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Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro,which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these,glutamic acid/alanine-rich protein (GARP),is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms,the variant surface glycoprotein (VSG) that protects the parasite membrane,and is involved in antigenic variation. Unlike VSG,however,the function of GARP is not known,which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP,we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG,the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively,these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal. View Publication

Mammary cancer stem cells (MaCSCs) have been identified as a rare population of cells capable of self-renewal to drive mammary tumorigenesis and metastasis. Nevertheless,relatively little is known about the intracellular signaling pathways regulating self-renewal and metastatic activities of MaCSCs in vivo. Using a recently developed breast cancer mouse model with focal adhesion kinase (FAK) deletion in mammary tumor cells (MFCKO-MT mice),here we present evidence suggesting a compensatory function of Pyk2,a FAK-related kinase,in the regulation of MaCSCs and metastasis in these mice. Increased expression of Pyk2 was found selectively in pulmonary metastatic nodules of MFCKO-MT mice,and its inhibition significantly reduced mammary tumor development and metastasis in these mice. Consistent with the idea of metastasis driven by MaCSCs,we detected selective up-regulation of Pyk2 in MaCSCs,but not bulk mammary tumor cells,of primary tumors developed in MFCKO-MT mice. We further showed that inhibition of Pyk2 in FAK-null MaCSCs significantly decreased their tumorsphere formation and migration in vitro as well as self-renewal,tumorigenicity,and metastatic activity in vivo. Last,we identified PI3K/Akt signaling as a major mediator of FAK regulation of MaCSCs as well as a target for the compensatory function of Pyk2 in FAK-null MaCSCs. Together,these results further advance our understanding of FAK and its related tyrosine kinase Pyk2 in regulation of MaCSCs in breast cancer and suggest that pharmaceutically targeting these kinases may hold promise as a novel treatment for the disease by targeting and eradicating MaCSCs. View Publication

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article,we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule,E-cadherin,to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin,but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase,suggesting that E-cadherin promotes rapid neuronal differentiation. Further,these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin,neurofilament,and neural cell adhesion molecule. Moreover,blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation,we are able to utilize a specific cell-cell adhesion molecule,E-cadherin,to influence the nature and degree of neural specialization. View Publication

O(6)-Alkylguanine-DNA alkyltransferase (AGT) is a widely distributed,unique DNA repair protein that acts as a single agent to directly remove alkyl groups located on the O(6)-position of guanine from DNA restoring the DNA in one step. The protein acts only once,and its alkylated form is degraded rapidly. It is a major factor in counteracting the mutagenic,carcinogenic,and cytotoxic effects of agents that form such adducts including N-nitroso-compounds and a number of cancer chemotherapeutics. This review describes the structure,function,and mechanism of action of AGTs and of a family of related alkyltransferase-like proteins,which do not act alone to repair O(6)-alkylguanines in DNA but link repair to other pathways. The paradoxical ability of AGTs to stimulate the DNA-damaging ability of dihaloalkanes and other bis-electrophiles via the formation of AGT-DNA cross-links is also described. Other important properties of AGTs include the ability to provide resistance to cancer therapeutic alkylating agents,and the availability of AGT inhibitors such as O(6)-benzylguanine that might overcome this resistance is discussed. Finally,the properties of fusion proteins in which AGT sequences are linked to other proteins are outlined. Such proteins occur naturally,and synthetic variants engineered to react specifically with derivatives of O(6)-benzylguanine are the basis of a valuable research technique for tagging proteins with specific reagents. View Publication

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane,the activated Akt is subjected to other regulatory events,including ubiquitination-mediated deactivation. Here,by identifying and characterizing an allosteric inhibitor,SC66,we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically,SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor,which stabilizes Akt,prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable,making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity,SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling,compared with other inhibitors of PIP3/Akt pathway. Finally,we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion,in this study,we not only identify a dual-function Akt inhibitor,but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation,thus identifying an avenue for pharmacological intervention in Akt signaling. View Publication

