Structural Basis for the Interaction of Human β-Defensin 6 and Its Putative Chemokine Receptor CCR2 and Breast Cancer Microvesicles

Human β-defensins (hBDs) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the hBD6 by NMR spectroscopy that contains a conserved β-defensin domain followed by an extended C-terminus. We use NMR to monitor the interaction of hBD6 with microvesicles shed by breast cancer cell lines and with peptides derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) possessing or not possessing sulfation on Tyr26 and Tyr28. The NMR-derived model of the hBD6/CCR2 complex reveals a contiguous binding surface on hBD6, which comprises amino acid residues of the α-helix and β2–β3 loop. The microvesicle binding surface partially overlaps with the chemokine receptor interface. NMR spin relaxation suggests that free hBD6 and the hBD6/CCR2 complex exhibit microsecond-to-millisecond conformational dynamics encompassing the CCR2 binding site, which might facilitate selection of the molecular configuration optimal for binding. These data offer new insights into the structure–function relation of the hBD6–CCR2 interaction, which is a promising target for the design of novel anticancer agents.     

Non-Canonical Interleukin 23 Receptor Complex Assembly: P40 PROTEIN RECRUITS INTERLEUKIN 12 RECEPTOR β1 VIA SITE II AND INDUCES P19/INTERLEUKIN 23 RECEPTOR INTERACTION VIA SITE III*

IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor β1 (IL-12Rβ1). Complex formation was hypothesized to follow the “site I-II-III” architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rβ1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rβ1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rβ1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rβ1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.     

Fusobacterium nucleatum-associated β-Defensin Inducer (FAD-I): IDENTIFICATION,ISOLATION, AND FUNCTIONAL EVALUATION*

Human β-defensins (hBDs) are small, cationic antimicrobial peptides, secreted by mucosal epithelial cells that regulate adaptive immune functions. We previously reported that Fusobacterium nucleatum, a ubiquitous Gram-negative bacterium of the human oral cavity, induces human β-defensin 2 (hBD2) upon contact with primary oral epithelial cells. We now report the isolation and characterization of an F. nucleatum (ATCC 25586)-associated defensin inducer (FAD-I). Biochemical approaches revealed a cell wall fraction containing four proteins that stimulated the production of hBD2 in human oral epithelial cells (HOECs). Cross-referencing of the N-terminal sequences of these proteins with the F. nucleatum genome revealed that the genes encoding the proteins were FadA, FN1527, FN1529, and FN1792. Quantitative PCR of HOEC monolayers challenged with Escherichia coli clones expressing the respective cell wall proteins revealed that FN1527 was most active in the induction of hBD2 and hence was termed FAD-I. We tagged FN1527 with a c-myc epitope on the C-terminal end to identify and purify it from the E. coli clone. Purified FN1527 (FAD-I) induced hBD2 mRNA and protein expression in HOEC monolayers. F. nucleatum cell wall and FAD-I induced hBD2 via TLR2. Porphorymonas gingivalis, an oral pathogen that does not induce hBD2 in HOECs, was able to significantly induce expression of hBD2 in HOECs only when transformed to express FAD-I. FAD-I or its derivates offer a potentially new paradigm in immunoregulatory therapeutics because they may one day be used to bolster the innate defenses of vulnerable mucosae.     

Caenorhabditis elegans and Human Dual Oxidase 1 (DUOX1) ��Peroxidase�� Domains: INSIGHTS INTO HEME BINDING AND CATALYTIC ACTIVITY*

