CD33 and clusterin interact biophysically and genetically to modulate Alzheimer risk.

We report the results of structural, functional and genetic studies on the CD33 sialic acid- binding receptor that reveal how non-coding variants in CD33 alter risk for Alzheimer's disease (AD). The full-length CD33 isoform, whose expression is upregulated by non-coding AD-risk alleles, preferentially forms dimers at the cell surface, where they interact with AD-related proteins (clusterin and Aβ). This interaction induces CD33 inhibitory signalling and downregulates protective microglial functions including phagocytic removal of amyloid plaques.

CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer's Disease Susceptibility.

The innate immune gene 33, encoding a myeloid inhibitory sialic acid-binding receptor, is associated with Alzheimer's disease (AD) susceptibility. The AD-associated risk variant reduces splicing of the sialic acid-binding domain and increases expression of the full-length (sialic acid-binding) CD33 isoform seven-fold compared to the protective genotype. Here we identify CD45 as an immune cell-specific sialic acid-dependent CD33 binding partner, whose phosphatase activity is inhibited by CD33.

A vital sugar code for ricin toxicity.

Ricin is one of the most feared bioweapons in the world due to its extreme toxicity and easy access. Since no antidote exists, it is of paramount importance to identify the pathways underlying ricin toxicity. Here, we demonstrate that the Golgi GDP-fucose transporter Slc35c1 and fucosyltransferase Fut9 are key regulators of ricin toxicity.

Deletions linked to TP53 loss drive cancer through p53-independent mechanisms.

Mutations disabling the TP53 tumour suppressor gene represent the most frequent events in human cancer and typically occur through a two-hit mechanism involving a missense mutation in one allele and a 'loss of heterozygosity' deletion encompassing the other. While TP53 missense mutations can also contribute gain-of-function activities that impact tumour progression, it remains unclear whether the deletion event, which frequently includes many genes, impacts tumorigenesis beyond TP53 loss alone. Here we show that somatic heterozygous deletion of mouse chromosome 11B3, a 4-megabase region syntenic to human 17p13.

Cooperative loss of RAS feedback regulation drives myeloid leukemogenesis.

RAS network activation is common in human cancers, and in acute myeloid leukemia (AML) this activation is achieved mainly through gain-of-function mutations in KRAS, NRAS or the receptor tyrosine kinase FLT3. We show that in mice, premalignant myeloid cells harboring a Kras(G12D) allele retained low levels of Ras signaling owing to negative feedback involving Spry4 that prevented transformation. In humans, SPRY4 is located on chromosome 5q, a region affected by large heterozygous deletions that are associated with aggressive disease in which gain-of-function mutations in the RAS pathway are rare.

Janus kinase inhibition by ruxolitinib extends dasatinib- and dexamethasone-induced remissions in a mouse model of Ph+ ALL.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is initiated and driven by the oncogenic fusion protein BCR-ABL, a constitutively active tyrosine kinase. Despite major advances in the treatment of this highly aggressive disease with potent inhibitors of the BCR-ABL kinase such as dasatinib, patients in remission frequently relapse due to persistent minimal residual disease possibly supported, at least in part, by salutary cytokine-driven signaling within the hematopoietic microenvironment. Using a mouse model of Ph+ ALL that accurately mimics the genetics, clinical behavior, and therapeutic response of the human disease, we show that a combination of 2 agents approved by the US Food and Drug Administration (dasatinib and ruxolitinib, which inhibit BCR-ABL and Janus kinases, respectively), significantly extends survival by targeting parallel signaling pathways.

Systemic blockade of sialylation in mice with a global inhibitor of sialyltransferases.

Sialic acid terminates glycans of glycoproteins and glycolipids that play numerous biological roles in health and disease. Although genetic tools are available for interrogating the effects of decreased or abolished sialoside expression in mice, pharmacological inhibition of the sialyltransferase family has, to date, not been possible. We have recently shown that a sialic acid analog, 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-NeuAc), added to the media of cultured cells shuts down sialylation by a mechanism involving its intracellular conversion to CMP-3F-NeuAc, a competitive inhibitor of all sialyltransferases.

Targeted delivery of mycobacterial antigens to human dendritic cells via Siglec-7 induces robust T cell activation.

Lipids from mycobacteria can be presented to human T cells by group 1 CD1 Ag-presenting molecules (CD1a, CD1b, and CD1c). Group 1 CD1-restricted T cells are activated by lipid Ags presented by myeloid dendritic cells (DCs), after which they generate antibacterial effector functions, including IFN-γ secretion and cytolysis. Thus, mycobacterial lipids are being investigated as components of novel vaccines for mycobacterial infections.

Disubstituted Sialic Acid Ligands Targeting Siglecs CD33 and CD22 Associated with Myeloid Leukaemias and B Cell Lymphomas.

The siglec family of sialic acid-binding proteins are endocytic immune cell receptors that are recognized as potential targets for cell directed therapies. CD33 and CD22 are prototypical members and are validated candidates for targeting acute myeloid leukaemia and non-Hodgkin's lymphomas due to their restricted expression on myeloid cells and B-cells, respectively. While nanoparticles decorated with high affinity siglec ligands represent an attractive platform for delivery of therapeutic agents to these cells, a lack of ligands with suitable affinity and/or selectivity has hampered progress.

On-chip synthesis and screening of a sialoside library yields a high affinity ligand for Siglec-7.

The Siglec family of sialic acid-binding proteins are differentially expressed on white blood cells of the immune system and represent an attractive class of targets for cell-directed therapy. Nanoparticles decorated with high-affinity Siglec ligands show promise for delivering cargo to Siglec-bearing cells, but this approach has been limited by a lack of ligands with suitable affinity and selectivity. Building on previous work employing solution-phase sialoside library synthesis and subsequent microarray screening, we herein report a more streamlined 'on-chip' synthetic approach.

Click and pick: identification of sialoside analogues for siglec-based cell targeting.

Click 'n' chips: Azide and alkyne-bearing sialic acids (purple diamond; see picture) were subjected to high-throughput click chemistry to generate a library of sialic acid analogues. Microarray printing of the library and screening with the siglec family of sialic-acid-binding proteins, led to the identification of high-affinity ligands for siglec-9 and siglec-10.

Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome.

Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate-based inhibitors inside the cell.

High-throughput screening for inhibitors of sialyl- and fucosyltransferases.

Sweet screens: A high-throughput screening platform for identification of inhibitors of sialyl- and fucosyltransferases based on fluorescence polarization (FP) has been developed. An analogue of the natural donor substrate carrying a fluorescent label (green star) is transferred to a glycoprotein acceptor, which results in robust FP. The screening of 16,000 compounds against different glycosyltransferases has identified various interesting inhibitors.

Glycan microarrays for decoding the glycome.

In the last decade, glycan microarrays have revolutionized the analysis of the specificity of glycan-binding proteins (GBPs), providing information that simultaneously illuminates the biology mediated by them and decodes the informational content of the glycome. Numerous methods have emerged for arraying glycans in a "chip" format, and glycan libraries have been assembled that address the diversity of the human glycome. Such arrays have been successfully used for analysis of GBPs, which mediate mammalian biology, host-pathogen interactions, and immune recognition of glycans relevant to vaccine production and cancer antigens.