Structural insight into the reaction mechanism and evolution of cytokinin biosynthesis

The phytohormone cytokinin regulates plant growth and development. This hormone is also synthesized by some phytopathogenic bacteria, such as Agrobacterium tumefaciens, and is as a key factor in the formation of plant tumors. The rate-limiting step of cytokinin biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (IPT). Agrobacterium IPT has a unique substrate specificity that enables it to increase trans-zeatin production by recruiting a metabolic intermediate of the host plant's biosynthetic pathway. Here, we show the crystal structures of Tzs, an IPT from A. tumefaciens, complexed with AMP and a prenyl-donor analogue, dimethylallyl S-thiodiphosphate. The structures reveal that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism. Site-directed mutagenesis was used to determine the amino acid residues, Asp-173 and His-214, which are responsible for differences in prenyl-donor substrate specificity between plant and bacterial IPTs. IPT and the p loop-containing nucleoside triphosphate hydrolases likely evolved from a common ancestral protein. Despite structural similarities, IPT has evolved a distinct role in which the p loop transfers a prenyl moiety in cytokinin biosynthesis.     

Information processing at the foxa node of the sea urchin endomesoderm specification network

The foxa regulatory gene is of central importance for endoderm specification across Bilateria, and this gene lies at an essential node of the well-characterized sea urchin endomesoderm gene regulatory network (GRN). Here we experimentally dissect the cis-regulatory system that controls the complex pattern of foxa expression in these embryos. Four separate cis-regulatory modules (CRMs) cooperate to control foxa expression in different spatial domains of the endomesoderm, and at different times. A detailed mutational analysis revealed the inputs to each of these cis-regulatory modules. The complex and dynamic expression of foxa is regulated by a combination of repressors, a permissive switch, and multiple activators. A mathematical kinetic model was applied to study the dynamic response of foxa cis-regulatory modules to transient inputs. This study shed light on the mesoderm–endoderm fate decision and provides a functional explanation, in terms of the genomic regulatory code, for the spatial and temporal expression of a key developmental control gene.     

Regulative recovery in the sea urchin embryo and the stabilizing role of fail-safe gene network wiring

Design features that ensure reproducible and invariant embryonic processes are major characteristics of current gene regulatory network models. New cis-regulatory studies on a gene regulatory network subcircuit activated early in the development of the sea urchin embryo reveal a sequence of encoded “fail-safe” regulatory devices. These ensure the maintenance of fate separation between skeletogenic and nonskeletogenic mesoderm lineages. An unexpected consequence of the network design revealed in the course of these experiments is that it enables the embryo to “recover” from regulatory interference that has catastrophic effects if this feature is disarmed. A reengineered regulatory system inserted into the embryo was used to prove how this system operates in vivo. Genomically encoded backup control circuitry thus provides the mechanism underlying a specific example of the regulative development for which the sea urchin embryo has long been famous.     

Evolutionary and functional insights into the mechanism underlying high-altitude adaptation of deer mouse hemoglobin

Adaptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. Here we investigate these possibilities by conducting a combined evolutionary and functional analysis of duplicated globin genes in natural populations of deer mice (Peromyscus maniculatus) that are adapted to different elevational zones. A multilocus analysis of nucleotide polymorphism and linkage disequilibrium revealed that high-altitude adaptation of deer mouse hemoglobin involves parallel functional differentiation at multiple unlinked gene duplicates: two α-globin paralogs on chromosome 8 and two β-globin paralogs on chromosome 1. Differences in O₂-binding affinity of the alternative β-chain hemoglobin isoforms were entirely attributable to allelic differences in sensitivity to 2,3-diphosphoglycerate (DPG), an allosteric cofactor that stabilizes the low-affinity, deoxygenated conformation of the hemoglobin tetramer. The two-locus β-globin haplotype that predominates at high altitude is associated with suppressed DPG-sensitivity (and hence, increased hemoglobin-O₂ affinity), which enhances pulmonary O₂ loading under hypoxia. The discovery that allelic differences in DPG-sensitivity contribute to adaptive variation in hemoglobin–O₂ affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. Investigation into the functional significance of the deer mouse β-globin polymorphism was motivated by the results of population genetic analyses which revealed evidence for a history of divergent selection between elevational zones. The experimental measures of O₂-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.     

Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB

Mucopolysaccharidosis III (MPS III) has four forms (A-D) that result from buildup of an improperly degraded glycosaminoglycan in lysosomes. MPS IIIB is attributable to the decreased activity of a lysosomal alpha-N-acetylglucosaminidase (NAGLU). Here, we describe the structure, catalytic mechanism, and inhibition of CpGH89 from Clostridium perfringens, a close bacterial homolog of NAGLU. The structure enables the generation of a homology model of NAGLU, an enzyme that has resisted structural studies despite having been studied for >20 years. This model reveals which mutations giving rise to MPS IIIB map to the active site and which map to regions distant from the active site. The identification of potent inhibitors of CpGH89 and the structures of these inhibitors in complex with the enzyme suggest small-molecule candidates for use as chemical chaperones. These studies therefore illuminate the genetic basis of MPS IIIB, provide a clear biochemical rationale for the necessary sequential action of heparan-degrading enzymes, and open the door to the design and optimization of chemical chaperones for treating MPS IIIB.     

Maternal transfer of xenobiotics and effects on larval striped bass in the San Francisco Estuary

Aquatic ecosystems around the world face serious threats from anthropogenic contaminants. Results from 8 years of field and laboratory investigations indicate that sublethal contaminant exposure is occurring in the early life stages of striped bass in the San Francisco Estuary, a population in continual decline since its initial collapse during the 1970s. Biologically significant levels of polychlorinated biphenyls, polybrominated diphenyl ethers, and current-use/legacy pesticides were found in all egg samples from river-collected fish. Developmental changes previously unseen with standard methods were detected with a technique using the principles of unbiased stereology. Abnormal yolk utilization, brain and liver development, and overall growth were observed in larvae from river-collected fish. Histopathological analyses confirmed and identified developmental alterations. Using this methodology enabled us to present a conclusive line of evidence for the maternal transfer of xenobiotics and their adverse effects on larval striped bass in this estuary.     

Structure of the phage TP901-1 1.8 MDa baseplate suggests an alternative host adhesion mechanism

Phages of the Caudovirales order possess a tail that recognizes the host and ensures genome delivery upon infection. The X-ray structure of the approximately 1.8 MDa host adsorption device (baseplate) from the lactococcal phage TP901-1 shows that the receptor-binding proteins are pointing in the direction of the host, suggesting that this organelle is in a conformation ready for host adhesion. This result is in marked contrast with the lactococcal phage p2 situation, whose baseplate is known to undergo huge conformational changes in the presence of Ca²⁺ to reach its active state. In vivo infection experiments confirmed these structural observations by demonstrating that Ca²⁺ ions are required for host adhesion among p2-like phages (936-species) but have no influence on TP901-1-like phages (P335-species). These data suggest that these two families rely on diverse adhesion strategies which may lead to different signaling for genome release.     

Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants

Plants produce a variety of toxic compounds, which are often used as anticancer drugs. The self-resistance mechanism to these toxic metabolites in the producing plants, however, remains unclear. The plant-derived anticancer alkaloid camptothecin (CPT) induces cell death by targeting DNA topoisomerase I (Top1), the enzyme that catalyzes changes in DNA topology. We found that CPT-producing plants, including Camptotheca acuminata, Ophiorrhiza pumila, and Ophiorrhiza liukiuensis, have Top1s with point mutations that confer resistance to CPT, suggesting the effect of an endogenous toxic metabolite on the evolution of the target cellular component. Three amino acid substitutions that contribute to CPT resistance were identified: Asn421Lys, Leu530Ile, and Asn722Ser (numbered according to human Top1). The substitution at position 722 is identical to that found in CPT-resistant human cancer cells. The other mutations have not been found to date in CPT-resistant human cancer cells; this predicts the possibility of occurrence of these mutations in CPT-resistant human cancer patients in the future. Furthermore, comparative analysis of Top1s of CPT-producing and nonproducing plants suggested that the former were partially primed for CPT resistance before CPT biosynthesis evolved. Our results demonstrate the molecular mechanism of self-resistance to endogenously produced toxic compounds and the possibility of adaptive coevolution between the CPT production system and its target Top1 in the producing plants.     

