Survey of the Proteolytic Activities Degrading the Kunitz Trypsin Inhibitor and Glycinin in Germinating Soybeans (Glycine max)

The cotyledons of the soybean (Glycine max [L.] Merrill cv Amsoy 71) were examined for proteolytic activities capable of degrading soybean seed proteins. Three distinct activities were identified that attack the native Kunitz soybean trypsin inhibitor of Amsoy 71, Ti^a. Protease K1 cleaves Ti^a to Ti^a_m, the inhibitor form lacking the five carboxyl-terminal amino acid residues relative to Ti^a. Protease K1 is a cysteine protease that peaks in activity on day 4 after the beginning of imbibition, with maximal activity toward Ti^a at pH 4. The characteristics of protease K1 are consistent with the involvement of this protease in the initial proteolysis of the Kunitz inhibitor during germination. Protease K2 also degrades Ti^a at pH 4 but produces no electrophoretically recognizable products. It peaks later in seedling growth, at day 8. Protease K3 degrades Ti^a to products other than Ti^a_m. However, it is active at pH 8. Two proteolytic activities were identified that attack the major storage protein, glycinin. Protease G1 (which appears by 4 days after imbibition) specifically cleaves the acidic polypeptides of glycinin at pH 4, yielding a product approximately 1.5 kilodaltons smaller. Protease G1 is inhibited by metal chelators as well as by reagents reactive toward thiols. Protease G2 also degrades the glycinin acidic chains at pH 4, but without the appearance of electrophoretically recognizable products. Protease G2, while present at low levels in the dry seed, is found primarily in the cotyledons after 8 days of growth.     

Characterization of the Major Protease Involved in the Soybean beta-Conglycinin Storage Protein Mobilization

Protease C1, the protease responsible for the initial degradation of the α′ and α subunits of the soybean β-conglycinin storage protein (Glycine max [L.] Merrill), has been purified. The enzyme was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a molecular weight of 70,000 and a pH optimum of 3.5 to 4.5. Susceptibility to protease inhibitors indicates that protease C1 is a serine protease. Study of the proteolytic intermediates generated suggests that the cleavage of the α′ and α subunits of β-conglycinin by protease C1 results in intermediates that are 1 or 2 kilodaltons smaller than the native α′ and α subunits. Following that, a succession of intermediates exhibiting molecular masses of 70.0 and 58.0 kilodaltons, then 63.0, 61.0, 55.0, and 53.5 kilodaltons, are observed. A 50.0- and a 48.0- kilodalton intermediate are the final products of protease C1 action. Comparison of these intermediates with the prominent anti-β-conglycinin cross-reacting bands that increase during the first few days of germination and early growth show that protease C1 plays an important physiological role, but not an exclusive one, in the living plant.     

Rapid release of protease inhibitors from soybeans: immunochemical quantitation and parallels with lectins

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.     

Differential expression of kunitz and bowman-birk soybean proteinase inhibitors in plant and callus tissue

Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred.     

In vivo and in vitro effects of wheat germ agglutinin and Bowman-Birk soybean trypsin inhibitor,two potential transgene products,on midgut esterase and protease activities from Apis mellifera

Wheat germ agglutinin (WGA) and Bowman-Birk soybean trypsin inhibitor represent potential transgene products for inducing pest resistance in plants. The effects of these molecules were studied on midgut esterase and protease activities from Apis mellifera L., a major insect pollinator. Trypsin inhibitor and WGA did not exhibit an acute toxicity in A. mellifera. In vivo, trypsin inhibitor caused a decrease in the amount of trypsin activity and did not have a significant effect on esterase activity. In vitro, trypsin inhibitor inhibited about 80% of non-specific protease activity and 100% of trypsin activity. In vivo, WGA at high concentration in food (1 mg/ml) elicited a large decrease in trypsin activity and did not have a significant effect on esterase activity. In vitro, WGA did not have any significant effect on trypsin and non-specific protease activities but slightly activated esterase activity.     

