PMC

Plaque stabilisation in acute coronary syndromes. ACE angiotensin converting enzyme; ACS, acute coronary syndrome; AMI, acute mycocardial infarction; GP, glycoprotein; LMWH, low molecular weight heparin; PCI, percutaneous coronary intervention.

Schematic representation of (a) magnetic particles, (b) activated with functional groups, and (c) conjugated to biological molecules []; (d) solution stability of antibody-modified magnetic particles over 3 months at 32°C using stabilisers from Applied Enzyme Technology Ltd. Activity tested by optical magneto-immunoassay using 15 ng/ml PSA, 0.1 mg/ml anti-PSA antibody-modified magnetic particles (Abcam, ab10184) and 1 μg/ml horseradish peroxidase (HRP)-labelled antibody (Abcam, ab24466).

Temperature stability. a Incubation of KdgDAb1 (100 % = 6.22 U/mg), KdgDAb2 (100 % = 6.56 U/mg), KdgDCt (100 % = 5.5 U/mg) and KdgDPn (100 % = 3.7 U/mg) at 37 °C; b Incubation of KdgDAb1 (100 % = 8.22 U/mg), KdgDAb2 (100 % = 8.1 U/mg), KdgDCt (100 % = 8.4 U/mg) and KdgDPn (100 % = 5.1 U/mg) at 65 °C; assay: volume 200 μl, 2.5 mM KP_i (pH 7.0), 2 mM MgCl₂, 25 μg/ml BTB and 5 mM 5-keto-4-D-deoxyglucarate; enzyme was incubated in 100 μl of 5 mM KP_i (pH 7.0); only KdgDAb1 differed, where additional 0.5 mM NH₄HCO₃ was used for enzyme stabilisation

Change in T _m of hexokinase in the presence of different sugars. The trend line was fitted to the modified Michaelis–Menten kinetics given in Eqn . The stabilisation effect could be used to estimate affinity of substrates or potential inhibitors (K _M or K _i). Symbols represent d‐mannose, d‐glucose, 2‐deoxy‐d‐glucose, N‐acetyl‐d‐glucosamine and d‐xylose.

(A) A model for substrate binding at the active site of GlpG. Surface representation of GlpG (coloured as in ) and TatA tetrapeptide shown as stick representation (yellow). (B) Possible interaction of P₂ and P₁′ side chains of the TatA substrate with loop-5. In our present best model, the side chains of residues from loop-5 (F245, M247 and M249) interact with substrate residues, in particular the side chains at the P₂ and P₁′ positions. It is possible that such interactions contribute to stabilisation of the substrate at the active site. (C) Possible substrate–enzyme interaction in the GlpG-TatA model. The TatA tetrapeptide consists of a sequence (T-A-A-F) corresponding to the P₂ to P₂′ residues. The model shows possible hydrogen bonding interaction between substrate and enzyme (red dashed lines).

Different domains of USP48 are required for stabilisation of ARL3 and UNC119a. Co-immunoprecipitation using different GFP-USP48 deletion constructs shows that (A) the C-terminal domains of USP48 are not required for the interaction with ARL3-HA, and (B) the most C-terminal domains (the regulatory CK2 and UBL domains) are responsible for the interaction of USP48 with HA-UNC119a. Note that ARL3 (approx. 23 kDa, indicated by a black asterisk) has a similar molecular weight to the light IgG chains (red asterisk). Lys, lysate; IP, immunoprecipitate.

Enzyme activity of truncated Fda1 mutants by C-PAGE. Enzyme activity of (c) Fda1Δ145 and (d) Fda1Δ395 on fucoidans from F. vesiculosus (F.ve), F. evanescens (F.ev), T. ornata (T.o), S. cichorioides (S.c) and U. pinnatifida (U.p), and standard (st). Both enzymes show activity on all the tested substrates to a comparable degree. The lowest band (**) of the standard (St), resulting from FFA2 treatment of fucoidan from F. evanescens, corresponds to a tetra-saccharide of (1→4)- and (1→3)-linked α-l-fucosyls with each fucosyl residue sulphated at C2; total mass has been calculated to be 972 Da [].

