Non-catalytic Domain Research Articles

The Arabidopsis Protein Disulfide Isomerase Subfamily M Isoform, PDI9, Localizes to the Endoplasmic Reticulum and Influences Pollen Viability and Proper Formation of the Pollen Exine During Heat Stress.

Plants adapt to heat via thermotolerance pathways in which the activation of protein folding chaperones is essential. In eukaryotes, protein disulfide isomerases (PDIs) facilitate the folding of nascent and misfolded proteins in the secretory pathway by catalyzing the formation and isomerization of disulfide bonds and serving as molecular chaperones. In Arabidopsis, several members of the PDI family are upregulated in response to chemical inducers of the unfolded protein response (UPR), including both members of the non-classical PDI-M subfamily, PDI9 and PDI10. Unlike classical PDIs, which have two catalytic thioredoxin (TRX) domains separated by two non-catalytic TRX-fold domains, PDI-M isoforms are orthologs of mammalian P5/PDIA6 and possess two tandem catalytic domains. Here, PDI9 accumulation was found to be upregulated in pollen in response to heat stress. Histochemical staining of plants harboring the PDI9 and PDI10 promoters fused to the gusA gene indicated they were actively expressed in the anthers of flowers, specifically in the pollen and tapetum. Immunoelectron microscopy revealed that PDI9 localized to the endoplasmic reticulum in root and pollen cells. transfer DNA (T-DNA) insertional mutations in the PDI9 gene disrupted pollen viability and development in plants exposed to heat stress. In particular, the pollen grains of pdi9 mutants exhibited disruptions in the reticulated pattern of the exine and an increased adhesion of pollen grains. Pollen in the pdi10 single mutant did not display similar heat-associated defects, but pdi9 pdi10 double mutants (DMs) completely lost exine reticulation. Interestingly, overexpression of PDI9 partially led to heat-associated defects in the exine. We conclude that PDI9 plays an important role in pollen thermotolerance and exine biogenesis. Its role fits the mechanistic theory of proteostasis in which an ideal balance of PDI isoforms is required in the endoplasmic reticulum (ER) for normal exine formation in plants subjected to heat stress.

In silico family-wide profiling and 3D modelling of the poly(ADP-ribose) polymerase superfamily.

Background: Due to the conserved nature of the poly(ADP-ribose) polymerase (PARP) catalytic domain, the identification of unique residues is critical for the design of selective inhibitors. With inhibitors of the DNA-dependent PARP members already clinically approved, new efforts lie in discovering selective inhibitors for PARP5a and beyond. Targeting the noncatalytic domains, such as the macro2 and WWE domains may also provide a way to achieve selectivity. Methodology &results: This paper details the in silico profiling of x-ray crystal structures and homology models of the PARP catalytic, WWE and macro2 domains. PARP10 was the least conserved catalytic domain, with the macro2 and WWE domains possessing more unique residues than their catalytic counterparts. Conclusion: Overall, we identify unique residues to target when designing selective PARP inhibitors including HIS1610, TYR1620, ALA1627 and ARG1658 of the PARP14 catalytic domain, along with multiple unique residues across the PARP WWE and macro2 domains.

Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1.

As a mitotic-specific target widely deregulated in various human cancers, polo-like kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT (Kd ∼ 450 nM). In addition, S-methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.

Denaturant Induced Equilibrium Unfolding and Conformational Transitional Studies of Germinated Fenugreek β-Amylase Revealed Molten Globule like State at Low pH.

β-Amylase (EC 3.2.1.2) is a maltogenic enzyme, which releases β-maltose from the non-reducing end of the substrates. The enzyme plays important roles for the production of vaccine, maltiol and maltose rich syrups. Apart from these applications the enzyme protects cells from abiotic as well as oxidative damage. The enzyme is βwell characterized in βplants and microbes and crystal structures of β-amylases βhave been βobtained from sweet potato, soybean and Bacillus cereus. Find out correlation between structural and functional stability induced by change in pH, temperature and chaotropes. Activity, intrinsic fluorescence, extrinsic fluorescence, near- and far- ultraviolet circular dichroism spectroscopic measurements were performed. Peaks about 208 nm and 222 nm obtained by near-ultraviolet circular dichroism correspond to α-helix whereas peak at 215 nm shows presence of β-sheet. At pH 2.0, absence of tertiary structures, exposed of hydrophobic regions and presence of substantial secondary structures, revealed the existence of molten globule like state. Temperature induced denaturation studies showed that the enzyme was stable up to 75 ºC and the process was found to be irreversible in nature. Chaotropes dependent equilibrium unfolding studies revealed that at low concentration of chaotropes, ellipticity and intrinsic fluorescence βintensity were βdecreased βwhereas βenzymatic activity remained unchanged, which revealed fenugreek β-amylase is multi-domains enzyme and catalytic βdomain βis more βstable compare to non-catalytic domain. Moreover, the transition was sigmoidal and non-coincidental. Results indicate the probable existence of intermediate states that might perform significant role in physiological process and biotechnological applications.