Structural basis for the in vitro efficacy of nirmatrelvir against SARS-CoV-2 variants

The COVID-19 pandemic continues to be a public health threat with emerging variants of SARS-CoV-2. Nirmatrelvir (PF-07321332) is a reversible, covalent inhibitor targeting the main protease (M^pro) of SARS-CoV-2 and the active protease inhibitor in PAXLOVID (nirmatrelvir tablets and ritonavir tablets). However, the efficacy of nirmatrelvir is underdetermined against evolving SARS-CoV-2 variants. Here, we evaluated the in vitro catalytic activity and potency of nirmatrelvir against the M^pro of prevalent variants of concern (VOCs) or variants of interest (VOIs): Alpha (α, B.1.1.7), Beta (β, B.1.351), Delta (δ, B1.617.2), Gamma (γ, P.1), Lambda (λ, B.1.1.1.37/C37), Omicron (ο, B.1.1.529), as well as the original Washington or wildtype strain. These VOCs/VOIs carry prevalent mutations at varying frequencies in the M^pro specifically for α, β, γ (K90R), λ (G15S), and ο (P132H). In vitro biochemical enzymatic assay characterization of the enzyme kinetics of the mutant M^pros demonstrates that they are catalytically comparable to wildtype. We found that nirmatrelvir has similar potency against each mutant M^pro including P132H that is observed in the Omicron variant with a Ki of 0.635 nM as compared to a Ki of 0.933 nM for wildtype. The molecular basis for these observations were provided by solution-phase structural dynamics and structural determination of nirmatrelvir bound to the ο, λ, and β M^pro at 1.63 to 2.09 Å resolution. These in vitro data suggest that PAXLOVID has the potential to maintain plasma concentrations of nirmatrelvir many-fold times higher than the amount required to stop the SARS-CoV-2 VOC/VOI, including Omicron, from replicating in cells.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE

The umr7–1 mutation, previously identified in a set of mutants that had been selected for defective UV-induced mutagenesis at CAN1, affects other cellular functions, including many of those regulated by the mating-type locus (MAT) in heterothallic Saccharomyces cerevisiae. The recessive umr7–1 allele, mapping approximately 20 cM distal to thr4 on chromosome III, causes clumpy growth in both a and α cells and has no apparent effect on a mating functions. However, α umr7 meiotic segregants fail to express several α-specific functions (e.g., high-frequency conjugation with a strains, secretion of the hormone α-factor and response to the hormone a-factor). In addition, α umr7 cells exhibit some a-specific characteristics, such as the barrier phenotype (Bar⁺) that prevents diffusion of α-factor and an increased mating frequency with α strains. The most striking property of α umr7 strains is their altered morphology, in which mitotic cells develop an asymmetric pear shape, like that of normal a cells induced to form "shmoos" by interaction with α-factor. Some a/α-specific diploid functions are also affected by umr7; instead of polar budding patterns, a/α umr7/umr7 diploids have medial budding like a/a, α/α and haploid strains. Moreover, a/α umr7/umr7 diploids have lost the ability to sporulate and are Bar⁺ like a or a/a strains. Revertant studies indicate that umr7–1 is a single point mutation. The umr7 mutant fails to complement mutants of both tup1 (selected for deoxythymidine monophosphate utilization) and cyc9 (selected for high iso-2-cytochrome c levels), and all three isolates have similar genetic and phenotypic properties. It is suggested that the product of this gene plays some common central role in the complex regulation of the expression of both MAT-dependent and MAT-independent functions.     

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.

The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.