Modeling early bactericidal activity in murine tuberculosis provides insights into the activity of isoniazid and pyrazinamide

Standard tuberculosis (TB) treatment includes an initial regimen containing drugs that are both rapidly bactericidal (isoniazid) and sterilizing (rifampin and pyrazinamide), and ethambutol to help prevent the emergence of drug resistance. Antagonism between isoniazid and pyrazinamide has been demonstrated in a TB treatment mouse model. Because isoniazid's bactericidal activity is greatest during the initial two treatment days, we hypothesized that removing isoniazid after the second day would increase the effectiveness of the standard regimen. To test this hypothesis, we developed a mouse model to measure the early bactericidal activity (EBA) of drug regimens designed to analyze the essentiality of both isoniazid and pyrazinamide during the first 14 d of therapy. Our results clearly indicate that discontinuation of isoniazid after the second day of treatment increases the EBA of standard therapy in the mouse model, whereas omitting pyrazinamide during the first 14 d was detrimental. Substitution of moxifloxacin for isoniazid on day 3 did not increase the EBA compared with only removing isoniazid after day 2. Our data show that a mouse model can be used to analyze the EBA of TB drugs, and our findings support pursuing clinical trials to evaluate the possible benefit of removing isoniazid after the first 2 treatment days.     

Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.     

Intestinal synthesis and secretion of bile salts as an adaptation to developmental biliary atresia in the sea lamprey

Bile salt synthesis is a specialized liver function in vertebrates. Bile salts play diverse roles in digestion and signaling, and their homeostasis is maintained by controlling input (biosynthesis) and intestinal conservation. Patients with biliary atresia (i.e., obliteration of the biliary tree) suffer liver fibrosis and cirrhosis. In contrast, sea lamprey thrives despite developmental biliary atresia. We discovered that the sea lamprey adapts to biliary atresia through a unique mechanism of de novo synthesis and secretion of bile salts in intestine after developmental biliary atresia, in addition to known mechanisms, such as the reduction of bile salt synthesis in liver. During and after developmental biliary atresia, expression of cyp7a1 in intestine increased by more than 100-fold (P < 0.001), whereas in liver it decreased by the same magnitude (P < 0.001). Concurrently, bile salt pools changed in similar patterns and magnitudes in these two organs and the composition shifted from C24 bile alcohol sulfates to taurine-conjugated C24 bile acids. In addition, both in vivo and ex vivo experiments showed that aductular sea lamprey secreted taurocholic acid into its intestinal lumen. Our results indicate that the sea lamprey, a jawless vertebrate, may be in an evolutionarily transitional state where bile salt synthesis occurs in both liver and intestine. Understanding the molecular basis of these mechanisms may shed light on the evolution of bile salt synthesis and possible therapy for infant biliary atresia.     

In silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen

The growing resistance to current first-line antimalarial drugs represents a major health challenge. To facilitate the discovery of new antimalarials, we have implemented an efficient and robust high-throughput cell-based screen (1,536-well format) based on proliferation of Plasmodium falciparum (Pf) in erythrocytes. From a screen of approximately 1.7 million compounds, we identified a diverse collection of approximately 6,000 small molecules comprised of >530 distinct scaffolds, all of which show potent antimalarial activity (<1.25 microM). Most known antimalarials were identified in this screen, thus validating our approach. In addition, we identified many novel chemical scaffolds, which likely act through both known and novel pathways. We further show that in some cases the mechanism of action of these antimalarials can be determined by in silico compound activity profiling. This method uses large datasets from unrelated cellular and biochemical screens and the guilt-by-association principle to predict which cellular pathway and/or protein target is being inhibited by select compounds. In addition, the screening method has the potential to provide the malaria community with many new starting points for the development of biological probes and drugs with novel antiparasitic activities.     

Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

The torque of the bacterial flagellum is generated by the rotor–stator interaction coupled with the ion flow through the channel in the stator. Anchoring the stator unit to the peptidoglycan layer with proper orientation around the rotor is believed to be essential for smooth rotation of the flagellar motor. The stator unit of the sodium-driven flagellar motor of Vibrio is composed of PomA and PomB, and is thought to be fixed to the peptidoglycan layer and the T-ring by the C-terminal periplasmic region of PomB. Here, we report the crystal structure of a C-terminal fragment of PomB (PomB_C) at 2.0-Å resolution, and the structure suggests a conformational change in the N-terminal region of PomB_C for anchoring the stator. On the basis of the structure, we designed double-Cys replaced mutants of PomB for in vivo disulfide cross-linking experiments and examined their motility. The motility can be controlled reproducibly by reducing reagent. The results of these experiments suggest that the N-terminal disordered region (121–153) and following the N-terminal two-thirds of α1(154-164) in PomB_C changes its conformation to form a functional stator around the rotor. The cross-linking did not affect the localization of the stator nor the ion conductivity, suggesting that the conformational change occurs in the final step of the stator assembly around the rotor.Motility is a fundamental function for bacterial survival in environmental change. The bacterial flagellum is a large filamentous macromolecular assembly for motility. A reversible motor, which is embedded at the base of each filament and powered by the electrochemical gradient of the coupling ion (H⁺ or Na⁺), rotates the filament like a screw to drive the cell toward a favorable environment (1–3).The torque generation unit of the flagellar motor consists of a rotor and stators. A dozen stators surround the rotor and work together, although a single stator can generate torque. Stepwise drops and restorations of the rotational speed have been reported, suggesting that each stator is able to associate into or dissociate from the functioning motor (4–6). The fluorescent photo-bleaching study has revealed a rapid turnover of the stator in the rotating motor (7). The rotor is made up of the MS-ring and the C-ring. The C-ring is formed just beneath the MS-ring and is essential for torque generation and switching the direction of rotation.The stator is a hetero-hexameric complex of two membrane proteins, A and B, with a stoichiometry of A₄B₂ (8, 9). The stator consists of MotA and MotB for the H⁺-driven motor of Escherichia coli and Salmonella enterica, and PomA and PomB for the Na⁺-driven motor of Vibrio and Shewanella spp. (10). The torque is generated by the interaction between rotor protein FliG in C-ring and the stator A subunit coupled with the ion flow through the channel in the stator (1). The stator B subunit consists of a short cytoplasmic segment, a single transmembrane helix and a C-terminal periplasmic region (11) (Fig. 1A and Fig. S1A). The transmembrane helix has an aspartate residue essential for ion translocation across the cytoplasmic membrane, and forms an ion channel with the transmembrane region of the A subunits (12–14). The periplasmic region of the B subunit (B_C) shows a sequence similarity to OmpA-like proteins and contains a putative peptidoglycan binding motif (PGB motif), which is thought to anchor the stator to the peptidoglycan layer (PG layer) (15). Many nonmotile mutation sites were found within or contiguous to the PGB motif, suggesting that proper anchoring of the stator to the PG layer is important to the motor function (16, 17). Ion-conductivity of the stator is coupled to its installation in the motor. Overproduction of the stator does not arrest cell growth, indicating that the ion-conductivity of the stator is activated only when it is installed around the rotor (13). A short segment following the transmembrane region of the B subunit, called the plug, is thought to suppress the ion leakage until the stator is incorporated into the motor (18, 19). However, it is not clear how the activation of the ion channel is coupled to the stator incorporation.Open in a separate windowFig. 1.Structure of the C-terminal fragment of PomB. (A) Schematic representation of the full-length PomB, PomB_ΔL, and the PomB_C variants used for crystallization. PomB_ΔL is the smallest construct that forms a stable functional stator unit derived from a systematic deletion experiment (19). The PEM region is highlighted in blue. Asp-24, which is essential for ion translocation across the cytoplasmic membrane, is shown as a solid red circle. (B) Ribbon representation of PomB_C5 dimer. One subunit is color coded from blue to red from the N to the C terminus, and the other is painted magenta. (C) Structure of Salmonella MotB_C2 dimer (PDB ID code 2ZVY) (21). The two subunits are colored in cyan and magenta.A systematic deletion study of the periplasmic region of MotB demonstrated that a MotB mutant with the deletion of residues 51–100 still forms a stable active stator. Moreover, another MotB mutant with the deletion of residues 51–110 and 271–309 is also functional, although unstable (20), indicating that the essential periplasmic region for motor function is only the residues 111–270, named periplasmic region essential for motility (PEM). The deleted dispensable region seems to work as a linker that connects the transmembrane segment and the PEM. Recently, the structure of a periplasmic fragment of Salmonella MotB (St-MotB_C) that covers the PEM has been solved (21). St-MotB_C forms a homo-dimer, the subunit of which is composed of a single OmpA-like domain, which contains the putative PGB site, with N-terminal long and short helices. The distance between the PG layer and the surface of the hydrophobic core layer of the cytoplasmic membrane is about 100 Å (22), whereas the height of St-MotB_C is only 50 Å. Thus, a long extension of MotB_C is expected to anchor the stator to the PG layer, but it is still obscure what conformational changes occur.The sodium-driven Vibrio motor rotates up to 1,700 Hz (23), in contrast to the proton-driven motors of E. coli and Salmonella, which typically revolve up to 300 Hz (24). The basal body of the Vibrio motor has two unique ring structures, the T-ring and the H-ring (25, 26). These extra rings are thought to reinforce the motor to resist the high-speed rotation. Recent structural study demonstrated that FlgT acts as an assembly base or scaffold for both the ring structures (27). The T-ring is made up of MotX and MotY, and is located beneath the P-ring, which is a part of a bushing structure for the rod, thereby believed not to rotate. The T-ring is an essential component to incorporate the stator into the motor (25). The periplasmic region of PomB is likely to bind to MotX (28), and MotX is connected to the basal body through the N-terminal domain of MotY (29). Thus, the stator of the sodium-driven motor is tightly fixed not only to the PG layer but also to the basal body through the interaction between PomB and the T-ring. Despite the rigid anchoring structure, the stator of the sodium-driven motor still shows a dynamic behavior dependent on the binding of sodium ion to PomB (30). In this study, we purified and crystallized various fragments of PomB_C (Table S1) and solved the structure of PomB_C4 (121–315), which covers the PEM of PomB, and PomB_C5 (135–315) (Fig. 1A). The crystal structures, following in vivo disulfide cross-link studies and motility assay, revealed novel insights into the molecular mechanism of the stator assembly and activation of the sodium-driven flagellar motor.     

