Jean Jakoncic

Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire

Idiosyncratic adverse drug reactions are unpredictable, dose-independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkages between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells that required HLA-B*57:01 molecules for their function; however, the mechanism by which abacavir induces this pathologic T-cell response remains unclear. Here we show that abacavir can bind within the F pocket of the peptide-binding groove of HLA-B*57:01, thereby altering its specificity. This provides an explanation for HLA-linked idiosyncratic adverse drug reactions, namely that drugs can alter the repertoire of self-peptides presented to T cells, thus causing the equivalent of an alloreactive T-cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir and that were recognized by T cells of hypersensitive patients. The assays that we have established can be applied to test additional compounds with suspected HLA-linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA-linked hypersensitivities, and guide the development of safer drugs.

Migrastatin analogues target fascin to block tumour metastasis

Tumour metastasis is the primary cause of death of cancer patients. Development of new therapeutics preventing tumour metastasis is urgently needed. Migrastatin is a natural product secreted by Streptomyces, and synthesized migrastatin analogues such as macroketone are potent inhibitors of metastatic tumour cell migration, invasion and metastasis. Here we show that these migrastatin analogues target the actin-bundling protein fascin to inhibit its activity. X-ray crystal structural studies reveal that migrastatin analogues bind to one of the actin-binding sites on fascin. Our data demonstrate that actin cytoskeletal proteins such as fascin can be explored as new molecular targets for cancer treatment, in a similar manner to the microtubule protein tubulin.

Structure and Hemimethylated CpG Binding of the SRA Domain from Human UHRF1.

Human UHRF1 (ubiquitin-like PHD and RING finger 1) functions to maintain CpG DNA methylation patterns through DNA replication by co-localizing with the DNA methyltransferase DNMT1 at chromatin in mammals. Recent studies show that UHRF1 binds selectively to hemimethylated CpG via its conserved SRA (SET- and RING finger-associated) domain. However, the underlying molecular mechanism is not known. Here, we report a 1.95Å resolution crystal structure of the SRA domain of human UHRF1. Using NMR structure-guided mutagenesis, electrophoretic mobility shift assay, and fluorescence anisotropy analysis, we determined key amino acid residues for methyl-DNA binding that are conserved in the SRA domain.

UHRF1 double tudor domain and the adjacent PHD finger act together to recognize K9me3-containing histone H3 tail

Human multi-domain-containing protein UHRF1 has recently been extensively characterized as a key epigenetic regulator for maintaining DNA methylation patterns. UHRF1 SRA domain preferentially binds to hemimethylated CpG sites, and double Tudor domain has been implicated in recognizing H3K9me3 mark, but the role of the adjacent PHD finger remains unclear. Here, we report the high-resolution crystal structure of UHRF1 PHD finger in complex with N-terminal tail of histone H3. We found that the preceding zinc-Cys4 knuckle is indispensable for the PHD finger of UHRF1 to recognize the first four unmodified residues of histone H3 N-terminal tail. Quantitative binding studies indicated that UHRF1 PHD finger (including the preceding zinc-Cys4 knuckle) acts together with the adjacent double Tudor domain to specifically recognize the H3K9me3 mark. Combinatorial recognition of H3K9me3-containing histone H3 tail by UHRF1 PHD finger and double Tudor domain may play a role in establishing and maintaining histone H3K9 methylation patterns during the cell cycle.

Molecular Mechanism of Fascin Function in Filopodial Formation

Background: Fascin is the main actin-bundling protein in filopodia. Results: Biochemical, cryo-electron tomographic, and x-ray crystal structural data reveal the unique actin-binding characteristics of fascin. Conclusion: There are two major actin-binding sites on fascin and there is a concerted conformational change between the actin-binding sites. Significance: These data will advance our understanding of the function of fascin in filopodial formation. Filopodia are cell surface protrusions that are essential for cell migration. This finger-like structure is supported by rigid tightly bundled actin filaments. The protein responsible for actin bundling in filopodia is fascin. However, the mechanism by which fascin functions in filopodial formation is not clear. Here we provide biochemical, cryo-electron tomographic, and x-ray crystal structural data demonstrating the unique structural characteristics of fascin. Systematic mutagenesis studies on 100 mutants of fascin indicate that there are two major actin-binding sites on fascin. Crystal structures of four fascin mutants reveal concerted conformational changes in fascin from inactive to active states in the process of actin bundling. Mutations in any one of the actin-binding sites impair the cellular function of fascin in filopodial formation. Altogether, our data reveal the molecular mechanism of fascin function in filopodial formation.