The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

     

Assimilate Unloading from Maize (Zea mays L.) Pedicel Tissues : I. Evidence for Regulation of Unloading by Cell Turgor

β-Glucan synthase activity in plant membranes can be markedly altered by a multiplicity of apparently unrelated factors. In pea epicotyl membranes it is enhanced by low and inhibited by high concentrations of added Ca²⁺, trypsin or soluble pea protease. Ca²⁺ stimulates preexisting synthase activity, particularly in the presence of polycations (spermidine), but protease treatments activate and, with time, inactivate synthase zymogen. Endogenous pea protease activity is also associated with washed pea membrane and appears to be responsible for the decay observed with time in the β-glucan synthase activity. Endogenous pea protease activity is inhibited by thiol inhibitors, e.g. iodoacetamide and Hg²⁺, and by a heat-stable peptide, molecular weight approximately 10,000, that is found in supernatants of pea extracts. These protease inhibitors have the capacity to protect β-glucan synthase activity from denaturation or its zymogen from activation due to endogenous or added protease activity. Evidence is described which supports the proposal that 1,4-β-glucan synthase is destroyed and possibly converted to 1,3-β-glucan synthase activity by protease action, and that the latter may then be greatly enhanced by Ca²⁺ and polycations.     

Evidence for Co-Dominance of the Homothallic Genes, HMalpha/hmalpha and HMa/hma, IN SACCHAROMYCES YEASTS

To investigate the dominance and recessiveness of the homothallism genes, HMα/hmα and HMa/hma, for mating-type conversion, we constructed hybrids with various configurations of the homothallic genes by fusion of protoplasts prepared from haploid strains having identical mating types. Eight different combinations of the homothallic genes were tested for their function by observing the mating and sporulation abilities of the fusion products. With few exceptions, nonmating and sporogenous fusion products were obtained from the following combinations: α HO hmα HMa + α ho hmα hma, α HO hmα HMa + α ho HMα hma, α HO hmα HMa + α ho HMα HMa, a HO HMα hma + a ho hmα hma, a HO HMα hma + a ho hmα HMa and a HO HMα hma + a ho HMα HMa. All the fusion products from the α HO hmα HMa + α ho hmα HMa and a HO HMα hma + a ho HMα hma combinations showed mating types identical to those of the respective haploid strains. These results clearly support the co-dominance of the HMα/hmα and HMa/hma alleles and indicate that the hmα allele has the same function as the HMa allele and that the hma allele has the same function as the HMα allele.     

Long-term Incubation with Proteasome Inhibitors (PIs) Induces IκBα Degradation via the Lysosomal Pathway in an IκB Kinase (IKK)-dependent and IKK-independent Manner

Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21^Waf1, and p27^Kip1. Moreover, the blocking of NF-κB nuclear translocation via the stabilization of IκB is an important mechanism of PI-induced apoptosis. However, we found that long-term incubation with PIs (PS-341 or MG132) increased NF-κB-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IκBα and its subsequent degradation via the alternative route, lysosome. Overexpression of the IκBα superrepressor (IκBα-SR) blocked PI-induced NF-κB activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FA-FMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IκBα degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IκBα degradation. Pretreatment with IKKβ specific inhibitor, SC-514, partially suppressed IκBα degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKKβ only delayed IκBα degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3β. Inactive GSK-3β accelerated PI-induced IκBα degradation. Overexpression of active GSK-3β (S9A) or knock-down of GSK-3β delayed PI-induced IκBα degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-κB, which is mediated by IκBα degradation via the lysosome in an IKK-dependent and IKK-independent manner.     

HV-BBI--a novel amphibian skin Bowman-Birk-like trypsin inhibitor

Here we describe the isolation of a novel C-terminally amidated octadecapeptide—SVIGCWTKSIPPRPCFVK-amide—that contains a disulphide loop between Cys⁵ and Cys¹⁵ that is consistent with a Bowman-Birk type protease inhibitor, from the skin secretion of the Chinese Bamboo odorous frog, Huia versabilis. Named HV-BBI, the peptide is encoded by a single precursor of 62 amino acid residues whose primary structure was deduced from cloned skin cDNA. The precursor exhibits the typical organization of that encoding an amphibian skin peptide with a highly-conserved signal peptide, an intervening acidic amino acid residue-rich domain and a single HV-BBI-encoding domain located towards the C-terminus. A synthetic replicate of HV-BBI, with the wild-type K (Lys-8) residue in the presumed P1 position, was found to be a potent inhibitor of trypsin with a K_i just slightly less than 19 nM. Substitution at this site with R (Arg) resulted in a significant reduction in potency (K_i 57 nM), whereas replacement of K with F (Phe) resulted in the complete abolition of trypsin inhibitory activity. Thus, HV-BBI is a potent inhibitor of trypsin and the lysyl (K) residue that occupies the P1 position appears to be optimal for potency of action against this protease.     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Isolation and partial characterization of two alkaline proteases of the greater wax moth Galleria mellonella (L.)

