinately neutralize the effects of immunosuppressive TGFß in the tumor microenvironment while empowering tumor immunity. Cancer Res; 76(19); 5683-95. ©2016 AACR.

GIFT4 fusokine converts leukemic B cells into immune helper cells.

BACKGROUND: Chronic lymphocytic leukemia (CLL) remains incurable with standard therapy, and is characterized by excessive expansion of monoclonal abnormal mature B cells and more regulatory immune properties of T cell compartment. Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL. METHODS: We generated a fusion cytokine (fusokine) arising from the combination of human GM-CSF and IL-4 (named GIFT4). Primary CLL cells were treated with GIFT4 or GM-CSG and IL-4 in vitro. GIFT4-triggered STAT5 signaling in CLL cells was examined by Western blot. The phenotype and secretome of GIFT4-treated CLL cells (GIFT4-CLL cells), and the immune stimulatory function of GIFT4-CLL cells on autologous T cells were analyzed by flow cytometry and luminex assay. RESULTS: GIFT4-CLL up-regulated the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54. GIFT4-CLL cells secreted IL-1ß, IL-6, ICAM-1 and substantial IL-2 relative to unstimulated CLL cells. GIFT4 treatment led to JAK1, JAK2 and JAK3-mediated hyper-phosphorylation of STAT5 in primary CLL cells, which is essential for GIFT4-triggered conversion of CLL cells. GIFT4-CLL cells directly propelled the expansion of autologous IFN-γ-producing CD314(+) cytotoxic T cells in vitro, and that these could lyse autologous CLL cells. Furthermore, administration of GIFT4 protein promoted the expansion of human T cells in NOD-scid IL2Rγ(null) immune deficient mice adoptively pre-transferred with peripheral blood mononuclear cells from subjects with CLL. CONCLUSION: GIFT4 has potent capability to converts primary CLL cells into APC-like immune helper cells that initiate a T cell driven anti-CLL immune response.

Maltose-binding protein fusion allows for high level bacterial expression and purification of bioactive mammalian cytokine derivatives.

Fusokines are chimeric proteins generated by the physical coupling of cytokines in a single polypeptide, resulting in proteins with highly pleiotropic activity and the potential to treat cancer and autoimmune ailments. For instance, the fusokine GIFT15 (GM-CSF and Interleukin 15 Fusion Transgene) has been shown to be a powerful immunosuppressive protein able to convert naïve B cells into IL-10-producing B cells. To date, the mammalian cell systems used for the expression of GIFT15 allow for secretion of the protein in the culturing media, an inefficient system for producing GMP-compliant fusokines. In this study we report the bacterial expression of bioactive recombinant GIFT15 (rGIFT15). Indeed, there is a constant demand to improve the expression systems for therapeutic proteins. Expression of a maltose-binding protein (MBP) fusion protein efficiently allowed the accumulation of soluble protein in the intracellular milieu. Optimizing the bacterial culture significantly increased the yield of recombinant protein. The biological activity of rGIFT15 was comparable to that of fusokine derived from a mammalian source. This approach led to the production of soluble, endotoxin-free functional protein, averaging 5 mg of rGIFT15 per liter of culture. This process is amenable to scale up for the development of Food and Drug Administration (FDA)-compliant immune-modulatory rGIFT15.

Engineered fusokine GIFT4 licenses the ability of B cells to trigger a tumoricidal T-cell response.

Engineered chimeric cytokines can generate gain-of-function activity in immune cells. Here, we report potent antitumor activity for a novel fusion cytokine generated by N-terminal coupling of GM-CSF to IL4, generating a fusokine termed GIFT4. B cells treated with GIFT4 clustered GM-CSF and IL4 receptors on the cell surface and displayed a pan-STAT hyperphosphorylation associated with acquisition of a distinct phenotype and function described to date. In C57BL/6J mice, administration of GIFT4 expanded endogenous B cells and suppressed the growth of B16F0 melanoma cells. Furthermore, B16F0 melanoma cells engineered to secrete GIFT4 were rejected immunologically in a B-cell-dependent manner. This effect was abolished when GIFT4-expressing B16F0 cells were implanted in B-cell-deficient mice, confirming a B-cell-dependent antitumor effect. Human GIFT4-licensed B cells primed cytotoxic T cells and specifically killed melanoma cells in vitro and in vivo. Taken together, our results demonstrated that GIFT4 could mediate expansion of B cells with potent antigen-specific effector function. GIFT4 may offer a novel immunotherapeutic tool and define a previously unrecognized potential for B cells in melanoma immunotherapy.

