Synthetic substrates for measuring activity of autophagy proteases: Autophagins (Atg4)

Atg4 cysteine proteases (autophagins) play crucial roles in autophagy by proteolytic activation of Atg8 paralogs for targeting to autophagic vesicles by lipid conjugation, as well as in subsequent deconjugation reactions. However, the means to measure the activity of autophagins is limited. Herein, we describe two novel substrates for autophagins suitable for a diversity of in vitro assays, including (i) fluorogenic tetrapeptide acetyl-Gly-L-Thr-L-Phe-Gly-AFC (Ac-GTFG-AFC) and (ii) a fusion protein comprised of the natural substrate LC3B appended to the N-terminus of phospholipase A₂ (LC3B-PLA₂), which upon cleavage releases active PLA₂ for fluorogenic assay. To generate the synthetic tetrapeptide substrate, the preferred tetrapeptide sequence recognized by autophagin-1/Atg4B was determined using a positional scanning combinatorial fluorogenic tetrapeptide library. With the LC3B-PLA₂ substrate, we show that mutation of the glycine proximal to the scissile bond in LC3B abolishes activity. Both substrates showed high specificity for recombinant purified autophagin-1/Atg4B compared to closely related proteases and the LC3B-PLA₂ substrate afforded substantially higher catalytic rates (k_cat/K_m 5.26 × 10⁵ M⁻¹/sec⁻¹) than Ac-GTFG-AFC peptide (0.92 M⁻¹/sec⁻¹), consistent with substrate-induced activation. Studies of autophagin-1 mutants were also performed, including the protease lacking a predicted autoinhibitory domain at residues 1 to 24 and lacking a regulatory loop at residues 259 to 262. The peptide and fusion protein substrates were also employed for measuring autophagin activity in cell lysates, showing a decrease in cells treated with autophagin-1/Atg4B siRNA or transfected with a plasmid encoding Atg4B (Cys74Ala) dominant-negative. Therefore, the synthetic substrates for autophagins reported here provide new research tools for studying autophagy.Key words: autophagin, fluorogenic assay, tetrapeptide, phospholipase A2, LC3     

Isolation of compound microsatellite markers for the endangered plant Neolitsea sericea (Lauraceae)

? Premise of the study: The development of compound microsatellite markers was conducted in Neolitsea sericea to investigate genetic diversity and population genetic structure of this endangered insular species. ? Methods and Results: Using the compound microsatellite marker technique, 10 compound microsatellite markers that were successfully amplified showed polymorphism when assessed in 55 individuals from two populations in East China and Japan. Overall, the number of alleles ranged from 3 to 17, with an average of 7.9 alleles per locus. In addition, these primers could be easily amplified in Neolitsea aurata var. paraciculata and N. aurata var. chekiangensis. ? Conclusions: The highly polymorphic markers developed and characterized in this study will be useful for population genetic studies of N. sericea.     

Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.     

Mutations in the Type II protein arginine methyltransferase AtPRMT5 result in pleiotropic developmental defects in Arabidopsis

Human PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) encodes a type II protein arginine (Arg) methyltransferase and its homologs in animals and yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe) are known to regulate RNA processing, signal transduction, and gene expression. However, PRMT5 homologs in higher plants have not yet been reported and the biological roles of these proteins in plant development remain elusive. Here, using conventional biochemical approaches, we purified a plant histone Arg methyltransferase from cauliflower (Brassica oleracea) that was nearly identical to AtPRMT5, an Arabidopsis (Arabidopsis thaliana) homolog of human PRMT5. AtPRMT5 methylated histone H4, H2A, and myelin basic protein in vitro. Western blot using symmetric dimethyl histone H4 Arg 3-specific antibody and thin-layer chromatography analysis demonstrated that AtPRMT5 is a type II enzyme. Mutations in AtPRMT5 caused pleiotropic developmental defects, including growth retardation, dark green and curled leaves, and FlOWERING LOCUS C (FLC)-dependent delayed flowering. Therefore, the type II protein Arg methyltransferase AtPRMT5 is involved in promotion of vegetative growth and FLC-dependent flowering time regulation in Arabidopsis.     

Fine mapping of S32(t), a new gene causing hybrid embryo sac sterility in a Chinese landrace rice (Oryza sativa L.)

Ketan Nangka, the donor of wide compatibility genes, showed sterility when crossed to Tuanguzao, a landrace rice from Yunnan province, China. Genetic and cytological analyses revealed that the semi-sterility was primarily caused by partial abortion of the embryo sac. Genome-wide analysis of the linkage map constructed from the backcross population of Tuanguzao/Ketan Nangka//Ketan Nangka identified two independent loci responsible for the hybrid sterility located on chromosomes 2 and 5, which explained 18.6 and 20.1% of phenotypic variance, respectively. The gene on chromosome 5 mapped to the previously reported sterility gene S31(t), while the gene on chromosome 2, a new hybrid sterility gene, was tentatively designated as S32(t). The BC₁F₂ was developed for further confirmation and fine mapping of S32(t). The gene S32(t) was precisely mapped to the same region as that detected in the BC₁F₁ but its position was narrowed down to an interval of about 1.9 cM between markers RM236 and RM12475. By assaying the recombinant events in the BC₁F₂, S32(t) was further narrowed down to a 64 kb region on the same PAC clone. Sequence analysis of this fragment revealed seven predicted open reading frames, four of which encoded known proteins and three encoded putative proteins. Further analyses showed that wide-compatibility variety Dular had neutral alleles at loci S31(t) and S32(t) that can overcome the sterilities caused by these two genes. These results are useful for map-based cloning of S32(t) and for marker-assisted transferring of the neutral allele in hybrid rice breeding.     