Expression and characterization of family 40 Carbohydrate Binding Module (CBM) from Vibrio cholerae Non-O1 sialidase

Carbohydrate Binding Module (CBM) is a non-catalytic protein domain found in carbohydrate-active enzyme (glycoside hydrolase) and its role is to bring carbohydrates in close proximity to the enzyme catalytic site for complete hydrolysis. The removal of this CBM from most protein domains often leads to reduced enzyme activity and efficiency. In this study, a gene encoding for family 40 CBM from Vibrio cholerae Non-O1 sialidase was cloned and successfully expressed in E. coli BL21 (DE3) strain. The CBM40 encoded 195 amino acids with 585 bp of nucleotide sequence. The protein was successfully expressed at 18°C when induced with 1 mM IPTG. Maximum expression was achieved at 20 hours after post-induction time. For purification of the protein, an anionic denaturing detergent method was used containing 1% SDS and 0.1% sarkosyl with gradient affinity elution at 50 mM imidazole concentrations. SDS-PAGE analysis of the purified CBM40 protein displayed a protein band with a molecular mass of 21 kDa. Protein characterization showed optimum stability in 100 mM citrate buffer pH 5.5, with the highest Tm value of 40 °C. The protein was stable between pH 5.5–6.2 and able to retain its activity at 27–56°C. The addition of Mn2+ and Mg2+ increased the protein melting temperature to 56°C. Meanwhile, the addition of reagents, such as 1% SDS and 1 M urea increased the protein melting temperature (Tm) to approximately 55°C. Protein stability can be influenced by many factors, including different buffers, pHs, temperatures, ionic strengths, and chemical reagents used in a study. The optimum characterization conditions established would further lead to the discovery of CBM40 protein true potential in enhancing substrate binding affinity and protein-carbohydrate recognition, which underpins its broad applications in biotechnology and protein engineering fields.

Crystal Structure of a Soluble APOBEC3G Variant Suggests ssDNA to Bind in a Channel that Extends between the Two Domains

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5 Å and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn2+, yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3′ to 5′ directional preference in processive deamination.

Compound heterozygous protein C variants undetectable by common laboratory testing causing purpura fulminans after the neonatal period.

Compound heterozygous protein C variants undetectable by common laboratory testing causing purpura fulminans after the neonatal period.

Extension of the taxonomic coverage of the family GH126 outside Firmicutes and in silico characterization of its non-catalytic terminal domains

The family GH126 is a family of glycoside hydrolases established in 2011. Officially, in the CAZy database, it counts ~ 1000 sequences originating solely from bacterial phylum Firmicutes. Two members, the proteins CPF_2247 from Clostridium perfringens and PssZ from Listeria monocytogenes have been characterized as a probable α-amylase and an exopolysaccharide-specific glycosidase, respectively; their three-dimensional structures being also solved as possessing catalytic (α/α)6-barrel fold. Previously, based on a detailed in silico analysis, the seven conserved sequence regions (CSRs) were identified for the family along with elucidating basic evolutionary relationships within the family members. The present study represents a continuation study focusing on two particular aims: (1) to find out whether the taxonomic coverage of the family GH126 might be extended outside the Firmicutes and, if positive, to deliver those out-of-Firmicutes proteins with putting them into the context of the family; and (2) to identify the family members containing the N- and/or C-terminal extensions of their polypeptide chain, additional to the catalytic (α/α)6-barrel domain, and perform the bioinformatics characterization of the extra domains. The main results could be summarized as follows: (1) 17 bacterial proteins caught by BLAST searches outside Firmicutes (especially from phyla Proteobacteria, Actinobacteria and Bacteroidetes) have been found and convincingly suggested as new family GH126 members; and (2) a thioredoxin-like fold and various leucine-rich repeat motifs identified by Phyre2 structure homology modelling have been recognized as extra domains occurring most frequently in the N-terminal extensions of family GH126 members possessing a modular organization.