The seven members of the NOX/DUOX family are responsible for generation of the superoxide and H₂O₂ required for a variety of host defense and cell signaling functions in nonphagocytic cells. Two members, the dual oxidase isozymes DUOX1 and DUOX2, share a structurally unique feature: an N-terminal peroxidase-like domain. Despite sequence similarity to the mammalian peroxidases, the absence of key active site residues makes their binding of heme and their catalytic function uncertain. To explore this domain we have expressed in a baculovirus system and purified the Caenorhabditis elegans (CeDUOX1_1–589) and human (hDUOX1_1–593) DUOX1 “peroxidase” domains. Evaluation of these proteins demonstrated that the isolated hDUOX1_1–593 does not bind heme and has no intrinsic peroxidase activity. In contrast, CeDUOX1_1–589 binds heme covalently, exhibits a modest peroxidase activity, but does not oxidize bromide ion. Surprisingly, the heme appears to have two covalent links to the protein despite the absence of a second conserved carboxyl group in the active site. Although the N-terminal dual oxidase motif has been proposed to directly convert superoxide to H₂O₂, neither DUOX1 domain demonstrated significant superoxide dismutase activity. These results strengthen the in vivo conclusion that the CeDUOX1 protein supports controlled peroxidative polymerization of tyrosine residues and indicate that the hDUOX1 protein either has a unique function or must interact with other protein factors to express its catalytic activity.The purposeful generation of reactive oxygen species within phagocytic cells has long been recognized as a component of their antibacterial defense system (1, 2). Reactive oxygen species generation is mediated by a membrane-bound NADPH oxidase (NOX)² and is activated by a diverse number of stimuli. The NOX enzymes catalyze the NADPH-dependent one-electron reduction of oxygen to superoxide (O₂^−̇) (3). It has long been debated whether the generation of similar species in other cell types is also an intentional, physiologically controlled process or is an accident of aerobic respiration. This controversy has been clarified by identification of the NOX/DUOX family of NADPH oxidases. The seven members of this family (NOX 1–5 and DUOX1 and 2) have been shown to produce the reactive oxygen species utilized for functions as varied as cellular signaling, host defense, and thyroid hormone biosynthesis (4–8). The latter function is specifically attributed to the DUOX members of this family.DUOX1 and 2 (formerly also known as ThOX1 and 2 for thyroid oxidase) were first identified in the mammalian thyroid gland (9, 10). This localization is not exclusive because both can also be found in nonthyroid tissues; DUOX1 is prominent in airway epithelial cells (11) and DUOX2 in the salivary glands and gastrointestinal tract (4, 12, 13). Homologs of each DUOX have also been identified in lower organisms, including Caenorhabditis elegans and Drosophila melanogaster (14). The human isoforms are 83% homologous, ∼190 kDa in size (after glycosylation), and are located in close proximity, because they are configured head-to-head on human chromosome 15 (15, 16). The glycosylation of both DUOX1 and 2 is extensive, contributing ∼30 kDa to the total apparent protein mass (17). Recent investigation has uncovered that maturation factors DUOXA1 and DUOXA2 are required to achieve heterologous expression of each DUOX in full-length, active form (18).Structurally, DUOX1 and 2 are characterized by a defining N-terminal, extracellular domain exhibiting considerable sequence identity with the mammalian peroxidases, a transmembrane (TM) segment appended to an EF-hand calcium-binding cytosolic region and a NOX2 homologous structure (six TMs tethered to NADPH oxidase; see Fig. 1A) (10). Both isoforms have a conserved calcium-binding site in the N-terminal peroxidase domain, mimicking that found in MPO, LPO, EPO, and TPO. Interestingly, although homologous to these heme-containing peroxidases, the peroxidase-like domains of the DUOX proteins lack some of the highly conserved amino acid residues that are thought to be essential for heme binding and/or peroxidase catalysis (see Fig. 1B) (16).Open in a separate windowFIGURE 1.Comparison of sequence and structural features of hDUOX1 and CeDUOX1. A, schematic view of the domain structures of hDUOX1 and CeDUOX1. Each DUOX1 protein contains an N-terminal extracellular peroxidase domain (dark gray rectangle), putative TM domains (light gray tubes), and cytosolic EF-hand and NADPH oxidase domains (light gray rectangles). Both proteins were truncated to generate soluble expression constructions focusing on the peroxidase domain, as shown. B, sequence alignment of classical peroxidase domains with the DUOX1 proteins. Highlighted segments shown focus on regions of active site residues (bold face) including the distal histidine (hMPO His²⁶¹), catalytic arginine (hMPO Arg⁴⁰⁵), proximal histidine (hMPO His⁵⁰²), and covalent heme-binding residues (hMPO Asp²⁶⁰ and Glu⁴⁰⁸). hEPO, human eosinophil peroxidase; bLPO, bovine lactoperoxidase; hTPO, human thyroid peroxidase; hVPO, human vascular peroxidase; hPxn, human peroxidasin.Functionally, mature DUOX enzymes appear to produce H₂O₂, in contrast to other NOX family members that produce superoxide. This activity is regulated by Ca²⁺ concentration through triggered dissociation of NOXA1 and possibly other as yet unidentified interacting proteins (19). Because the N-terminal peroxidase domain is the structural feature that differentiates the dual oxidases from the NOX proteins, it may be directly responsible for the conversion of superoxide to H₂O₂. To investigate this crucial domain, we report here the first expression, purification, and characterization of the Homo sapiens (hDUOX1_1–593) and C. elegans (CeDUOX1_1–589) DUOX1 peroxidase domains. We demonstrate that heme is covalently bound to CeDUOX1_1–589 (two covalent bonds are suggested by heme hydroxylation studies), whereas hDUOX1_1–593 does not stably bind this co-factor. Both domains share overall sequence similarity with the mammalian peroxidases (specifically LPO), but only CeDUOX1_1–589 exhibits peroxidase activity, as measured with either ABTS or tyrosine ethyl ester as the substrate. We also demonstrate that neither DUOX1 domain has significant superoxide dismutase or halide oxidizing activity.     