Two alkaline proteases were isolated from whole-body extracts of Galleria mellonella larvae. The two proteases were separated by cation-exchange chromatography on CM-Sepharose CL6B and further purified by gel filtration on Ultrogel ACA 54. The optimal pH of activity using Azocoll as substrate was 10.5 for protease P-1 and 11.2 for protease P-2. The molecular weights of the two enzymes determined by gel filtration were respectively 12,500 and 10,500. Protease P-1 was inhibited by soybean trypsin inhibitor, TPCK, TLCK and activated by non-ionic detergents. Protease P-2 was inhibited by soybean trypsin inhibitor, 4-aminobenzamidine, ovomucoid and activated by dithiothreitol. Both enzymes were partially inhibited by PMSF.Distribution studies suggested that the two proteases were digestive enzymes.     

Secondary-site binding of Glu-plasmin, Lys-plasmin and miniplasmin to fibrin

Active-site-inhibited plasmin was prepared by inhibition with d-valyl-l-phenylalanyl-l-lysylchloromethane or by bovine pancreatic trypsin inhibitor (Kunitz inhibitor). Active-site-inhibited Glu-plasmin binds far more strongly to fibrin than Glu-plasminogen [native human plasminogen with N-terminal glutamic acid (residues 1–790)]. This binding is decreased by α₂-plasmin inhibitor and tranexamic acid, and is, in the latter case, related to saturation of a strong lysine-binding site. In contrast, α₂-plasmin inhibitor and tranexamic acid have only weak effects on the binding of Glu-plasminogen to fibrin. This demonstrates that its strong lysine-binding site is of minor importance to its binding to fibrin. Active-site-inhibited Lys-plasmin and Lys-plasminogen (Glu-plasminogen lacking the N-terminal residues Glu¹–Lys⁷⁶, Glu¹–Arg⁶⁷ or Glu¹–Lys⁷⁷)display binding to fibrin similar to that of active-site inhibited Glu-plasmin. In addition, α₂-plasmin inhibitor or tranexamic acid similarly decrease their binding to fibrin. Glu-plasminogen and active-site-inhibited Glu-plasmin have the same gross conformation, and conversion into their respective Lys- forms produces a similar marked change in conformation [Violand, Sodetz & Castellino (1975) Arch. Biochem. Biophys. 170, 300–305]. Our results indicate that this change is not essential to the degree of binding to fibrin or to the effect of α₂-plasmin inhibitor and tranexamic acid on this binding. The conversion of miniplasminogen (Glu-plasminogen lacking the N-terminal residues Glu¹–Val⁴⁴¹) into active-site-inhibited miniplasmin makes no difference to the degree of binding to fibrin, which is similarly decreased by the addition of tranexamic acid and unaffected by α₂-plasmin inhibitor. Active-site-inhibited Glu-plasmin, Lys-plasmin and miniplasmin have lower fibrin-binding values in a plasma system than in a purified system. Results with miniplasmin(ogen) indicate that plasma proteins other than α₂-plasmin inhibitor and histidine-rich glycoprotein decrease the binding of plasmin(ogen) to fibrin.     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

DCM-Related Tropomyosin Mutants E40K/E54K Over-Inhibit the Actomyosin Interaction and Lead to a Decrease in the Number of Cycling Cross-Bridges

