Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Background

Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.

Methods

To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.

Results

Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.

Conclusion

We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.

General significance

Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Disulfide reduction abolishes tissue factor cofactor function

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.     

Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

Matrix metalloproteinases in inflammation

Background

Matrix metalloproteinases (MMPs) are a family of ubiquitously expressed zinc-dependent endopeptidases with broad substrate specificity and strictly regulated tissue specific expression. They are expressed in physiological situations and pathological conditions involving inflammation. MMPs regulate several functions related to inflammation including bioavailability and activity of inflammatory cytokines and chemokines. There is also evidence that MMPs regulate inflammation in tumor microenvironment, which plays an important role in cancer progression.

Scope of review

Here, we discuss the current view on the role of MMPs in the regulation of inflammation.

Major conclusions

MMPs modulate inflammation by regulating bioavailability and activity of cytokines, chemokines, and growth factors, as well as integrity of physical tissue barriers. MMPs are also involved in immune evasion of tumor cells and in regulation of inflammation in tumor microenvironment.

General significance

There is increasing evidence for non-matrix substrates of MMPs that are related to regulation of inflammatory processes. New methods have been employed for identification of the substrates of MMPs in inflammatory processes in vivo. Detailed information on the substrates of MMPs may offer more specific and effective ways of inhibiting MMP function by blocking the cleavage site in substrate or by inhibition of the bioactivity of the substrate. It is expected, that more precise information on the MMP–substrate interaction may offer novel strategies for therapeutic intervention in inflammatory diseases and cancer without blocking beneficial actions of MMPs. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Structure–function studies on jaburetox,a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease

Background

Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10 kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation.

Methods

Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61–74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1–44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45–93), Jbtx C-ter.

Results

1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure–function analysis.

Major conclusions

The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx.

General significance

Jbtx represents a new type of insecticidal and membrane-active peptide.     

Alternative cell death of Apaf1-deficient neural progenitor cells induced by withdrawal of EGF or insulin

Background

Various forms of cell death, such as apoptotic, autophagic and non-lysosomal types, are implicated in normal physiological processes. Apoptotic protease activating factor 1 (Apaf1) is an important component of the intrinsic apoptotic pathway. Deficiency of Apaf1 results in an accumulation of neural progenitor cells (NPCs) in the developing central nervous system and thus, in perinatal lethality. A small percentage of the mutant mice, however, are viable and grow to maturity. The occurrence of such normal mutants implicates alternative cell death pathways during neurogenesis.

Methods

NPCs prepared from wild-type or Apaf1-deficient embryos were cultured in growth factor-deprived medium and examined for cell death, caspase activation and morphological alterations. Generation of reactive oxygen species (ROS) and the effects of antioxidants were examined.

Results

Wild-type NPCs underwent apoptosis within 24 hours of withdrawal of epidermal growth factor (EGF) or insulin, whereas Apaf1-deficient NPCs underwent cell death but showed no signs of apoptosis. Autophagy was not necessarily accompanied by cell death. Cell death of the Apaf1-deficient NPCs resembled necroptosis—necrosis-like programmed cell death. The necroptosis inhibitor necrostatin-1, however, failed to inhibit the cell death. ROS accumulation was detected in NPCs deprived of growth factors, and an antioxidant partially suppressed the non-apoptotic cell death of Apaf1-deficient NPCs.

Conclusions

These data indicate that after withdrawal EGF or insulin withdrawal, the Apaf1-deficient cells underwent non-apoptotic cell death. ROS generation may partially participate in the cell death.

General Significance

Non-apoptotic cell death in NPCs may be a compensatory mechanism in the developing CNS of Apaf1-deficient embryos.     

Inhibition of gingipains by their profragments as the mechanism protecting Porphyromonas gingivalis against premature activation of secreted proteases

Background

Arginine-specific (RgpB and RgpA) and lysine-specific (Kgp) gingipains are secretory cysteine proteinases of Porphyromonas gingivalis that act as important virulence factors for the organism. They are translated as zymogens with both N- and C-terminal extensions, which are proteolytically cleaved during secretion. In this report, we describe and characterize inhibition of the gingipains by their N-terminal prodomains to maintain latency during their export through the cellular compartments.

