Extracellular Binding Protein Research Articles

The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research

A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations (IVIVEs) of toxicity (efficacy) at the cellular level from data generated in cell assays. Overall, this present study may help to optimize the human dose prediction in preclinical and clinical studies, while prescribing drugs with narrow therapeutic windows that are highly bound to extracellular proteins and/or highly ionized at the physiological pH. This may facilitate building a more accurate safety (efficacy) profile for such drugs.

The Fibroblast Growth Factor signaling pathway.

The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc.

Schwann cell-derived Apolipoprotein D controls the dynamics of post-injury myelin recognition and degradation

Management of lipids, particularly signaling lipids that control neuroinflammation, is crucial for the regeneration capability of a damaged nervous system. Knowledge of pro- and anti-inflammatory signals after nervous system injury is extensive, most of them being proteins acting through well-known receptors and intracellular cascades. However, the role of lipid binding extracellular proteins able to modify the fate of lipids released after injury is not well understood. Apolipoprotein D (ApoD) is an extracellular lipid binding protein of the Lipocalin family induced upon nervous system injury. Our previous study shows that axon regeneration is delayed without ApoD, and suggests its participation in early events during Wallerian degeneration. Here we demonstrate that ApoD is expressed by myelinating and non-myelinating Schwann cells and is induced early upon nerve injury. We show that ApoD, known to bind arachidonic acid (AA), also interacts with lysophosphatidylcholine (LPC) in vitro. We use an in vivo model of nerve crush injury, a nerve explant injury model, and cultured macrophages exposed to purified myelin, to uncover that: (i) ApoD regulates denervated Schwann cell-macrophage signaling, dampening MCP1- and Tnf-dependent macrophage recruitment and activation upon injury; (ii) ApoD controls the over-expression of the phagocytosis activator Galectin-3 by infiltrated macrophages; (iii) ApoD controls the basal and injury-triggered levels of LPC and AA; (iv) ApoD modifies the dynamics of myelin-macrophage interaction, favoring the initiation of phagocytosis and promoting myelin degradation. Regulation of macrophage behavior by Schwann-derived ApoD is therefore a key mechanism conditioning nerve injury resolution. These results place ApoD as a lipid binding protein controlling the signals exchanged between glia, neurons and blood-borne cells during nerve recovery after injury, and open the possibility for a therapeutic use of ApoD as a regeneration-promoting agent.

The DAN family: modulators of TGF-β signaling and beyond.

Extracellular binding proteins or antagonists are important factors that modulate ligands in the transforming growth factor (TGF-β) family. While the interplay between antagonists and ligands are essential for developmental and normal cellular processes, their imbalance can lead to the pathology of several disease states. In particular, recent studies have implicated members of the differential screening-selected gene in neuroblastoma (DAN) family in disease such as renal fibrosis, pulmonary arterial hypertension, and reactivation of metastatic cancer stem cells. DAN family members are known to inhibit the bone morphogenetic proteins (BMP) of the TGF-β family. However, unlike other TGF-β antagonist families, DAN family members have roles beyond ligand inhibition and can modulate Wnt and vascular endothelial growth factor (VEGF) signaling pathways. This review describes recent structural and functional advances that have expanded our understanding of DAN family proteins with regards to BMP inhibition and also highlights their emerging roles in the modulation of Wnt and VEGF signaling pathways.