Purified recombinantly expressed fucoidan-modifying enzymes. (A) SDS-PAGE, and (B) Western blot of purified FcnA2, FdlA, FdlB, and Fda2. (St) is the protein plus molecular weight marker. The expected molecular weights of the recombinant enzymes FcnA2, FdlA, FdlB, and Fda2 were 87, 75, 76, and 94 kDa, respectively. The multiple bands seen for FcnA2 and Fda2, notably in the Western blot, indicate partial degradation of the proteins. Expression of recombinant Fda1 resulted in insoluble enzyme material which is not shown in this figure.

Schematic of methods utilised to increase NO bioavailability in dystrophic skeletal muscle and the downstream effects. Increasing NO bioavailability through (1) restoration of nNOS, (2) ˪-arginine supplementation, (3) NO donation and (4) inhibition of the enzyme phosphodiesterase (PDE) has led to increases in mitochondrial function, exercise capacity and stabilisation of the membrane in dystrophin-deficient skeletal muscle. A potential consequence of increased NO bioavailability, as observed through nitrate supplementation (5), is peroxynitrite (ONOO⁻) formation which can lead to further muscle damage and is undesirable in dystrophic skeletal muscle

(A) Model of Ac-SDKP (grey) bound to N-domain ACE (cyan) prepared as above. N-domain ACE residues involved in the zinc-mediated activity and stabilisation of the potential transition state intermediates are shown as stick. The water involved in the nucleophilic reaction is shown in red (sphere). (B) Potential role of the chloride ion in activity. Chloride ion is shown in green, with potential hydrogen bond and water mediated-interactions with the catalytic site highlighted as green dashes.

(A) p.P187S primarily affects three structural regions (highlighted in red with arrows): (i) the dynamics of the loop 57–66 in the apo-state; (ii) the dynamics of the region 127–134 in the holo-state; (iii) the C-terminal domain (CTD) is partially unfolded and highly dynamic in the holo-state; (B) dicoumarol binding induces the folding of the CTD in the holo-state (highlighted in green); (C) the suppressor mutation p.H80R partially corrects FAD binding affinity by dynamic stabilisation of the loop 57–66 in the apo-state (in orange); (D) the suppressor mutation p.E247Q stabilises the CTD in the holo-state and dynamically stabilises the loop 57–66 in the apo-state (highlighted in orange); (E) the suppressor mutations in cis lead to additive correction of both FAD binding and CTD stability. DIC, dicoumarol.

Comparison of photochemical inactivation of CvFAP as purified enzyme (•) and crude cell extract preparation (○). Incubation conditions: [CvFAP]=18 μM, buffer: 100 mM Tris‐HCl (pH 8.5), light intensity of blue light=14.5 μE L⁻¹ s⁻¹, T=30 °C. Activity assay conditions: [palmitic acid]₀=13 mM, [DMSO]=30 vol %, buffer: 100 mM Tris‐HCl (pH 8.5), [CvFAP]=3–6 μM, light intensity of blue light=14.5 μE L⁻¹ s⁻¹, T=37 °C, reaction time=30 min. Data represent the mean±SD of two independent experiments.

Scheme 1. (A) Role of Q63, Y66, N123 and H187 in cleavage of the N-glycosidic bond between uracil and the deoxyribose sugar (adapted from 36). (B) Possible effects of mutations at N123 residue. Two resonance forms (a and b) of uracilate anion are shown. Note that the mutation of N123 to D123 results in loss of one hydrogen bond. Additionally, the repulsive force between the partially negatively charged O4 of the leaving group and the carboxylate anion (form b) would adversely affect stabilisation of the leaving group (for details see Discussion).

