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Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. Bacterial asparaginases are used in cancer chemotherapy to deplete asparagine from the blood, since several hematological malignancies depend on extracellular asparagine for growth. To avoid the immune response against the bacterial enzymes it would be beneficial to replace them with human asparaginases. However, unlike the bacterial asparaginases, the human enzymes have a millimolar Km value for asparagine, making them inefficient in depleting the amino acid from blood. To facilitate the development of human variants suitable for therapeutic use, we solved the structure of human L-asparaginase (hASNase3). This asparaginase is an N-terminal nucleophile (Ntn) family member that requires autocleavage between Gly167 and Thr168 to become catalytically competent. For most Ntn-hydrolases this autoproteolytic activation occurs efficiently. In contrast, hASNas3 is relatively stable in its uncleaved state, and this allowed us to observe the structure of the enzyme prior to cleavage. To determine the structure of the cleaved state we exploited our discovery that the free amino acid glycine promotes complete cleavage of hASNase3. Both enzyme states were elucidated in the absence and presence of the product aspartate. Together, these structures provide insight into the conformational changes required for cleavage, and on the precise enzyme-substrate interactions. The new understanding of hASNase3 will serve to guide the design of variants that possess a decreased Km value for asparagine, making the human enzyme a suitable replacement for the bacterial asparaginases in cancer therapy.

Background: ZO-1 is a scaffolding protein implicated in the assembly of tight junctions. Results: Structures of core PDZ-SH3-GUK, plus and minus JAM-A peptide, and isolated PDZ are presented. Conclusion: The SH3 domain is required for JAM-A binding to PDZ3. Significance: This is the first demonstration for the role of an adjacent domain to the binding of ligands to PDZ domains in the MAGUK family.

Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile (Ntn) hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form, and undergoes self-cleavage to the active form at a very slow rate. Here we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated post-translational processing, and explain why no other amino acid can substitute for glycine.

Our long-term goal is the design of a human L-asparaginase (hASNase3) variant, suitable for use in cancer therapy without the immunogenicity problems associated with the currently used bacterial enzymes. Asparaginases catalyze the hydrolysis of the amino acid asparagine to aspartate and ammonia. The key property allowing for the depletion of blood asparagine by bacterial asparaginases is their low micromolar KM value. In contrast, human enzymes have a millimolar KM for asparagine. Towards the goal of engineering an hASNase3 variant with micromolar KM, we conducted a structure/function analysis of the conserved catalytic threonine triad of this human enzyme. As a member of the N-terminal nucleophile (Ntn) family, to become enzymatically active, hASNase3 must undergo autocleavage between residues Gly167 and Thr168. To determine the individual contribution of each of the three conserved active site threonines (threonine triad, Thr168, Thr186, Thr219) for the enzyme-activating autocleavage and asparaginase reactions, we prepared the T168S, T186V and T219A/V mutants. These mutants were tested for their ability to cleave and to catalyze asparagine hydrolysis, in addition to being examined structurally. We also elucidated the first Ntn plant-type asparaginase structure in the covalent intermediate state. Our studies indicate that while not all the triad threonines are required for the cleavage reaction, all are essential for the asparaginase activity. The increased understanding of hASNase3 function resulting from these studies reveals the key regions that govern cleavage and the asparaginase reaction, which may inform the design of variants that attain a low KM for asparagine.

Co-targeting of both de novo and salvage pathways for dCTP biosynthesis shows efficacy in T-ALL and B-ALL.

Background:The interaction between the C terminus of claudin proteins and the ZO-1 PDZ1 domain regulates tight junction assembly. Results: Solved structures of PDZ1 in complex with claudin-1 and claudin-2 and determined binding affinities. Conclusion: Phosphorylation state of the tyrosine at position-6 regulates claudin/ZO-1 interaction. Significance: Revealed how post-translational modifications could affect the claudin/ZO-1 interaction and thereby tight junction barrier properties.

Human Rad51 is a key element of recombinational DNA repair and is related to the resistance of cancer cells to chemo‐ and radiotherapies. The protein is thus a potential target of anti‐cancer treatment. The crystallographic analysis shows that the BRC‐motif of the BRCA2 tumor suppressor is in contact with the subunit–subunit interface of Rad51 and could thus prevent filament formation of Rad51. However, biochemical analysis indicates that a BRC‐motif peptide of 69 amino acids preferentially binds to the N‐terminal part of Rad51. We show experimentally that a short peptide of 28 amino acids derived from the BRC4 motif binds to the subunit–subunit interface and dissociates its filament, both in the presence and absence of DNA, certainly by binding to dissociated monomers. The inhibition is efficient and specific for Rad51: the peptide does not even interact with Rad51 homologs or prevent their interaction with DNA. Neither the N‐terminal nor the C‐terminal half of the peptide interacts with human Rad51, indicating that both parts are involved in the interaction, as expected from the crystal structure. These results suggest the possibility of developing inhibitors of human Rad51 based on this peptide.