Methods

Recombinant forms of various prodomains (PD) were analyzed for their interaction with mature gingipains. The kinetics of their inhibition of proteolytic activity along with the formation of stable inhibitory complexes with native gingipains was studied by gel filtration, native PAGE and substrate hydrolysis.

Results

PD_RgpB and PD_RgpA formed tight complexes with arginine-specific gingipains (Ki in the range from 6.2 nM to 0.85 nM). In contrast, PD_Kgp showed no inhibitory activity. A conserved Arg-102 residue in PD_RgpB and PD_RgpA was recognized as the P1 residue. Mutation of Arg-102 to Lys reduced inhibitory potency of PD_RgpB by one order of magnitude while its substitutions with Ala, Gln or Gly totally abolished the PD inhibitory activity. Covalent modification of the catalytic cysteine with tosyl-l-Lys-chloromethylketone (TLCK) or H-D-Phe-Arg-chloromethylketone did not affect formation of the stable complex.

Conclusion

Latency of arginine-specific progingipains is efficiently exerted by N-terminal prodomains thus protecting the periplasm from potentially damaging effect of prematurely activated gingipains.

General significance

Blocking progingipain activation may offer an attractive strategy to attenuate P. gingivalis pathogenicity.     

Glycolysis–respiration relationships in a neuroblastoma cell line

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.     

Enteral calcium infusion used successfully as treatment for a patient with hereditary vitamin D resistant rickets (HVDRR) without alopecia: A novel mutation

Background

We report a novel mutation in a case of hereditary vitamin D resistant rickets (HVDRR) without alopecia and successful management of this condition with the intravenous formulation of calcium chloride delivered via gastric tube.

Clinical Case

A 22 month old male (length − 3.4 SDS; weight − 2.1 SDS) presented with failure to thrive, short stature, severe hypocalcemia and gross motor delay. He did not have alopecia. Initial blood tests and history were thought possibly to suggest vitamin D deficiency rickets: calcium 5.1 mg/dL, (8.8–10.8); phosphorus 4.1 mg/dL, (4.5–5.5); alkaline phosphatase 1481 U/L (80–220); intact PTH 537.1 pg/mL (10–71). Subsequently, vitamin D studies returned that were consistent with HVDRR: 25-hydroxyvitamin D 34 ng/mL (20–100); 1,25-dihydroxyvitamin D 507 pg/mL. This diagnosis was confirmed by DNA sequencing. His subsequent clinical course was complicated by the fact that IV calcium was not a viable option for this patient, and his calcium levels could not be well controlled on oral calcium citrate or calcium glubionate therapy. Eventually, we were able to maintain calcium levels above 8 mg/dL using the intravenous preparation of calcium chloride administered via gastric tube.

Genetic Studies

A unique homozygous T to C base substitution was found in exon 6 in the vitamin D receptor (VDR) gene. This mutation causes leucine to be converted to proline at position 227 in helix 3 in the VDR ligand binding domain (LBD). The mutation rendered the VDR non-functional, leading to HVDRR, with absence of alopecia.

Conclusion

HVDRR should be considered in a patient with profound hypocalcemia which is refractory to conventional therapy of vitamin D deficiency rickets even without evidence of alopecia. We report the first case of HVDRR with a novel mutation in the LBD that was successfully treated with enteral treatment using a calcium chloride infusion.     

Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain,and is consistent with Michaelis–Menten mechanism

Background

It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease.

Methods

Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis.

Results

The CHS4 domain comprised of sequence residues 116–263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis–Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94–170) within CHS4 is indispensable.

Conclusion

The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does.

Significance

Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.     

Cooperation of binding sites at the hydrophilic domain of cell-surface sulfatase Sulf1 allows for dynamic interaction of the enzyme with its substrate heparan sulfate

Background

Sulf1 is a cell-surface sulfatase removing internal 6-O-sulfate groups from heparan sulfate (HS) chains. Thereby it modulates the activity of HS-dependent growth factors. For HS interaction Sulf1 employs a unique hydrophilic domain (HD).