The Sensor Kinase DctS Forms a Tripartite Sensor Unit with DctB and DctA for Sensing C4-Dicarboxylates in Bacillus subtilis

The DctSR two-component system of Bacillus subtilis controls the expression of the aerobic C4-dicarboxylate transporter DctA. Deletion of DctA leads to an increased dctA expression. The inactivation of DctB, an extracellular binding protein, is known to inhibit the expression of dctA. Here, interaction between the sensor kinase DctS and the transporter DctA as well as the binding protein DctB was demonstrated in vivo using streptavidin (Strep) or His protein interaction experiments (mSPINE or mHPINE), and the data suggest that DctA and DctB act as cosensors for DctS. The interaction between DctS and DctB was also confirmed by the bacterial two-hybrid system (BACTH). In contrast, no indication was obtained for a direct interaction between the transporter DctA and the binding protein DctB. Activity levels of uptake of [(14)C]succinate by bacteria that expressed DctA from a plasmid were similar in the absence and the presence of DctB, demonstrating that the binding protein DctB is not required for transport. Thus, DctB is involved not in transport but in cosensing with DctS, highlighting DctB as the first example of a TRAP-type binding protein that acts as a cosensor. The simultaneous presence of DctS/DctB and DctS/DctA sensor pairs and the lack of direct interaction between the cosensors DctA and DctB indicate the formation of a tripartite complex via DctS. It is suggested that the DctS/DctA/DctB complex forms the functional unit for C4-dicarboxylate sensing in B. subtilis.

Staphylococcal Ecb Protein and Host Complement Regulator Factor H Enhance Functions of Each Other in Bacterial Immune Evasion

Staphylococcus aureus is a major human pathogen causing more than a tenth of all septicemia cases and often superficial and deep infections in various tissues. One of the immune evasion strategies of S. aureus is to secrete proteins that bind to the central complement opsonin C3b. One of these, extracellular complement binding protein (Ecb), is known to interfere directly with functions of C3b. Because C3b is also the target of the physiological plasma complement regulator, factor H (FH), we studied the effect of Ecb on the complement regulatory functions of FH. We show that Ecb enhances acquisition of FH from serum onto staphylococcal surfaces. Ecb and FH enhance mutual binding to C3b and also the function of each other in downregulating complement activation. Both Ecb and the C-terminal domains 19-20 of FH bind to the C3d part of C3b. We show that the mutual enhancing effect of Ecb and FH on binding to C3b depends on binding of the FH domain 19 to the C3d part of C3b next to the binding site of Ecb on C3d. Our results show that Ecb, FH, and C3b form a tripartite complex. Upon exposure of serum-sensitive Haemophilus influenzae to human serum, Ecb protected the bacteria, and this effect was enhanced by the addition of the C-terminal domains 19-20 of FH. This finding indicates that the tripartite complex formation could give additional protection to bacteria and that S. aureus is thereby able to use host FH and bacterial Ecb in a concerted action to eliminate C3b at the site of infection.

Chemokine contribution to neuropathic pain: Respective induction of CXCL1 and CXCR2 in spinal cord astrocytes and neurons

Recent studies have indicated an important role of chemokines such as CCL2 in the development of chronic pain. However, the distinct roles of different chemokines in the development and maintenance of neuropathic pain and in their interactions with neurons have not been clearly elucidated. We found that spinal nerve ligation (SNL) not only induced persistent neuropathic pain symptoms, including mechanical allodynia and heat hyperalgesia, but also produced sustained CXCL1 upregulation in the spinal cord. Double staining of immunofluorescence and in situ hybridization revealed that CXCL1 was primarily induced in spinal astrocytes. In cultured astrocytes, tumor necrosis factor-α induced robust CXCL1 expression via the activation of the c-jun N-terminal kinase. Intrathecal administration of CXCL1 neutralizing antibody transiently reduced SNL-induced pain hypersensitivity, suggesting an essential role of CXCL1 in neuropathic pain sensitization. In particular, intraspinal delivery of CXCL1 shRNA lentiviral vectors, either before or after SNL, persistently attenuated SNL-induced pain hypersensitivity. Spinal application of CXCL1 not only elicited pain hypersensitivity but also induced rapid neuronal activation, as indicated by the expression of phosphorylated extracellular signal-regulated kinase and cAMP response element binding protein, and c-Fos in spinal cord neurons. Interestingly, CXCR2, the primary receptor of CXCL1, was upregulated in dorsal horn neurons after SNL, and the CXCR2 antagonist SB225002 completely blocked the CXCL1-induced heat hyperalgesia. SB225002 also attenuated SNL-induced pain hypersensitivity. Collectively, our results have demonstrated a novel form of chemokine-mediated glial-neuronal interaction in the spinal cord that can drive neuropathic pain. Inhibition of the CXCL1-CXCR2 signaling may offer a new therapy for neuropathic pain management.

Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis

The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a ‘gate’ by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function.

Extracellular conversion of silver ions into silver nanoparticles by protozoan Tetrahymena thermophila

In the current study, cell-free exudates of the ciliated protozoan Tetrahymena thermophila were shown to progressively convert silver nitrate to silver nanoparticles (Ag NPs) under illumination at ambient temperature. The formation of Ag NPs in the reaction mixture was evidenced by gradual colour changes, appearance of a specific absorbance peak (420–450 nm) and visualization using scanning electron microscopy coupled to an energy-dispersive X-ray spectrometer. After 2 h of incubation the mean hydrodynamic size of the Ag NPs was 70 nm. Seven days of incubation resulted in larger agglomerates and a significant decrease in silver toxicity to T. thermophila, accompanied by about 100-fold reduction in the silver ion concentration. Protein analysis indicated an extensive extracellular protein binding by the Ag NPs formed in the protozoan exudates. As protozoa are important components in wastewater treatment, their ability to sequester silver ions into a less bioavailable and less toxic form of silver (e.g. NPs) may be one of the adaption mechanisms of ciliate survival in contaminated environments.

Magnetic resonance diffusion and relaxation characterization of water in the unfrozen vein network in polycrystalline ice and its response to microbial metabolic products

Polycrystalline ice, as found in glaciers and the ice sheets of Antarctica, is a low porosity porous media consisting of a complicated and dynamic pore structure of liquid-filled intercrystalline veins within a solid ice matrix. In this work, Nuclear Magnetic Resonance measurements of relaxation rates and molecular diffusion, useful for probing pore structure and transport dynamics in porous systems, were used to physically characterize the unfrozen vein network structure in ice and its response to the presence of metabolic products produced by V3519-10, a cold tolerant microorganism isolated from the Vostok ice core. Recent research has found microorganisms that can remain viable and even metabolically active within icy environments at sub-zero temperatures. One potential mechanism of survival for V3519-10 is secretion of an extracellular ice binding protein that binds to the prism face of ice crystals and inhibits ice recrystallization, a coarsening process resulting in crystal growth with ice aging. Understanding the impact of ice binding activity on the bulk vein network structure in ice is important to modeling of frozen geophysical systems and in development of ice interacting proteins for biotechnology applications, such as cryopreservation of cell lines, and manufacturing processes in food sciences. Here, we present the first observations of recrystallization inhibition in low porosity ice containing V3519-10 extracellular protein extract as measured with Nuclear Magnetic Resonance and Magnetic Resonance Imaging.

Synthesis of Silk Fibroin–Glycopolypeptide Conjugates and Their Recognition with Lectin

Silk fibroin (SF), the natural fibrous protein created by the Bombyx mori silk worm, is being increasingly explored as a biomaterial for tissue engineering due to its excellent mechanical strength, high oxygen/water permeability, and biocompatibility. It is also well known that surface modification of SF with organic ligands such as the extracellular protein binding Arg-Gly-Asp (RGD) peptides help adhesion and proliferation of cells better-a key requirement for it to function as extracellular matrices. In this work, we have conjugated synthetic glycopolypeptides (GPs) that were synthesized by controlled ring-opening polymerization of α-manno-lys N-carboxyanhydrides (NCAs) onto SF by using Cu catalyzed click reaction to synthesize a new hybrid material (SF-GP), which we believe will have both the mechanical properties of native SF and the molecular recognition property of the carbohydrates in the GP. By controlling the amount of GP grafted onto SF, we have made three SF-GP conjugates that differ in their ability to assemble into films. SF-GP conjugates having a very high content of GP formed completely water-soluble brush-like polymer that displayed very high affinity toward the lectin concanavalin-A (Con-A). Films cast from SF-GP conjugates using lower amounts of grafted GP were more stable in water, and the stability can be modulated by varying the amount of GP grafted. The water-insoluble film SF-GP(25) was also found to bind to fluorescently labeled Con-A, as was seen by confocal microscopy. Such SF-GP hybrid films may be useful as mimics of extracellular matrices for tissue engineering.