The In Situ Structure of Parkinson’s Disease-Linked LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson's disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.

Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences

Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation, and organ growth. O-Glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of GalNAc onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain two functional domains: a catalytic domain and a C-terminal ricin-like lectin domain comprised of three potential GalNAc recognition repeats termed α, β, and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, whereas the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. We previously demonstrated a novel role for the α repeat of lectin domain in influencing charged peptide preferences. Here, we further interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, we find that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the noncatalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, our results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.

Crystal structure of Arabidopsis thaliana cytidine deaminase

Cytidine deaminase (CDA) catalyzes the (deoxy)cytidine deamination to (deoxy)uridine, which involves in the catabolic and salvage pathways of pyrimidine nucleotides in plants. CDA serves as a prototype of the cytidine deaminase superfamily that contains a number of RNA editing enzymes. Arabidopsis thaliana has only one functional CDA, AtCDA1. We solved the crystal structures of AtCDA1, which is a dimeric zinc-containing enzyme and each protomer consists of an N-terminal zinc-binding catalytic domain and a C-terminal non-catalytic domain. Both domains adopt a typical α/β/α sandwich fold. In vitro biochemical assays showed that the ribose moiety of cytidine is required for ligand binding, and structural analyses revealed a conserved catalytic mechanism is adopted by AtCDA1.

A novel AA10 from Paenibacillus curdlanolyticus and its synergistic action on crystalline and complex polysaccharides.

Lytic polysaccharide monooxygenases (LPMOs) play an important role in the degradation of complex polysaccharides in lignocellulosic biomass. In the present study, we characterized a modular LPMO (PcAA10A), consisting of a family 10 auxiliary activity of LPMO (AA10) catalytic domain, and non-catalytic domains including a family 5 carbohydrate-binding module, two fibronectin type-3 domains, and a family 3 carbohydrate-binding module from Paenibacillus curdlanolyticus B-6, which was expressed in a recombinant Escherichia coli. Comparison of activities between full-length PcAA10A and the catalytic domain polypeptide (PcAA10A_CD) indicates that the non-catalytic domains are important for the deconstruction of crystalline cellulose and complex polysaccharides contained in untreated lignocellulosic biomass. Interestingly, PcAA10A_CD acted not only on cellulose and chitin, but also on xylan, mannan, and xylan and cellulose contained in lignocellulosic biomass, which has not been reported for the AA10 family. Mutation of the key residues, Trp51 located at subsite - 2 and Phe171 located at subsite +2, in the substrate-binding site of PcAA10A_CD revealed that these residues are substantially involved in broad substrate specificity toward cellulose, xylan, and mannan, albeit with a low effect toward chitin. Furthermore, PcAA10A had a boosting effect on untreated corn hull degradation by P. curdlanolyticus B-6 endo-xylanase Xyn10D and Clostridium thermocellum endo-glucanase Cel9A. These results suggest that PcAA10A is a unique LPMO capable of cleaving and enhancing lignocellulosic biomass degradation, making it a good candidate for biotechnological applications. KEY POINTS: • PcAA10A is a novel modular LPMO family 10 from Paenibacillus curdlanolyticus. • PcAA10A showed broad substrate specificity on β-1,4 glycosidic linkage substrates. • Non-catalytic domains are important for degrading complex polysaccharides. • PcAA10A is a unique LPMO capable of enhancing lignocellulosic biomass degradation.

Functional Features of TREHALOSE-6-PHOSPHATE SYNTHASE1, an Essential Enzyme in Arabidopsis.

In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.

Identifying the Substrate Interactions of KDAC7

Protein post‐translational modifications are chemical adaptations that take place in the human body to control behavior of proteins. The acetylation of lysine is one type of protein post‐translational modification. Lysine deacetylases (KDACs or HDACs), are enzymes that help regulate proteins by reversing this modification. It has been shown that KDAC7 is essential in many biological processes, but KDAC7 has no identified substrate proteins. Therefore, better understanding of KDAC7 substrate preferences could lead to the improved treatment of diseases. Using a gain‐of‐function variant of KDAC7 that significantly increases enzyme activity, we have identified several possible substrates using peptides derived from identified acetylated proteins. Molecular dynamics simulations suggest possible important interactions between these peptides and the enzyme. We are also examining the effect of the second, non‐catalytic domain of KDAC7 on substrate preferences and overall activity. Possible substrates identified by these experiments provide leads for cell‐based assays to determine in vivo substrates and/or binding partners. This work provides a greater understanding of how KDAC7 interacts with potential substrate proteins and the biological activity of the enzyme.Support or Funding InformationNIH TL4GM118968, UL1GM118967, 5G12MD007595, and R15GM129682; NSF CHE 1625993 and MCB 1817358; U. S. Army Research Laboratory and the U. S. Army Research Office W911NF1810450; and the Louisiana Cancer Research Consortium.