Overexpression of high mobility group AT-hook 2 (HMGA2) is found in a number of benign and malignant tumors,including the clonal PIGA(-) cells in 2 cases of paroxysmal nocturnal hemoglobinuria (PNH) and some myeloproliferative neoplasms (MPNs),and recently in hematopoietic cell clones resulting from gene therapy procedures. In nearly all these cases overexpression is because of deletions or translocations that remove the 3' untranslated region (UTR) which contains binding sites for the regulatory micro RNA let-7. We were therefore interested in the effect of HMGA2 overexpression in hematopoietic tissues in transgenic mice (ΔHmga2 mice) carrying a 3'UTR-truncated Hmga2 cDNA. ΔHmga2 mice expressed increased levels of HMGA2 protein in various tissues including hematopoietic cells and showed proliferative hematopoiesis with increased numbers in all lineages of peripheral blood cells,hypercellular bone marrow (BM),splenomegaly with extramedullary erythropoiesis and erythropoietin-independent erythroid colony formation. ΔHmga2-derived BM cells had a growth advantage over wild-type cells in competitive repopulation and serial transplantation experiments. Thus overexpression of HMGA2 leads to proliferative hematopoiesis with clonal expansion at the stem cell and progenitor levels and may account for the clonal expansion in PNH and MPNs and in gene therapy patients after vector insertion disrupts the HMGA2 locus. View Publication

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms,however,underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite,retinoic acid (RA),as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism,we investigated the ability of HSC to function as regulatory APC. Different from previous reports,we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function,HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast,HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells,whereas they greatly inhibited the Th17 differentiation. Furthermore,the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders,and therefore,by promoting Tregs and suppressing Th17 differentiation,they might represent key players in the mechanism that drives liver-induced tolerance. View Publication

The demonstration of the brain's ability to initiate repair in response to disease or injury has sparked considerable interest in therapeutic strategies to stimulate adult neurogenesis. In this study we examined the effect of a progressive neurodegenerative condition on neural precursor activity in the subventricular zone (SVZ) and hippocampus of the R6/1 transgenic mouse model of Huntington's disease (HD). Our results revealed an age-related decline in SVZ precursor numbers in both wild-type (WT) and HD mice. Interestingly,hippocampal precursor numbers declined with age in WT mice,although we observed maintenance in hippocampal precursor number in the HD animals in response to advancement of the disease. This maintenance was consistent with activation of a recently identified latent hippocampal precursor population. We found that the small latent stem cell population was also maintained in the HD hippocampus at 33 weeks,whereas it was not present in the WT. Our findings demonstrate that,despite a loss of neurogenesis in the HD hippocampus in vivo,there is a unique maintenance of the precursor and stem cells,which may potentially be activated to ameliorate disease symptoms. View Publication

Nature Methods 8,293 (2011). doi:10.1038/nmeth0411-293 View Publication

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity,we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo,activated and memory B cells expressed lower levels of CD1d compared with resting,naive,and marginal zone-like B cells. In vitro,CD1d was downregulated by all forms of B cell activation,leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone,whereas activation via the BCR significantly upregulated CD1c,particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes,an effect that was reversed by RARα agonists. However,BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells,in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity. View Publication

The stem cell factor (SCF)/Kit system has served as a classic model in deciphering molecular signaling events in the hematopoietic compartment,and Kit expression is a most critical marker for hematopoietic stem cells (HSCs) and progenitors. However,it remains to be elucidated how Kit expression is regulated in HSCs. Herein we report that a cytoplasmic tyrosine phosphatase Shp2,acting downstream of Kit and other RTKs,promotes Kit gene expression,constituting a Kit-Shp2-Kit signaling axis. Inducible ablation of PTPN11/Shp2 resulted in severe cytopenia in BM,spleen,and peripheral blood in mice. Shp2 removal suppressed the functional pool of HSCs/progenitors,and Shp2-deficient HSCs failed to reconstitute lethally irradiated recipients because of defects in homing,self-renewal,and survival. We show that Shp2 regulates coordinately multiple signals involving up-regulation of Kit expression via Gata2. Therefore,this study reveals a critical role of Shp2 in maintenance of a functional HSC/progenitor pool in adult mammals,at least in part through a kinase-phosphatase-kinase cascade. View Publication

Although regulation of histone methylation is believed to contribute to embryonic stem cell (ESC) self-renewal,the mechanisms remain obscure. We show here that the histone H3 trimethyl lysine 4 (H3K4me3) demethylase,KDM5B,is a downstream Nanog target and critical for ESC self-renewal. Although KDM5B is believed to function as a promoter-bound repressor,we find that it paradoxically functions as an activator of a gene network associated with self-renewal. ChIP-Seq reveals that KDM5B is predominantly targeted to intragenic regions and that it is recruited to H3K36me3 via an interaction with the chromodomain protein MRG15. Depletion of KDM5B or MRG15 increases intragenic H3K4me3,increases cryptic intragenic transcription,and inhibits transcriptional elongation of KDM5B target genes. We propose that KDM5B activates self-renewal-associated gene expression by repressing cryptic initiation and maintaining an H3K4me3 gradient important for productive transcriptional elongation. View Publication