Two DCM mutants (E40K and E54K) of tropomyosin (Tm) were examined using the thin-filament extraction/reconstitu­tion technique. The effects of the Ca²⁺, ATP, phos­phate (Pi), and ADP concentrations on isometric tension and its transients were studied at 25°C, and the results were com­pared to those for the WT protein. Our results indicate that both E40K and E54K have a significantly lower T _HC (high Ca²⁺ ten­sion at pCa 4.66) (E40K: 1.21±0.06 T _a, ±SEM, N = 34; E54K: 1.24±0.07 T _a, N = 28), a significantly lower T _LC (low- Ca²⁺ tension at pCa 7.0) (E40K: 0.07±0.02 T _a, N = 34; E54K: 0.06±0.02 T _a, N = 28), and a significantly lower T _act (Ca²⁺ activatable tension) (T _act = T _HC–T_LC, E40K: 1.15±0.08 T _a, N = 34; E54K: 1.18±0.06 T _a, N = 28) than WT (T _HC = 1.53±0.07 T _a, T _LC = 0.12±0.01 T _a, T _act = 1.40±0.07 T _a, N = 25). All tensions were normalized to T _a ( = 13.9±0.8 kPa, N = 57), the ten­sion of actin-filament reconstituted cardiac fibers (myocardium) under the standard activating conditions. The Ca²⁺ sensitivity (pCa₅₀) of E40K (5.23±0.02, N = 34) and E54K (5.24±0.03, N = 28) was similar to that of the WT protein (5.26±0.03, N = 25). The cooper­a­tivity increased significantly in E54K (3.73±0.25, N = 28) compared to WT (2.80±0.17, N = 25). Seven kinetic constants were deduced using sinusoidal analysis at pCa 4.66. These results enabled us to calculate the cross-bridge distribution in the strongly attached states, and thereby deduce the force/cross-bridge. The results indicate that the force/cross-bridge is ∼15% less in E54K than WT, but remains similar to that of the WT protein in the case of E40K. We conclude that over-inhibition of the actomyosin interaction by E40K and E54K Tm mutants leads to a decreased force-generating ability at systole, which is the main mechanism underlying the early pathogenesis of DCM.     

Domain Organization of the Polymerizing Mannosyltransferases Involved in Synthesis of the Escherichia coli O8 and O9a Lipopolysaccharide O-antigens

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA^O9a contains two separable and functionally active domains, whereas WbdA^O8 possesses three. In WbdC^O9a and WbdB^O9a, substitution of the first Glu of the EX₇E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX₇E motif of the N-terminal WbdA^O9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA^O9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA^O9a possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.     

Selective autophagic receptor NbNBR1 prevents NbRFP1-mediated UPS-dependent degradation of βC1 to promote geminivirus infection

Autophagy is an evolutionarily conserved, lysosomal/vacuolar degradation mechanism that targets cell organelles and macromolecules. Autophagy and autophagy-related genes have been studied for their antiviral and pro-viral roles in virus-infected plants. Here, we demonstrate the pro-viral role of a selective autophagic receptor NbNBR1 in geminivirus-infected Nicotiana benthamiana plants. The βC1 protein encoded by tomato yellow leaf curl China betasatellite (TYLCCNB) that is associated with tomato yellow leaf curl China virus (TYLCCNV) enhanced the expression level of NbNBR1. Then NbNBR1 interacted with βC1 to form cytoplasmic granules. Interaction of NbNBR1 with βC1 could prevent degradation of βC1 by the NbRFP1, an E3 ligase. Overexpression of NbNBR1 in N. benthamiana plants increased βC1 accumulation and promoted virus infection. In contrast, silencing or knocking out NbNBR1 expression in N. benthamiana suppressed βC1 accumulation and inhibited virus infection. A single amino acid substitution in βC1 (βC1^K4A) abolished its interaction with NbNBR1, leading to a reduced level of βC1^K4A. The TYLCCNV/TYLCCNB^K4A mutant virus caused milder disease symptoms and accumulated much less viral genomic DNAs in the infected plants. Collectively, the results presented here show how a viral satellite-encoded protein hijacks host autophagic receptor NbNBR1 to form cytoplasmic granules to protect itself from NbRFP1-mediated degradation and facilitate viral infection.