Differential Splicing of the Lectin Domain of an O‐glycosyltransferase Influences Charged Peptide Preferences

Mucin‐type O‐glycosylation is an essential post‐translational modification that is required for secretion, extracellular matrix composition and organ growth. O‐glycosylation is initiated by a large family of enzymes (GalNAcTs in mammals and PGANTs in Drosophila) that catalyze the addition of the sugar N‐acetylgalactosamine (GalNAc), onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes have distinct domains, including the catalytic domain and the C‐terminal ricin‐like lectin domain. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing the transfer of GalNAc, while the lectin domain is thought to recognize extant GalNAc on previously glycosylated substrates to position the catalytic domain for further glycosylation of unmodified serines and threonines. Previous work from our group has demonstrated that one member of this family in Drosophila undergoes differential splicing within the lectin domain, leading to variants that contain either a positively or negatively charged subdomain. Interestingly, while this splicing event affects only a subdomain of the lectin region, we find that it influences peptide specificity. We demonstrate that each charged enzyme variant has a preference for oppositely charged peptide substrates, and that this preference can be predictably altered by altering peptide charge. We further show that the expression of each splice variant is associated with the expression of appropriately charged mucins in secretory tissues of Drosophila, suggesting that differential splicing may be employed in vivo to ensure O‐glycosylation of highly charged substrates. Our results elucidate a mechanism for modulating substrate preferences of O‐glycosyltransferases based on the needs of the cell through alternative splicing within a specific subregion of the non‐catalytic domain.

Functional Alterations in Ciliogenesis-Associated Kinase 1 (CILK1) that Result from Mutations Linked to Juvenile Myoclonic Epilepsy.

Ciliopathies are a group of human genetic disorders associated with mutations that give rise to the dysfunction of primary cilia. Ciliogenesis-associated kinase 1 (CILK1), formerly known as intestinal cell kinase (ICK), is a conserved serine and threonine kinase that restricts primary (non-motile) cilia formation and length. Mutations in CILK1 are associated with ciliopathies and are also linked to juvenile myoclonic epilepsy (JME). However, the effects of the JME-related mutations in CILK1 on kinase activity and CILK1 function are unknown. Here, we report that JME pathogenic mutations in the CILK1 N-terminal kinase domain abolish kinase activity, evidenced by the loss of phosphorylation of kinesin family member 3A (KIF3A) at Thr672, while JME mutations in the C-terminal non-catalytic domain (CTD) have little effect on KIF3A phosphorylation. Although CILK1 variants in the CTD retain catalytic activity, they nonetheless lose the ability to restrict cilia length and also gain function in promoting ciliogenesis. We show that wild type CILK1 predominantly localizes to the base of the primary cilium; in contrast, JME variants of CILK1 are distributed along the entire axoneme of the primary cilium. These results demonstrate that JME pathogenic mutations perturb CILK1 function and intracellular localization. These CILK1 variants affect the primary cilium, independent of CILK1 phosphorylation of KIF3A. Our findings suggest that CILK1 mutations linked to JME result in alterations of primary cilia formation and homeostasis.

RNase II binds to RNase E and modulates its endoribonucleolytic activity in the cyanobacterium Anabaena PCC 7120.

In Escherichia coli, the endoribonuclease E (RNase E) can recruit several other ribonucleases and regulatory proteins via its noncatalytic domain to form an RNA degradosome that controls cellular RNA turnover. Similar RNA degradation complexes have been found in other bacteria; however, their compositions are varied among different bacterial species. In cyanobacteria, only the exoribonuclease PNPase was shown to bind to the noncatalytic domain of RNase E. Here, we showed that Alr1240, a member of the RNB family of exoribonucleases, could be co-isolated with RNase E from the lysate of the cyanobacterium Anabaena PCC 7120. Enzymatic analysis revealed that Alr1240 is an exoribonuclease II (RNase II), as it only degrades non-structured single-stranded RNA substrates. In contrast to known RNase E-interacting ribonucleases, which bind to the noncatalytic domain of RNase E, the Anabaena RNase II was shown to associate with the catalytic domain of RNase E. Using a strain in which RNase E and RNase II were tagged in situ with GFP and BFP, respectively, we showed that RNase E and RNase II form a compact complex in vivo by a fluorescence resonance energy transfer (FRET) assay. RNase E activity on several synthetic substrates was boosted in the presence of RNase II, suggesting that the activity of RNase E could be regulated by RNase II-RNase E interaction. To our knowledge, Anabaena RNase II is an unusual ribonuclease that interacts with the catalytic domain of RNase E, and it may represent a new type of RNA degradosome and a novel mechanism for regulating the activity of the RNA degradosome. As Anabaena RNase E interacts with RNase II and PNPase via different regions, it is very likely that the three ribonucleases form a large complex and cooperatively regulate RNA metabolism in the cell.