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.Although the ubiquitin proteasome pathway plays essential roles in eukaryotes (reviewed in refs. 1 and 2), most bacterial species do not have proteasome systems and instead degrade proteins using ATP-dependent proteases like ClpP, Lon, and HslUV (reviewed in refs. 3 and 4). However, bacteria of the orders Actinomycetales and Nitrospirales also encode proteasomes that are structurally highly similar to eukaryotic and archaeal proteasomes (reviewed in refs. 5 and 6). Importantly, the human pathogen Mycobacterium tuberculosis (Mtb), an Actinomycete, requires proteasomal function to cause lethal infections in mice (7). Ablation of proteasomal degradation sensitizes bacteria to nitric oxide, an antimicrobial free radical made by macrophages and other cell types, and attenuates bacterial growth in mice (7–9). The potential to target persistent or latent bacteria has made the Mtb proteasome system a prioritized target for the development of antituberculosis drugs (10, 11). Indeed, Mtb-specific proteasome inhibitors have been identified that may provide a promising lead for new drugs to treat tuberculosis (12, 13).There are numerous similarities and differences between eukaryotic and bacterial proteasomes. The 20S proteasome core particle (20S CP), which consists of two seven-membered β-rings between two seven-membered α-rings, is highly conserved structurally between prokaryotes and eukaryotes (14–16). However, the accessory factors that associate with the 20S CPs quickly diverge among the domains of life. Both bacteria and eukaryotes use a covalent small protein modification to mark substrate proteins for degradation; however, the eukaryotic ubiquitin tag is a well-folded protein whereas the Mtb Pup (prokaryotic ubiquitin-like protein) tag is intrinsically disordered (17, 18). Furthermore, degradation of ubiquitylated proteins by eukaryotic 20S CPs largely relies on a complex regulatory particle that caps one or both ends of the 20S CP and includes a heterohexameric ring of adenosine triphosphatases (ATPases) for substrate recognition and unfolding (reviewed in refs. 19 and 20). In contrast, the mycobacterial 20S CP uses a homohexameric ATPase ring called Mpa (mycobacterial proteasome ATPase) for both the recognition and unfolding of pupylated proteins (18, 21, 22).In addition to the ATPase activators, proteolysis by eukaryotic proteasomes can also be stimulated by several ATP-independent factors, such as the 11S activators PA26 and PA28, as well as Blm10 (23–28). We and another group recently discovered that Mtb has an analogous factor encoded by Rv3780 that we call PafE (proteasome accessory factor E; also known as Bpa for bacterial proteasome activator), which stimulates the degradation of small peptides and β-casein in vitro (29, 30). Both studies also showed that a carboxyl (C)-terminal glycine-glutamine-tyrosine-leucine (GQYL) motif is essential for interacting with and activating 20S CPs, and the penultimate tyrosine residue contributes to activation similarly to tyrosines observed in the “HbYX” (hydrophobic-tyrosine-any amino acid) motif in other characterized proteasome activators (reviewed in ref. 28). Our work further showed that PafE promotes the degradation of at least one native Mtb protein substrate, heat-shock protein repressor (HspR), and that an Mtb pafE mutant is sensitive to heat shock and is attenuated for growth in mice (30). Importantly, PafE-mediated degradation does not require pupylation. Thus, there appear to be at least two independent paths for targeting proteins to the mycobacterial proteasome for degradation.Like the eukaryotic 11S proteasome activators, PafE does not require ATP to stimulate proteolysis. However, it was unknown if PafE formed heptameric complexes like PA26 or PA28. In this work, we show that PafE monomers assume a four-helix bundle structure that is similar to that found in 11S activators, but assemble differently into an unprecedented dodecameric ring structure with 12-fold symmetry. We used isothermal titration calorimetry, cryo-electron microscopy (cryo-EM), and X-ray crystallography to analyze interactions between PafE and 20S core particles, and found that PafE binding induces a larger gate-opening change than has been described for other organisms. We also found that PafE has an extended C terminus that limits the ability of PafE to activate proteasomal degradation in vitro and in vivo.     