Purification and activity of enzyme FcnAΔ229. (A) SDS-PAGE indicating the expected molecular weight of 80 kDa and purity; (B) Western blot of purified FcnAΔ229. (St) is the protein plus molecular weight marker; and (C) enzyme activity by C-PAGE of (a) FcnA2 and (b) FcnAΔ229 on fucoidans from S. mcclurei, F. vesiculosus and F. evanescens. FcnA2 and FcnAΔ229 have similar profiles on F. vesiculosus and F. evanescens fucoidans. The reaction time was 24 h. The lowest band (**) of the standard (St), resulting from FFA2 treatment of fucoidan from F. evanescens, corresponds to a tetra-saccharide of (1→4)- and (1→3)-linked α-l-fucosyls with each fucosyl residue sulphated at C2; total mass has been calculated to be approximate 972 Da []. (*) An oligosaccharide of lower molecular weight or higher charge than the lowest band in the standard (**).

Thyroid hormone-dependent activation of HIF-1α: T3 and T4 induce HIF-1α activity by both genomic and nongenomic mechanisms. Genomically, T3 indirectly increases HIF-1α mRNA by increasing expression of the transcription factor hepatic leukemia factor (HLF), which initiates transcription of HIF-1α. Non-genomically, T3 stimulates PI3K signalling by promoting the interaction of both TRα and β with the PI3K regulatory subunit p85 leading to enhanced PI3K/AKT/mTOR activity and translation of HIF-1α mRNA. T3-induced PI3K promotes nuclear shuttling of TRα leading to increased HIF-1α expression. T3-induced PI3K signalling by either TRα or β is cell specific. T3 inhibits the enzyme fumarate dehydrogenase resulting in the accumulation of fumarate. Fumarate inhibits PHD2 leading to reduced hydroxylation of HIF-1α and increased protein stabilisation. T4 increases HIF-1α by stimulating MAPK signalling, leading to enhanced T4/TRβ activity and expression of HIF-1α. Activated HIF-1α by T3/T4 results in the upregulation of target genes, known to promote tumour cell survival and progression. These include GLUT-1, PFK, MCT-4, and VEGF. Additionally, HIF-1 upregulates DIO3, which inhibits T3 by catalysing the conversion of T3 to the metabolically inactive T2.

(A) In the presence of wildtype ubiquitin, K3 recruits Ub-charged Ubc13 for transfer of the charged donor ubiquitin from Ubc13 to the acceptor ubiquitin. (B) In the presence of the I44A mutant ubiquitin, Uev1a is unable to bind the I44 patch on the acceptor ubiquitin and orient its K63 for conjugation by Ubc13. Uev1a dissociates and in the absence of polyubiquitination the monoubiquitin on MHC I is likely removed by a deubiquinating enzyme. As Ubc13 is unable to transfer the donor ubiquitin to an acceptor ubiquitin it cannot dissociate from K3 stabilising the otherwise transient MHC I-E3-E2-I44A complex.

The pyrrole chain elongation catalysed by hydroxymethylbilane synthase. Hydroxymethylbilane synthase (HMBS) and the pyrrole chain elongation. (a) Cartoon representation of HMBS crystal structure (blue; PDB ID: 3ECR) with the dipyrromethane (DPM) cofactor in the centre. The housekeeping HMBS is a monomeric protein of 39.3 KDa. (b) HMBS (blue) with the DPM cofactor (2 × PBG molecules; green) constitute the holoenzyme (E) and bind four PBG substrates (S; red), sequentially, producing enzyme intermediates ES (=ES₁), ES₂, ES₃ and ES₄. Hydroxymethylbilane (HMB), the linear tetrapyrrole product, is released by hydrolysis. The substrate sidechains are acetate (A) and propionate (P). Figure modified from [,].