Selective targeting of TET catalytic domain promotes somatic cell reprogramming

Ten-eleven translocation (TET) family enzymes (TET1, TET2, and TET3) oxidize 5-methylcytosine (5mC) and generate 5-hydroxymethylcytosine (5hmC) marks on the genome. Each TET protein also interacts with specific binding partners and partly plays their role independent of catalytic activity. Although the basic role of TET enzymes is well established now, the molecular mechanism and specific contribution of their catalytic and noncatalytic domains remain elusive. Here, by combining in silico and biochemical screening strategy, we have identified a small molecule compound, C35, as a first-in-class TET inhibitor that specifically blocks their catalytic activities. Using this inhibitor, we explored the enzymatic function of TET proteins during somatic cell reprogramming. Interestingly, we found that C35-mediated TET inactivation increased the efficiency of somatic cell programming without affecting TET complexes. Using high-throughput mRNA sequencing, we found that by targeting 5hmC repressive marks in the promoter regions, C35-mediated TET inhibition activates the transcription of the BMP-SMAD-ID signaling pathway, which may be responsible for promoting somatic cell reprogramming. These results suggest that C35 is an important tool for inducing somatic cell reprogramming, as well as for dissecting the other biological functions of TET enzymatic activities without affecting their other nonenzymatic roles.

Structure and properties of the oyster mushroom (Pleurotus ostreatus) lectin.

Pleurotus ostreatus Lectin (POL) is a 353 amino acid chain lectin that can be purified from the fruiting bodies of the very well-known and widely diffused edible oyster mushrooms (P. ostreatus). The lectin has been partially characterized by different groups and, although it was crystallized about 20years ago, its 3D structure and the details of its interactions with carbohydrates are still unknown. This paper reports the 3D structure and ligand-binding properties of POL. We have determined the X-ray structure of the apo-protein purified from the fruiting bodies of the mushroom and that of the recombinant protein in complex with melibiose to a resolution of about 2Å. The lectin is a homodimer in which the two polypeptide chains are linked by a disulfide bridge. A POL monomer is composed of two highly homologous β-jellyroll domains each of which containing a calcium-dependent carbohydrate-binding site. A high degree of sequence similarity is observed between the two carbohydrate-binding modules present in each monomer. The structure of the lectin in complex with melibiose reveals that a POL dimer has four calcium-dependent carbohydrate-binding sites. The interaction with sugars in solution has been characterized by isothermal titration calorimetry and saturation transfer difference NMR and it sheds new light on the molecular determinants of POL specificity. The lectin exhibits in vitro antiproliferative effects against human cancer cell lines and presents structural similarity with the prototype member of the CBM67 family, the noncatalytic domain of Streptomyces avermitilis α-rhamnosidase.

Dissecting contributions of catalytic and reader domains in regulation of histone demethylation.

Histone demethylases catalyze the removal of methyl marks from histones, an activity associated with transcriptional regulation and DNA damage repair. As these processes are critical for normal physiology, deregulation of histone demethylases is disease causative, and their function and regulation are targets for therapeutic intervention. The larger of two histone demethylase families are Jumonji C (JmjC) demethylases. The members of the JmjC family share a conserved catalytic domain, and often contain non-catalytic domains that "read" the modification state of chromatin. By binding to specific histone modifications, reader domains assist in recruitment and promote accumulation of demethylases at their targets, as well as regulate their activity and substrate specificity. Here, we present protocols for the investigation of this functional coupling between reader and catalytic domains in human histone demethylase KDM5A. Although we use KDM5A and its PHD1 domain as our model system, the procedures presented herein can be applied for the biochemical characterization of other JmjC demethylases and chromatin readers.