Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase

Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors(ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Strikingly, loss of this protection results in the loss of K63-linked di-ubiquitin from the adaptors and diverts the adaptors for K48-linked poly-ubiquitination and proteasome-mediated degradation. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.

Distinct Regulations of HO-1 Gene Expression for Stress Response and Substrate Induction

Heme oxygenase-1 (HO-1) is the key enzyme for heme catabolism and cytoprotection. Whereas HO-1 gene expression in response to various stresses has been investigated extensively, the precise mechanisms by which HO-1 gene expression is regulated by the HO-1 substrate heme remain elusive. To systematically examine whether stress-mediated induction and substrate-mediated induction of HO-1 utilize similar or distinct regulatory pathways, we developed an HO-1-DsRed-knock-in reporter mouse in which the HO-1 gene is floxed by loxP sites and the DsRed gene has been inserted. Myeloid lineage-specific recombination of the floxed locus led to fluorescence derived from expression of the HO-1-DsRed fusion protein in peritoneal macrophages. We also challenged general recombination of the locus and generated mice harboring heterozygous recombinant alleles, which enabled us to monitor HO-1-DsRed expression in the whole body in vivo and ex vivo . HO-1 inducers upregulated HO-1-DsRed expression in myeloid lineage cells isolated from the mice. Notably, analyses of peritoneal macrophages from HO-1-DsRed mice lacking NRF2, a major regulator of the oxidative/electrophilic stress response, led us to identify NRF2-dependent stress response-mediated HO-1 induction and NRF2-independent substrate-mediated HO-1 induction. Thus, the HO-1 gene is subjected to at least two distinct levels of regulation, and the available lines of evidence suggest that substrate induction in peritoneal macrophages is independent of CNC family-based regulation.

Screening of bacterial mutants for improved production of amylase

Amylase is one of the most widely used enzymes in many industrial sectors (starch decomposition, bakery, fermentation, biofuel, detergent, paper, textile, etc.), thus isolating pure cultures of amylase producing microorganisms from natural sources and improving their activity is important in biotechnology. Enzyme preparations with high activity can be obtained only by improved synthesis of biologically active substances of microorganisms by mutagenesis. In the present investigation was enhanced the amylase productivity of some Bacillus sp. (assigned as 1,2,3) isolated from soil sample by substrate induction and mutagenesis. 3 isolates are subcultured in the medium with starch (10 mg/ml) as only carbon source, to improve amylase production. Enzyme activity of parental strains increased 50-58% by substrate induction. The highest productive strain (Bacillus sp. 2) screened and selected. Then it was subjected to 4 period mutagenesis using UV irradiation and еthidium bromide. Amylase activity of Bacillus sp. 2 increased after first period of mutagenesis to:0.305, second:0.514, third:0.579 and fourth:0.592 U/ml. In the result our experiment, amylase activity of parental strain increased from 0.138 to 0.592 U/ml, which means 4.3 times more enzyme. Амилаза нийлэгжүүлэгч бактерийн мутант омог гарган авсан дүн Хураангуй: Амилаза нь үйлдвэрлэлийн олон салбарт (цардуул задлах, талх нарийн боов, исгэлт, биотүлш, угаалгын нунтаг, цаас, нэхмэл гэх мэт) өргөнөөр хэрэглэгддэг фермент тул түүнийг нийлэгжүүлэгч бичил биетний өсгөврийг байгалийн эх үүсвэрээс ялган авч, идэвхийг нь сайжруулах явдал биотехнологийн салбарт чухал ач холбогдолтой юм. Мутагенезийн аргаар бичил биетний биологийн идэвхтэй бодисын нийлэгжилтийг сайжруулснаар ферментийн бэлдмэл гарган авах боломжтой болно. Судалгаагаар Монгол орны биосферээс ялган авсан цардуул задлах идэвхтэй бактерийн цэвэр өсгөврийн амилаза ферментийн идэвхийг субстратаар өдөөх болон мутагенезийн аргаар сайжруулахыг зорив. Хөрснөөс ялган авсан цардуул задлах идэвхтэй цэвэр өсгөврүүдийг (Bacillus sp. 1, 2, 3) нүүрс-усны эх үүсвэрээр зөвхөн цардуул агуулсан (10 г/л) тэжээлт орчинд өсгөвөрлөх замаар амилаза ферментийн идэвхийг нэмэгдүүлэх туршилт хийж үр дүнг үндэслэн хамгийн идэвхтэй нэг өсгөврийг сонгон шалгаруулж, хэт ягаан туяа, этидиум бромид, хэт ягаан туяа, этидиум бромид гэсэн дарааллаар зориудын мутагенезид 4 үе шаттайгаар оруулав. Субстратаар өдөөхөд бактерийн амилаза ферментийн идэвх анхдагч өсгөврийнхөөс 50-58 хувиар нэмэгдсэн. Bacillus sp. 2 өсгөврийг сонгон шалгаруулж, мутагенезид оруулахад амилаза ферментийн идэвх нь I шатны мутагенезээр:0,305, II-оор:0,514, III-аар:0,579, IV-өөр:0,592 н/мл болж нэмэгдэв. Бидний судалгааны үр дүнд байгалийн анхдагч өсгөврийн (Bacillus sp. 2) амилаза ферментийн идэвх 0,138-аас 0,592 н/мл хүртэл буюу 4,3 дахин нэмэгдсэн байна. Түлхүүр үг: амилаза, Bacillus sp., субстратын өдөөлт, UV-мутагенез, EtBr- мутангенез