Ligand-Specific Interactions Modulate Kinetic, Energetic, and Mechanical Properties of the Human β(2) Adrenergic Receptor

G protein-coupled receptors (GPCRs) are a class of versatile proteins that transduce signals across membranes. Extracellular stimuli induce inter- and intramolecular interactions that change the functional state of GPCRs and activate intracellular messenger molecules. How these interactions are established and how they modulate the functional state of GPCRs remain to be understood. We used dynamic single-molecule force spectroscopy to investigate how ligand binding modulates the energy landscape of the human β(2) adrenergic receptor (β(2)AR). Five different ligands representing either agonists, inverse agonists or neutral antagonists established a complex network of interactions that tuned the kinetic, energetic, and mechanical properties of functionally important structural regions of β(2)AR. These interactions were specific to the efficacy profile of the ligands investigated and suggest that the functional modulation of GPCRs follows structurally well-defined interaction patterns.     

Functional Analysis of the Transmembrane Domains of Presenilin 1: PARTICIPATION OF TRANSMEMBRANE DOMAINS 2 AND 6 IN THE FORMATION OF INITIAL SUBSTRATE-BINDING SITE OF γ-SECRETASE*

γ-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2, which mediates intramembrane proteolysis of a range of type I transmembrane proteins. We previously analyzed the functional roles of the N-terminal transmembrane domains (TMDs) 1–6 of PS1 in the assembly and proteolytic activity of the γ-secretase using a series of TMD-swap PS1 mutants. Here we applied the TMD-swap method to all the TMDs of PS1 for the structure-function analysis of the proteolytic mechanism of γ-secretase. We found that TMD2- or -6-swapped mutant PS1 failed to bind the helical peptide-based, substrate-mimic γ-secretase inhibitor. Cross-linking experiments revealed that both TMD2 and TMD6 of PS1 locate in proximity to the TMD9, the latter being implicated in the initial substrate binding. Taken together, our data suggest that TMD2 and the luminal side of TMD6 are involved in the formation of the initial substrate-binding site of the γ-secretase complex.     

Co-fibrillogenesis of Wild-type and D76N β2-Microglobulin: THE CRUCIAL ROLE OF FIBRILLAR SEEDS*

The amyloidogenic variant of β₂-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β₂-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β₂-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β₂-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β₂-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β₂-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β₂-microglobulin fibrils.     

The Role of Mg(II) in DNA Cleavage Site Recognition in Group II Intron Ribozymes: SOLUTION STRUCTURE AND METAL ION BINDING SITES OF THE RNA·DNA COMPLEX*

Group II intron ribozymes catalyze the cleavage of (and their reinsertion into) DNA and RNA targets using a Mg²⁺-dependent reaction. The target is cleaved 3′ to the last nucleotide of intron binding site 1 (IBS1), one of three regions that form base pairs with the intron''s exon binding sites (EBS1 to -3). We solved the NMR solution structure of the d3′ hairpin of the Sc.ai5γ intron containing EBS1 in its 11-nucleotide loop in complex with the dIBS1 DNA 7-mer and compare it with the analogous RNA·RNA contact. The EBS1·dIBS1 helix is slightly flexible and non-symmetric. NMR data reveal two major groove binding sites for divalent metal ions at the EBS1·dIBS1 helix, and surface plasmon resonance experiments show that low concentrations of Mg²⁺ considerably enhance the affinity of dIBS1 for EBS1. Our results indicate that identification of both RNA and DNA IBS1 targets, presentation of the scissile bond, and stabilization of the structure by metal ions are governed by the overall structure of EBS1·dIBS1 and the surrounding loop nucleotides but are irrespective of different EBS1·(d)IBS1 geometries and interstrand affinities.     

Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET): UNIQUE AND SHARED EFFECTS ON SIGNALING AND DEATH*

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance ¹³C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

Synthesis of β-D-fructopyranosyl-(2→6)-D-glucopyranose from D-glucose and D-fructose by a thermal treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Synthesis of β-D-Fructopyranosyl-(2→6)-D-glucopyranose from D-Glucose and D-Fructose by a Thermal Treatment

The synthesis is reported of β-D-fructopyranosyl-(2→6)-D-glucopyranose that had previously been isolated from a fermented plant extract as a new saccharide. A disaccharide was predominately formed from an equal amount of D-glucose and D-fructose under melting conditions at 140 °C for 60 to 90 min. This saccharide was isolated from the reaction mixture by carbon-Celite column chromatography and preparative HPLC, and was confirmed to be β-D-fructopyranosyl-(2→6)-D-glucopyranose by TOF-MS and NMR analyses.     

Synthesis of Some New α- and β-(l→2) Linked Disaccharides Containing L-Quinovose (6-Deoxy L-Glucose)

Hepta-O-acetyl-2-0-β-l-quinovopyranosyl-α-d-glucose (VI) and hepta-O-acetyl-2-O-α-l-quinovopyranosyl-β-d-gIucose (VIII) were prepared by the coupling of 2,3,4-tri-O-acetyl-α-l-quinovopyranosyl bromide (IV) with l,3,4,6-tetra-O-acetyl-α-D-glucose (V) in the presence of mercuric cyanide and mercuric bromide in absolute acetonitrile.

Similarly, hepta-O-acetyW-O-α-l-quinovopyranosyl-α-d-galactose (X) and hepta-O-acetyl-2-O-β-L-quinovopyranosyl-α-d-galactose (XI) were prepared by the reaction of IV with 1,3,4,6-tetra-O-acetyl-α-d-galactose (IX).

Removal of the protecting groups of VI, VIII, X and XI afforded the corresponding disaccharides. On treatment with hydrogen bromide, VI, VIII, X and XI gave the corresponding acetobromo derivatives.     

Cyclic (1→2)-β-d-Glucan-hydrolyzing Enzymes from Acremonium sp. 15 Purification and Some Properties of Endo-( 1 → 2)-α-d-glucanase and β-d-Glucosidase

Acremonium sp. 15 a fungus isolated from soil, produces an extracellular enzyme system degrading cyclic (1→2)-β-d-glucan. This enzyme was found to be a mixture of endo-(1→2)-β-d-glucanase and β-d-glucosidase. The (1→2)-β-d-glucanase was purified to homogeneity shown by disc-electrophoresis after SP-Sephadex column chromatography, Sephadex G-75 gel filtration, and rechromatography on SP-Sephadex. The molecular weight of the enzyme was 3.6 × 10⁴ by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was pH 9.6. The enzyme was most active at pH 4.0—4.5, and stable up to 40°C in 20 mm acetate buffer (pH 5.0) for 2 hr of incubation. This enzyme hydrolyzed only (l→2)-β-d-glucan and did not hydrolyze laminaran, curdlan, or CM-cellulose. The hydrolysis products from cyclic (1→2)-β-d-glucan were mainly sophorose.

The β-d-glucosidase was purified about 4000-fold. The rate of hydrolysis of the substrates by this β-d-glucosidase decreased in the following order: β-nitrophenyl-β-d-glucoside, sophorose, phenyl-β-d-glucoside, laminaribiose, and salicin. This enzyme has strong transfer action even at the low concentration of 0.75 mm substrate.     

Kinetics of β-N-methylamino-L-alanine (BMAA) and 2, 4-diaminobutyric acid (DAB) production by diatoms: the effect of nitrogen

The neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) are produced by cyanobacteria, diatoms and dinoflagellates and have been detected in seafood worldwide. Our present knowledge of their metabolism or biosynthesis is limited. In this study, the production of BMAA and DAB as a function of time was monitored in five strains representing four species of diatoms, i.e. Phaeodactylum tricornutum, Thalassiosira weissflogii, Thalassiosira pseudonana and Navicula pelliculosa, previously identified as BMAA and DAB producers. Subsequently, three strains were selected and exposed to three nitrogen treatments – starvation, control (the standard concentration in f/2 medium) and enrichment, because BMAA metabolism has been suggested to be closely associated with cellular nitrogen metabolism in both cyanobacteria and diatoms. Chlorophyll a and total protein concentrations were also determined. Our results indicate that BMAA and DAB production in diatoms is species- and strain-specific. However, production might also be affected by stress, particularly as related to nitrogen starvation and cell density. Furthermore, this study shows a significant correlation between the production of the two neurotoxins which might further suggest common steps in the metabolic pathways.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were