Small-Molecule Modulators of Methyl-Lysine Binding for the CBX7 Chromodomain.

Chromobox homolog 7 (CBX7) plays an important role in gene transcription in a wide array of cellular processes, ranging from stem cell self-renewal and differentiation to tumor progression. CBX7 functions through its N-terminal chromodomain (ChD), which recognizes trimethylated lysine 27 of histone 3 (H3K27me3), a conserved epigenetic mark that signifies gene transcriptional repression. In this study, we report the discovery of small molecules that inhibit CBX7ChD binding to H3K27me3. Our crystal structures reveal the binding modes of these molecules that compete against H3K27me3 binding through interactions with key residues in the methyl-lysine binding pocket of CBX7ChD. We further show that a lead compound, MS37452, derepresses transcription of Polycomb repressive complex target gene p16/CDKN2A by displacing CBX7 binding to the INK4A/ARF locus in prostate cancer cells. These small molecules have the potential to be developed into high-potency chemical modulators that target CBX7 functions in gene transcription in different disease pathways.

Ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immune-type receptor in amphioxus

Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region–containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 Å) and a pair of V-type domains (to 1.85 Å) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.

A bony fish immunological receptor of the NITR multigene family mediates allogeneic recognition.

Novel immune-type receptors (NITRs) comprise an exceptionally large, diversified family of activating and inhibitory receptors that has been identified in bony fish. Here, we characterized the structure of an activating NITR that is expressed by a cytotoxic natural killer (NK)-like cell line and that specifically binds an allogeneic B cell target. A single amino acid residue within the NITR immunoglobulin variable (V)-type domain accounts for specificity of the interaction. Structures solved by X-ray crystallography revealed that the V-type domains of NITRs form homodimers resembling rearranging antigen-binding receptor heterodimers. CDR1 elements of both subunits of NITR dimers form ligand-binding surfaces that determine specificity for the nonself target. In the evolution of immune function, it appears that a specific NK type of innate recognition may be mediated by a complex germline multigene family of V structures resembling those that are somatically diversified in adaptive immunological responses.

The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

The ring‐hydroxylating dioxygenase (RHD) from Sphingomonas CHY‐1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four‐ and five‐fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY‐1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 Å by molecular replacement revealed the enzyme to share the same global α3β3 structural pattern. The catalytic domain distinguishes itself from other bacterial non‐heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 Å. An 11 Å long water channel extends from the interface between the α and β subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY‐1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.

Potent elastase inhibitors from cyanobacteria: structural basis and mechanisms mediating cytoprotective and anti-inflammatory effects in bronchial epithelial cells.

We discovered new structural diversity to a prevalent, yet medicinally underappreciated, cyanobacterial protease inhibitor scaffold and undertook comprehensive protease profiling to reveal potent and selective elastase inhibition. Structure-activity relationship (SAR) studies and X-ray cocrystal structure analysis allowed a detailed assessment of critical and tunable structural elements. To realize the therapeutic potential of these cyclodepsipeptides, we probed the cellular effects of a novel and representative family member, symplostatin 5 (1), which attenuated the downstream cellular effects of elastase in an epithelial lung airway model system, alleviating clinical hallmarks of chronic pulmonary diseases such as cell death, cell detachment, and inflammation. This compound attenuated the effects of elastase on receptor activation, proteolytic processing of the adhesion protein ICAM-1, NF-κB activation, and transcriptomic changes, including the expression of pro-inflammatory cytokines IL1A, IL1B, and IL8. Compound 1 exhibited activity comparable to the clinically approved elastase inhibitor sivelestat in short-term assays and demonstrated superior sustained activity in longer-term assays.