Ionic basis of ischemia-induced bradycardia in the rabbit sinoatrial node

To investigate the basis of ischemia-induced bradycardia (<60 beats/min), we isolated pacemaker cells from the rabbit sinoatrial node and exposed them to ischemic-like conditions, including omission of glucose, pH 6.6, and either 5.4 or 10 mM KCl to evaluate the role of increased serum [K]. A perforated-patch technique was employed to test the hypothesis that the arrhythmia is caused by attenuation of inward currents that contribute to the diastolic depolarization. After exposure to "ischemic" Tyrode containing 5.4 mM KCl, the pacemaker cells exhibited 13% slower beat rates and action potentials with 6-mV greater overshoots and 44% longer durations. In contrast, after exposure to "ischemic" Tyrode containing 10 mM KCl, the pacemaker cells exhibited a 7-mV depolarization of the maximum diastolic potential but no significant change in the overshoot. Beat rates were slowed by 43%, and the action potentials were prolonged by 46%. "Ischemic" Tyrode containing 5.4 mM KCl increased L-type Ca current, decreased T-type Ca current and reduced Ni-sensitive inward current tails (presumably Na-Ca exchange current), even after treatment with 40 muM ryanodine to block Ca release from the sarcoplasmic reticulum. "Ischemic" Tyrode containing 10 mM KCl increased hyperpolarization-activated inward current at diastolic potentials and reduced the slowly activating component, but not the rapidly activating component, of delayed rectifier K current. Our results suggest that reductions of inward Na-Ca exchange current and T-type Ca current contribute to "ischemia"-induced "bradycardia" in sinoatrial node pacemaker cells.     