Kinetics of heme transfer by the Shr NEAT domains of Group A Streptococcus.

The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface.

Pathway of glycine betaine biosynthesis in Aspergillus fumigatus.

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD(+) to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.

Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments.

Quantitative analysis of Ca(2+) fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca(2+)-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca(2+) binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca(2+) release kinetics with an off-rate estimated at around 700 s(-1) and appropriate Ca(2+) binding affinity, likely attributable to local Ca(2+)-induced conformational changes around the designed Ca(2+) binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca(2+) dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca(2+) in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca(2+) release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca(2+) homeostasis.

Stabilization of an intermediate in the oxidative half-reaction of human liver glycolate oxidase.

Glycolate oxidase is a flavin-dependent enzyme that catalyzes the oxidation of α-hydroxy acids to the corresponding α-keto acids, with reduction of molecular oxygen to hydrogen peroxide. A number of probes have been used to investigate the oxidative half-reaction catalyzed by the enzyme, including steady state and rapid kinetics, pH studies, solvent kinetic isotope effects, and solvent viscosity effects. Here we present the first spectroscopic evidence of the formation of an intermediate with absorbance features resembling those of a flavosemiquinone in the oxidative half-reaction of glycolate oxidase.

Rescuing of the hydride transfer reaction in the Glu312Asp variant of choline oxidase by a substrate analogue.

In the active site of choline oxidase, Glu312 participates in binding the trimethylammonium group of choline, thereby positioning the alcohol substrate properly for efficient hydride transfer to the enzyme-bound flavin. Previous studies have shown that substitution of Glu312 with aspartate results in a perturbed mechanism of hydride transfer, with a 260-fold decrease in the rate associated with the mutation. Here, the reaction of alcohol oxidation catalyzed by the Glu312Asp enzyme has been investigated with 3-hydroxypropyl-trimethylamine (3-HPTA), a choline analogue with an extra methylene, as substrate. The results of the kinetic investigation using steady state and rapid reaction approaches showed that the impaired ability of the Glu312Asp enzyme to catalyze a hydride transfer reaction can be effectively, but not completely, rescued in the presence of an extra methylene group on the substrate that compensates for the equivalent shortening of the side chain on residue 312. This observation is consistent with choline oxidase having evolved to optimally catalyze the oxidation of choline.

Involvement of ionizable groups in catalysis of human liver glycolate oxidase.

Glycolate oxidase is a flavin-dependent, peroxisomal enzyme that oxidizes alpha-hydroxy acids to the corresponding alpha-keto acids, with reduction of oxygen to H(2)O(2). In plants, the enzyme participates in photorespiration. In humans, it is a potential drug target for treatment of primary hyperoxaluria, a genetic disorder where overproduction of oxalate results in the formation of kidney stones. In this study, steady-state and pre-steady-state kinetic approaches have been used to determine how pH affects the kinetic steps of the catalytic mechanism of human glycolate oxidase. The enzyme showed a Ping-Pong Bi-Bi kinetic mechanism between pH 6.0 and 10.0. Both the overall turnover of the enzyme (k(cat)) and the rate constant for anaerobic substrate reduction of the flavin were pH-independent at pH values above 7.0 and decreased slightly at lower pH, suggesting the involvement of an unprotonated group acting as a base in the chemical step of glycolate oxidation. The second-order rate constant for capture of glycolate (k(cat)/K(glycolate)) and the K(d)((app)) for the formation of the enzyme-substrate complex suggested the presence of a protonated group with apparent pK(a) of 8.5 participating in substrate binding. The k(cat)/K(oxygen) values were an order of magnitude faster when a group with pK(a) of 6.8 was unprotonated. These results are discussed in the context of the available three-dimensional structure of GOX.