Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.     

Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.     

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser²²/Ser²³, reduces myofilament Ca²⁺ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser²²/Ser²³ remains to be established. To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser²²/Ser²³ are substituted by nonphosphorylatable Ala (cTnI-Ala₂). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser²²/Ser²³ and decreased the Ca²⁺ sensitivity of force. In contrast, PKD had no effect on the Ca²⁺ sensitivity of force in myocardium from cTnI-Ala₂ mice, in which Ser²²/Ser²³ were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala₂ mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser²⁷³, Ser²⁸², and Ser³⁰², and revealed that PKD phosphorylates only Ser³⁰². Furthermore, PKD phosphorylated Ser³⁰² selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala₂ mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser³⁰², which may mediate the latter effect.     

Heart-specific Small Subunit of Myosin Light Chain Phosphatase Activates Rho-associated Kinase and Regulates Phosphorylation of Myosin Phosphatase Target Subunit 1

Phosphorylation of myosin regulatory light chain (MLC) plays a regulatory role in muscle contraction, and the level of MLC phosphorylation is balanced by MLC kinase and MLC phosphatase (MLCP). MLCP consists of a catalytic subunit, a large subunit (MYPT1 or MYPT2), and a small subunit. MLCP activity is regulated by phosphorylation of MYPTs, whereas the role of small subunit in the regulation remains unknown. We previously characterized a human heart-specific small subunit (hHS-M₂₁) that increased the sensitivity to Ca²⁺ in muscle contraction. In this study, we investigated the role of hHS-M₂₁ in the regulation of MLCP phosphorylation. Two isoforms of hHS-M₂₁, hHS-M₂₁A and hHS-M₂₁B, preferentially bound the C-terminal one-third region of MYPT1 and MYPT2, respectively. Amino acid substitutions at a phosphorylation site of MYPT1, Ser-852, impaired the binding of MYPT1 and hHS-M₂₁. The hHS-M₂₁ increased the phosphorylation level of MYPT1 at Thr-696, which was attenuated by Rho-associated kinase (ROCK) inhibitors and small interfering RNAs for ROCK. In addition, hHS-M₂₁ bound ROCK and enhanced the ROCK activity. These findings suggest that hHS-M₂₁ is a heart-specific effector of ROCK and plays a regulatory role in the MYPT1 phosphorylation at Thr-696 by ROCK.     

Smad7 Inhibits Transforming Growth Factor-β Family Type I Receptors through Two Distinct Modes of Interaction

The inhibitory Smads (I-Smads), i.e. Smad6 and Smad7, are negative regulators of transforming growth factor-β (TGF-β) family signaling. I-Smads inhibit TGF-β family signaling principally through physical interaction with type I receptors (activin receptor-like kinases), so as to compete with receptor-regulated Smads (R-Smads) for activation. However, how I-Smads interact with type I receptors is not well understood. In the present study, we found that Smad7 has two modes of interaction with type I receptors. One is through a three-finger-like structure in the MH2 domain, consisting of residues 331–361, 379–387, and the L3 loop. The other is through a basic groove in the MH2 domain (Mochizuki, T., Miyazaki, H., Hara, T., Furuya, T., Imamura, T., Watabe, T., and Miyazono, K. (2004) J. Biol. Chem. 279, 31568–31574). We also found that Smad6 principally utilizes a basic groove in the MH2 domain for interaction with type I receptors. Smad7 thus has an additional mode of interaction with TGF-β family type I receptors not possessed by Smad6, which may play roles in mediating the inhibitory effects unique to Smad7.     