Fluorescent Fatty Acid Transfer from Bovine Serum Albumin to Phospholipid Vesicles: Collision or Diffusion Mediated Uptake

The extent of palmitate uptake by hepatocytes is dependent upon the surface charge of the extracellular binding protein. Specifically, hepatocyte uptake is greater when palmitate is bound to cationic binding proteins than when it is bound to anionic proteins. To further understand the role of protein surface charge on the uptake process of protein-bound ligands, we examined the rate of transfer of fluorescent anthroyloxy palmitic acid (AOPA) in the presence of anionic and cationic extracellular proteins to model membranes containing different surface charged groups. AOPA transfer rate in the presence of bovine serum albumin (ALB; isoelectric point pI = 4.8-5.0) or modified ALB (ALBe; pI = 7.0-7.5) to negative, positive and neutral lipid vesicles was investigated using a fluorescence resonance energy transfer assay. The rate of AOPA transfer from both proteins was decreased when ionic strength was increased; directly dependent on the concentration of acceptor lipid vesicles; and was affected by both the lipid membrane surface charge and protein-bound concentration. The data support the notion that AOPA transfer from binding proteins to lipid membranes occurred through two concomitant processes, aqueous diffusion of the unbound ligand (diffusion-mediated process) and a collisional interaction between the protein-ligand complex and acceptor membrane. The contribution of diffusional mediated transfer to the overall uptake process was determined to be 3 to 4 times less than the contribution of a collisional interaction. This study strengthened the hypothesis that charged amino acid residues on proteins are important for effective collisional interaction between protein-ligand complexes and cell membranes through which more free ligand could be supplied for the uptake process.

Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.

Volatile pheromone signalling in Drosophila

Once captured by the antenna, 11-cis vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH-cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only neurones that detect cVA pheromone. This mechanism explains the single molecule sensitivity of insect pheromone detection systems. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co-receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Drosophila melanogaster Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species.

Biglycan Is an Extracellular MuSK Binding Protein Important for Synapse Stability

The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for prepatterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn(-/o)) muscle is indistinguishable from wild-type controls. However, by 5 weeks after birth, nerve-muscle synapses in bgn(-/o) mice are abnormal as judged by the presence of perijunctional folds, increased segmentation, and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied presynaptic and postsynaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn(-/o) synapses. In bgn(-/o) myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability.

Label-free Quantitative Proteomics for the Extremely Thermophilic Bacterium Caldicellulosiruptor obsidiansis Reveal Distinct Abundance Patterns upon Growth on Cellobiose, Crystalline Cellulose, and Switchgrass

Mass spectrometric analysis of Caldicellulosiruptor obsidiansis cultures grown on four different carbon sources identified 65% of the cells' predicted proteins in cell lysates and supernatants. Biological and technical replication together with sophisticated statistical analysis were used to reliably quantify protein abundances and their changes as a function of carbon source. Extracellular, multifunctional glycosidases were significantly more abundant on cellobiose than on the crystalline cellulose substrates Avicel and filter paper, indicating either disaccharide induction or constitutive protein expression. Highly abundant flagellar, chemotaxis, and pilus proteins were detected during growth on insoluble substrates, suggesting motility or specific substrate attachment. The highly abundant extracellular binding protein COB47_0549 together with the COB47_1616 ATPase might comprise the primary ABC-transport system for cellooligosaccharides, while COB47_0096 and COB47_0097 could facilitate monosaccharide uptake. Oligosaccharide degradation can occur either via extracellular hydrolysis by a GH1 β-glycosidase or by intracellular phosphorolysis using two GH94 enzymes. When C. obsidiansis was grown on switchgrass, the abundance of hemicellulases (including GH3, GH5, GH51, and GH67 enzymes) and certain sugar transporters increased significantly. Cultivation on biomass also caused a concerted increase in cytosolic enzymes for xylose and arabinose fermentation.

Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Evidence supports local roles for transforming growth factor β superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signalling receptors is likely modulated by extracellular binding proteins (BP). In this study, we compared ex vivo expression of four BPs (chordin, gremlin, noggin and follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1-18 mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P < 0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P < 0.01) gremlin and follistatin mRNA abundance, with a significant cell-type × follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P < 0.05) in GC of large 'E-active' than 'E-inactive' follicles while follistatin mRNA level was higher (P < 0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP-BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin and gremlin mRNA levels up to 20-fold (P < 0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signalling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signalling.

A derivative of 2-aminothiazole inhibits melanogenesis in B16 mouse melanoma cells via glycogen synthase kinase 3β phosphorylation

We have investigated whether KHG25855 (2-cyclohexylamino-1,3-thiazole hydrochloride) affected melanogenesis in B16 mouse melanoma cells, and the mechanisms involved. Melanin content and tyrosinase activity were measured using an ELISA reader after cells were treated with KHG25855. KHG25855-induced signalling pathways were examined using Western blot analysis. KHG25855 decreased melanin production in a dose-dependent fashion, but KHG25855 did not directly inhibit tyrosinase, the rate-limiting melanogenic enzyme. The expression of microphthalmia-associated transcription factor, tyrosinase, and the related signal transduction pathways were also investigated. The effects of KHG25855 on the extracellular signal-regulated kinase and cAMP response element binding protein signalling pathways were determined, and KHG25855 was shown to have no effect on these signalling pathways. The Wnt signalling pathway is also deeply involved in melanogenesis, and so glycogen synthase kinase 3β (GSK3β) phosphorylation was assessed after KHG25855 treatment; KHG25855 caused GSK3β phosphorylation (inactivation), but the level of β-catenin was not changed by KHG25855. Furthermore, α-melanocyte stimulating hormone-induced tyrosinase expression was downregulated by KHG25855. We propose that KHG25855 showed hypopigmentary activity through tyrosinase downregulation via GSK3β phosphorylation.

Use of Label-Free Quantitative Proteomics To Distinguish the Secreted Cellulolytic Systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.

Laminin database: a tool to retrieve high-throughput and curated data for studies on laminins

The Laminin(LM)-database, hosted at http://www.lm.lncc.br, is the first database focusing a non-collagenous extracellular matrix protein family, the LMs. Part of the knowledge available in this website is automatically retrieved, whereas a significant amount of information is curated and annotated, thus placing LM-database beyond a simple repository of data. In its home page, an overview of the rationale for the database is seen and readers can access a tutorial to facilitate navigation in the website, which in turn is presented with tabs subdivided into LMs, receptors, extracellular binding and other related proteins. Each tab opens into a given LM or LM-related molecule, where the reader finds a series of further tabs for ‘protein’, ‘gene structure’, ‘gene expression’ and ‘tissue distribution’ and ‘therapy’. Data are separated as a function of species, comprising Homo sapiens, Mus musculus and Rattus novergicus. Furthermore, there is specific tab displaying the LM nomenclatures. In another tab, a direct link to PubMed, which can be then consulted in a specific way, in terms of the biological functions of each molecule, knockout animals and genetic diseases, immune response and lymphomas/leukemias. LM-database will hopefully be a relevant tool for retrieving information concerning LMs in health and disease, particularly regarding the hemopoietic system.