Methods

Affinity-chromatography, AFM-single-molecule force spectroscopy (SMFS) and immunofluorescence on living cells were used to analyze specificity, kinetics and structural basis of this interaction.

Results

Full-length Sulf1 interacts broadly with sulfated glycosaminoglycans (GAGs) showing, however, higher affinity toward HS and heparin than toward chondroitin sulfate or dermatan sulfate. Strong interaction depends on the presence of Sulf1-substrate groups, as Sulf1 bound significantly weaker to HS after enzymatic 6-O-desulfation by Sulf1 pretreatment, hence suggesting autoregulation of Sulf1/substrate association. In contrast, HD alone exhibited outstanding specificity toward HS and did not interact with chondroitin sulfate, dermatan sulfate or 6-O-desulfated HS. Dynamic SMFS revealed an off-rate of 0.04/s, i.e., ~ 500-fold higher than determined by surface plasmon resonance. SMFS allowed resolving the dynamics of single dissociation events in each force–distance curve. HD subdomain constructs revealed heparin interaction sites in the inner and C-terminal regions of HD.

Conclusions

Specific substrate binding of Sulf1 is mediated by HD and involves at least two separate HS-binding sites. Surface plasmon resonance K_D-values reflect a high avidity resulting from multivalent HD/heparin interaction. While this ensures stable cell–surface HS association, the dynamic cooperation of binding sites at HD and also the catalytic domain enables processive action of Sulf1 along or across HS chains.

General significance

HD confers a novel and highly dynamic mode of protein interaction with HS.     

A pentapeptide signature motif plays a pivotal role in Leishmania DNA topoisomerase IB activity and camptothecin sensitivity

Background

Leishmania donovani – the causative agent of visceral leishmaniasis – has several evolutionary characteristics that make the disease difficult to combat. Among these differences, a rare heterodimeric DNA topoisomerase IB has been reported thus opening a new promising field in the therapy of leishmaniasis. Several studies of the human enzyme have pointed to the importance of the linker domain in respect to camptothecin sensitivity. At present, it has been impossible to pinpoint the regions that make up the linker domain in Leishmania.

Methods

Several site-directed mutations as well as internal and linear truncations involving both subunits were assayed on both, relaxation activity and sensitivity to camptothecin.

Results

Truncations performed on the trypanosomatids conserved motif (RPPVVRS) of the small subunit of leishmanial DNA topoisomerase IB demonstrated that elimination of pentapeptide RPPVV produced a nonfunctional enzyme. However, the removal of the dipeptide RS led to an enzyme with reduced relaxation activity and less sensitivity to camptothecin. The basic structure, both sensitive to camptothecin and able to fully relax DNA, composed of amino acids 1–592 and 175–262 in the large and small subunits, respectively.

Conclusion

It has been established that the region between amino acids 175 and 180 (RPPVV) of the small subunit plays a pivotal role in both interaction with the large subunit and sensitivity to camptothecin in Leishmania.

General significance

The present report describes a functional analysis of the leishmanial DNA topoisomerase IB regions directly involved both in sensitivity to poisons and in the conformation of the linker domain.     

Effects of passive smoking on breast cancer risk in pre/post-menopausal women as modified by polymorphisms of PARP1 and ESR1

Objectives

The association between passive smoking and breast cancer risk differs in pre- and post-menopausal women. We aimed to explore the modification effects of PARP1 rs1136410 and ESR1 rs2234693 on the association between passive smoking and breast cancer risk among pre- and post-menopausal women.

Design and methods

A case–control study of 839 breast cancer cases and 863 controls was conducted. The gene–environment interactions were tested after adjusting for potential breast cancer risk factors with unconditional logistic regression models.