Two novel loci for pollen sterility in hybrids between the weedy strain Ludao and the Japonica variety Akihikari of rice (Oryza sativa L.)

Partial pollen sterility has been observed in hybrid progeny derived from a japonica cultivar, Akihikari and a weedy strain, Ludao, which naturally grows in Jiangsu province of east China. Cytological and histological analyses revealed that pollen abortion occurred largely at the bicellular pollen stage, primarily due to the gradual disaggregation of generative and vegetative cells. A genome-wide analysis was further carried out in a backcross population of Akihikari //Ludao/Akihikari using a total of 118 simple sequence repeat (SSR) markers and an expressed sequence tag (EST) marker distributed on the entire rice linkage map. Two loci controlling hybrid pollen sterility, designated as S33(t) and S34(t), were located on chromosomes 3 and 11, respectively. Both loci were putatively different from all the previously reported gametophyte genes and hybrid pollen sterility loci. Interaction between the Ludao and Akihikari alleles at each of the two loci resulted in reduction of fertility in the pollens carring the Ludao alleles. To map the precise location of the major locus, S33(t), we selected 165 plants of the backcross population with pollen fertility higher than 80.0%, and assayed the recombinant events surrounding the S33(t) locus using newly developed SSR markers. The S33(t) was delimited to an 86 kb region between SSR markers RM15621 and RM15627. Sequence analysis of this region indicated that there were ten open reading frames. These results will be valuable for cloning this gene and marker-assisted transferring of the corresponding neutral allele in rice breeding programs. Furthermore, the origin of the weedy strain Ludao is discussed.     

Quantitative trait loci mapping of resistance to Laodelphax striatellus (Homoptera: Delphacidae) in rice using recombinant inbred lines

Laodelphax striatellus Fallén (Homoptera: Delphacidae), is a serious pest in rice, Oryza sativa L., production. A mapping population consisting of 81 recombinant inbred lines (RILs), derived from a cross between japonica' Kinmaze' and indica' DV85' rice, was used to detect quantitative trait loci (QTLs) for the resistance to L. striatellus. Seedbox screening test (SST), antixenosis test, and antibiosis test were used to evaluate the resistance response of the two parents and 81 RILs to L. striatellus at the seedling stage, and composite interval mapping was used for QTL analysis. When the resistance was measured by SST method, two QTLs conferring resistance to L. striatellus were mapped on chromosome 11, namely, Qsbph11a and Qsbph11b, with log of odds scores 2.51 and 4.38, respectively. The two QTLs explained 16.62 and 27.78% of the phenotypic variance in this population, respectively. In total, three QTLs controlling antixenosis against L. striatellus were detected on chromosomes 3, 4, and 11, respectively, accounting for 37.5% of the total phenotypic variance. Two QTLs expressing antibiosis to L. striatellus were mapped on chromosomes 3 and 11, respectively, explaining 25.9% of the total phenotypic variance. The identified QTL located between markers XNpb202 and C1172 on chromosome 11 was detected repeatedly by three different screening methods; therefore, it may be important to confer the resistance to L. striatellus. Once confirmed in other mapping populations, these QTLs should be useful in breeding for resistance to L. striatellus by marker-assisted selection of different resistance genes in rice varieties.     

Defective alloreactive CD8 T cell function and memory response in allograft recipients in the absence of CD4 help

We have shown that alloreactive CD8 T cell activation may proceed via CD4-dependent and CD4-independent pathways, and that CD8 T cell activation in Ag-primed animals is independent of CD154 costimulation. In this report, we further analyzed the activation and function of alloreactive CD8 CTL effectors in CD4 knockout (KO) skin/cardiac allograft recipients. FACS analysis showed that alloreactive CD8 T cells were activated at a significantly reduced level in CD4 KO mice. Importantly, these helpless CD8 T cells failed to develop CD154 blockade resistance following reactivation by the same alloantigen, indicative of defective memory formation. Only transient CD4 help was required, as short-term CD4 blockade at the time of first skin graft challenge only delayed alloreactive CD8 activation, without affecting the CD8 T cell memory response to a second skin graft. Moreover, postoperative CD4 blockade had no effect on alloreactive CD8 activation. Alloreactive CD8 cells generated in the absence of CD4 help exhibited decreased effector responses. Interestingly, intragraft induction of T cell-targeted chemokines early after transplant was also dependent on CD4 help, as the induction kinetics of CXCL9 and CCL5 in CD4 KO recipients was significantly delayed, coupled with similarly delayed infiltration by CD3/CD8 cells. Remarkably, helpless CD8 cells ultimately entering the graft still displayed significantly diminished T cell effector molecules (IFN-gamma, granzyme B). Thus, CD4 help is critical for alloreactive CD8 activation, function, and memory formation.