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

The receptor kinase EFR of Arabidopsis thaliana detects the microbe-associated molecular pattern elf18, a peptide that represents the N terminus of bacterial elongation factor Tu. Here, we tested subdomains of EFR for their importance in receptor function. Transient expression of tagged versions of EFR and EFR lacking its cytoplasmic domain in leaves of Nicotiana benthamiana resulted in functional binding sites for elf18. No binding of ligand was found with the ectodomain lacking the transmembrane domain or with EFR lacking the first 5 of its 21 leucine-rich repeats (LRRs). EFR is structurally related to the receptor kinase flagellin-sensing 2 (FLS2) that detects bacterial flagellin. Chimeric receptors with subdomains of FLS2 substituting for corresponding parts of EFR were tested for functionality in ligand binding and receptor activation assays. Substituting the transmembrane domain and the cytoplasmic domain resulted in a fully functional receptor for elf18. Replacing also the outer juxtamembrane domain with that of FLS2 led to a receptor with full affinity for elf18 but with a lower efficiency in response activation. Extending the substitution to encompass also the last two of the LRRs abolished binding and receptor activation. Substitution of the N terminus by the first six LRRs from FLS2 reduced binding affinity and strongly affected receptor activation. In summary, chimeric receptors allow mapping of subdomains relevant for ligand binding and receptor activation. The results also show that modular assembly of chimeras from different receptors can be used to form functional receptors.     

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H₂O₂-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.     

Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi

Bacillus subtilis DC33 producing a novel fibrinolytic enzyme was isolated from Ba-bao Douchi, a traditional soybean-fermented food in China. The strong fibrin-specific enzyme subtilisin FS33 was purified to electrophoretic homogeneity using the combination of various chromatographic steps. The optimum temperature, pH value, and pI of subtilisin FS33 were 55°C, 8.0, and 8.7, respectively. The molecular weight was 30 kDa measured by SDS–PAGE under both reducing and non-reducing conditions. The enzyme showed a level of fibrinolytic activity that was about six times higher than that of subtilisin Carlsberg. The first 15 amino acid residues of N-terminal sequence of the enzyme were A-Q-S-V-P-Y-G-I-P-Q-I-K-A-P-A, which are different from that of other known fibrinolytic enzymes. The amidolytic activities of subtilisin FS33 were inhibited completely by 5 mM phenylmethanesulfonyl fluoride (PMSF) and 1 mM soybean trypsin inhibitor (SBTI), but 1,4-dithiothreitol (DTT), β-mercaptoethanol, and p-hydroxymercuribenzoate (PHMB) did not affect the enzyme activity; serine and tryptophan are thus essential in the active site of the enzyme. The highest affinity of subtilisin FS33 was towards N-Succ-Ala-Ala-Pro-Phe-pNA. Therefore, the enzyme was considered to be a subtilisin-like serine protease. The fibrinolytic enzyme had a high degrading activity for the Bβ-chains and Aα-chain of fibrin(ogen), and also acted on thrombotic and fibrinolytic factors of blood, such as plasminogen, urokinase, thrombin, and kallikrein. So subtilisin FS33 was able to degrade fibrin clots in two ways, i.e., (a) by forming active plasmin from plasminogen and (b) by direct fibrinolysis.     

The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica)

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the “α/β hydrolase fold”. The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.     

Metabolic pathways and biotechnological production of l-cysteine

l-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are commercially produced by fermentation, cysteine is mainly produced by protein hydrolysis. However, synthetic or biotechnological products have been preferred in the market. Biotechnological processes for cysteine production, both enzymatic and fermentative processes, are discussed. Enzymatic process, the asymmetric hydrolysis of dl-2-amino-Δ²-thiazoline-4-carboxylic acid to l-cysteine, has been developed and industrialized. The l-cysteine biosynthetic pathways of Escherichia coli and Corynebacterium glutamicum, which are used in many amino acid production processes, are also described. These two bacteria have basically same l-cysteine biosynthetic pathways. l-Cysteine-degrading enzymes and l-cysteine-exporting proteins both in E. coli and C. glutamicum are also described. In conclusion, for the effective fermentative production of l-cysteine directly from glucose, the combination of enhancing biosynthetic activity, weakening the degradation pathway, and exploiting the export system seems to be effective.