Structural and functional analysis of the yeast N-acetyltransferase Mpr1 involved in oxidative stress tolerance via proline metabolism

Mpr1 (sigma1278b gene for proline-analog resistance 1), which was originally isolated as N-acetyltransferase detoxifying the proline analog l-azetidine-2-carboxylate, protects yeast cells from various oxidative stresses. Mpr1 mediates the l-proline and l-arginine metabolism by acetylating l-Δ¹-pyrroline-5-carboxylate, leading to the l-arginine–dependent production of nitric oxide, which confers oxidative stress tolerance. Mpr1 belongs to the Gcn5-related N-acetyltransferase (GNAT) superfamily, but exhibits poor sequence homology with the GNAT enzymes and unique substrate specificity. Here, we present the X-ray crystal structure of Mpr1 and its complex with the substrate cis-4-hydroxy-l-proline at 1.9 and 2.3 Å resolution, respectively. Mpr1 is folded into α/β-structure with eight-stranded mixed β-sheets and six α-helices. The substrate binds to Asn135 and the backbone amide of Asn172 and Leu173, and the predicted acetyl-CoA–binding site is located near the backbone amide of Phe138 and the side chain of Asn178. Alanine substitution of Asn178, which can interact with the sulfur of acetyl-CoA, caused a large reduction in the apparent k_cat value. The replacement of Asn135 led to a remarkable increase in the apparent K_m value. These results indicate that Asn178 and Asn135 play an important role in catalysis and substrate recognition, respectively. Such a catalytic mechanism has not been reported in the GNAT proteins. Importantly, the amino acid substitutions in these residues increased the l-Δ¹-pyrroline-5-carboxylate level in yeast cells exposed to heat stress, indicating that these residues are also crucial for its physiological functions. These studies provide some benefits of Mpr1 applications, such as the breeding of industrial yeasts and the development of antifungal drugs.     

Ontogeny and distribution of cholecystokinin-immuno reactive cells in the digestive tract of sharpsnout sea bream, Diplodus puntazzo (Cetti, 1777), during larval development

The appearance and regional distribution of cholecystokinin-immuno reactive cells (CCK-IR) in the developing gut of larval Diplodus puntazzo were studied by means of immunohistochemistry, with the aim of understanding the role of this peptide hormone in the acquisition of digestive capacity. Immunohistochemical reaction showed CCK-IR cells from 10 days after hatching (DAH), near the pyloric sphincter and past the first bend in the midgut, as well as in the hindgut. At 25 DAH CCK-IR cells were scattered throughout the midgut, as well as in the hindgut. Since gastric glands appeared at 30 DAH, CCK-IR cells were most abundant in the anterior midgut, near and including the pyloric caeca, and just afore the ileo-rectal sphincter in the posterior midgut, as well as in the hindgut. In older larvae (39 DAH), CCK-IR cells were mainly distributed in the anterior midgut, including the pyloric caeca, as well as in the hindgut. No CCK-IR cells were detected in the foregut at any stage. The distribution pattern of CCK-IR cells differed from other species which also possess a rotated gut as D. puntazzo. In fact, although cells were abundant in regions where the ingested food is retained, so that they can be stimulated to modulating the release of digestive enzymes, a large number of cells occurred also in the hindgut.