(Summary illustration): Three USPs regulate Imd activation or stability and downstream signalling. Three ubiquitin specific proteases regulate the Imd pathway, USP34 is putatively acting at the level of NF-κB like factors associated complexes (Rel in the case of Imd pathway) while USP2 and USP36 differentially targets the scaffolding molecule Imd by hydrolysing Ub^K48 (this study) and Ub^K63 [[]] chains, respectively. Imd is subjected to a permanent turn-over via the linkage of Ub^K48 ubiquitin chains and subsequent proteasomal degradation. This prevents Imd accumulation and constitutive signal activation in unchallenged conditions. The deubiquitinating enzyme USP2 is required for both Ub^K48 hydrolysis and Imd degradation at the level of the proteasome. In response to an immune challenge, the activity of the E3 ligase would be prevented resulting in the observed Imd stabilisation and accumulation. It has been previously described that Imd activation in response to immune challenge results in its cleavage by Dredd and linkage of Ub^K63 by Iap2 [[]]. USP36 deubiquitinates Imd linked Ub^K63 thus preventing inappropriate or excessive signal transduction and putatively promoting ubiquitin chain editing through the replacement of activating Ub^K63 by Ub^K48 degradative chains [[]].

(A) Schematic of human HIF-1α (hsHIF-1α) showing the regions involved in DNA-binding and dimerisation with ARNT (bHLH-PAS), oxygen-dependent degradation (ODD), and coactivator binding (the N- and C-terminal transactivation domains, NAD and CAD, respectively). The asparaginyl residue (Asn803) hydroxylated by FIH is shown in red with residues constituting the remainder of the “FIH preferred target sequence” shown in blue above the CAD. The PHD-targeted prolyl residues which are central to the N- and C-terminal ODDs (Pro402 (NODD) and Pro564 (CODD), respectively) are similarly indicated above the ODD. (B) Schematic showing the consequences of different oxygen levels (from “adequate” or normoxic at the top of the schematic to severely hypoxic at the bottom) on FIH/PHD enzyme and hsHIF-1α activity. When adequate oxygen is present, the PHDs and FIH are both active, resulting in hydroxylation of their target residues in HIF-1α (coloured as in part A). Prolyl hydroxylation results in efficient VHL-mediated ubiquitination and rapid proteasomal degradation of HIF-1α, thus ensuring minimal HIF-1 target gene activation. At intermediate levels of oxygen, the PHDs are inactive, resulting in HIF-1α stabilisation, translocation to the nucleus, and partnering with ARNT on hypoxia response elements (HREs). Ongoing FIH-mediated hydroxylation at this oxygen tension, however, precludes CBP binding to the CAD, thus only the NAD recruits CBP for target gene activation. Under more severe hypoxia, both PHDs and FIH are inactive, thus both the NAD and CAD of HRE-bound HIF-1α can recruit CBP for target gene activation.

In the absence of dNTPs Apo-SAMHD1 is found in a monomer-dimer equilibrium regardless of the phosphorylation state (1). At high dNTP levels, typically in cycling cells, constitutively abundant GTP combines with dNTPs to fill allosteric sites. In both phosphorylated and un-phosphorylated SAMHD1 this results in the formation of an activated tetramer (2) that in the non-phosphorylated protein also includes additional intra-tetramer CtD interactions forming a stable activated tetramer (3). Under these conditions, both activated and stable activated tetramers hydrolyse the dNTP pool at comparable rates. At lower dNTP levels, the stabilisation afforded by the CtD interactions maintains enzyme activity in non-phosphorylated SAMHD1 by preventing the loss of dNTP-activator from the allosteric site. However, in phospho-SAMHD1, activating dNTPs dissociate from the allosteric site (4) resulting in disassembly of the tetramer and down-regulation of triphosphohydrolase activity. At very low levels, such as in differentiated myeloid cells, CtD-stabilised tetramers still retain activating dNTPs in the allosteric site (5) and SAMHD1 remains catalytically competent. It can therefore rapidly respond to any increase in intracellular dNTPs to maintain the dNTP levels below the threshold required for HIV-1 replication.