Results

We found that the effect of passive smoking was modified by the genotypes in both pre- and post-menopausal women, but in opposite directions. The combination of the TC/CC genotypes of ESR1 rs2234693 and passive smoking significantly increased the risk of breast cancer [OR (95%CI): 2.06 (1.39–3.05)] in pre-menopausal women. A significant association was observed between TT genotype and passive smoking [OR (95%CI): 2.40 (1.27–4.53)] in postmenopausal women. For PARP1 rs1136410, similar differential associations were observed, but the interactions were not significant.

Conclusions

These results imply that the risk of breast cancer from passive smoking may be influenced by genetic factors, and that the association may differ depending on menopausal status.     

Immune signal transduction in leishmaniasis from natural to artificial systems: Role of feedback loop insertion

Background

Modulated immune signal (CD14–TLR and TNF) in leishmaniasis can be linked to EGFR pathway involved in wound healing, through crosstalk points. This signaling network can be further linked to a synthetic gene circuit acting as a positive feedback loop to elicit a synchronized intercellular communication among the immune cells which may contribute to a better understanding of signaling dynamics in leishmaniasis.

Methods

Network reconstruction with positive feedback loop, simulation (ODE 15s solver) and sensitivity analysis of CD14–TLR, TNF and EGFR was done in SimBiology (MATLAB 7.11.1). Cytoscape and adjacency matrix were used to calculate network topology. PCA was extracted by using sensitivity coefficient in MATLAB. Model reduction was done using time, flux and sensitivity score.

Results

Network has five crosstalk points: NIK, IκB–NFκB and MKK (4/7, 3/6, 1/2) which show high flux and sensitivity. PI3K in EGFR pathway shows high flux and sensitivity. PCA score was high for cytoplasmic ERK1/2, PI3K, Atk, STAT1/3 and nuclear JNK. Of the 125 parameters, 20% are crucial as deduced by model reduction.

Conclusions

EGFR can be linked to CD14–TLR and TNF through the MAPK crosstalk points. These pathways may be controlled through Ras and Raf that lie upstream of signaling components ERK ½ (c) and JNK (n) that have a high PCA score via a synthetic gene circuit for activating cell–cell communication to elicit an inflammatory response. Also a disease resolving effect may be achieved through PI3K in the EGFR pathway.

General significance

The reconstructed signaling network can be linked to a gene circuit with a positive feedback loop, for cell–cell communication resulting in synchronized response in the immune cell population, for disease resolving effect in leishmaniasis.     

Variants of NLRP3 gene are associated with insulin resistance in Chinese Han population with type-2 diabetes

Aims

Nod like receptor pyrin domain containing 3 (NLRP3) is the best characterized member of nod like receptor family. Recent studies suggest that NLRP3 plays a crucial role in the pathogenesis of type-2 diabetes (T2DM), and variants in NLRP3 affect its mRNA stability and expression. Therefore, we hypothesize that the variants in NLRP3 gene may contribute to T2DM susceptibility. The aim of this study is to evaluate the association of NLRP3 SNPs with T2DM in Chinese Han patients.

Methods

Two common variants in NLRP3 gene, rs10754558 and rs4612666, were detected using the polymerase chain reaction–restriction fragment length polymorphism procedure in 952 unrelated T2DM patients and 871 healthy controls. All participants were unrelated Chinese Hans.

Results

The GG genotype and G allele frequencies of rs10754558 were significantly higher in T2DM patients than those in controls (for GG genotype, 19.6% vs. 14.5%, p = 0.019; for G allele, 43.9% vs. 39.8%, p = 0.013). The GG genotype of rs10754558 was significantly associated with higher LDL-C levels and more prone to insulin resistance, as evaluated by HOMA-IR or QUICK indexes.

Conclusions

The variant (rs10754558) in NLRP3 is related to insulin resistance and increased risk of T2DM in Chinese Han population.     

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains

Background

A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs — complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes.

Methods

An original algorithmic approach (Global Sequence Signature—GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences.

Results

Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation.

Conclusions

Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop.

General significance

In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.     

Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III

Background

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca^2 +-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores.

Methods

Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined.

Results

K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane.

Conclusions

Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III.

General significance

The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.