KLHL12 can form large COPII structures in the absence of CUL3 neddylation.

CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric-a-brac, Tramtrack, and Broad-complex (BTB)-domain proteins. KLHL12, a BTB-domain protein, is suggested to play an essential role in the export of large cargo molecules such as procollagen from the endoplasmic reticulum (ER). CRL3KLHL12 monoubiquitylates SEC31, leading to an increase in COPII vesicle dimension. Enlarged COPII vesicles can accommodate procollagen molecules. Thus, CRL3KLHL12 is essential for the assembly of large COPII structures and collagen secretion. CRL3s are activated by CUL3 neddylation. Here, we evaluated the importance of CUL3 neddylation in COPII assembly and collagen secretion. Unexpectedly, the assembly of large COPII-KLHL12 structures persisted and cellular collagen levels decreased on treatment with MLN4924, a potent inhibitor of NEDD8-activating enzyme. When we introduced mutations into KLHL12 at the CUL3 interface, these KLHL12 variants did not interact with neddylated CUL3, but one of them (Mut A) still supported large COPII-KLHL12 structures. Overexpression of wild-type KLHL12, but not Mut A, lowered cellular collagen levels most likely via lysosomal degradation. Our results suggest that CUL3 neddylation is not necessary for the formation of large COPII-KLHL12 structures, but active CRL3KLHL12 contributes to the maintenance of collagen levels in the cell.

Evaluation of antimicrobial, antioxidant and cytotoxic properties of bioactive compounds produced from endophytic fungi of Himalayan yew ( Taxus wallichiana) in Nepal.

Background: Endophytic fungi are largely underexplored in the discovery of natural bioactive products though being rich sources of novel compounds with promising pharmaceutical potential. In this study, Taxus wallichiana, which has huge medicinal value, was investigated for its endophytic diversity and capability to produce bioactive secondary metabolites by analyzing antioxidant, antimicrobial and cytotoxic properties. Methods: The endophytes were identified by ITS-PCR using genomic DNA samples. The secondary metabolites were extracted by solvent extraction method using ethyl acetate. The antioxidant activity was analyzed by Thin Layer Chromatography, Total Phenol Content (TPC), Total Flavonoid Content (TFC) and DPPH assay, and the antimicrobial activity was analyzed by agar-well diffusion method. Brine shrimp lethality assay was used to analyze the cytotoxicity of the fungal extracts. Results: Out of 16 different Taxus trees sampled from different locations of Dhorpatan, 13 distinctive endophytic fungi were isolated and grouped into 9 different genera: Bjerkandera, Trichoderma, Preussia, Botrytis, Arthrinium, Alternaria, Cladosporium, Sporormiella and Daldinia. The ethyl acetate extracts isolated from three endophytic fungi: Alternaria alternata, Cladosporium cladosporioides and Alternaria brassicae showed significant TPC values of 204±6.144, 312.3±2.147 and 152.7±4.958µg GAE/mg of dry extract, respectively, and TFC values of 177.9±2.911, 644.1±4.202 and 96.38±3.851µg RE/mg of dry extract, respectively. Furthermore, these three extracts showed a dose dependent radical scavenging activity with IC 50 concentration of 22.85, 22.15 and 23.001 µg/ml, respectively. The extracts of C. cladosporioides and A. brassicae also showed promising antimicrobial activity against Escherichia coli, Staphylococcus aureus and Bacillus subtilis with a minimum inhibitory concentration of 250µg/ml for all bacteria. Both the samples showed cytotoxic property against shrimp nauplii with LC 50 of 104.2 and 125.9µg/ml, respectively. Conclusions: The crude fungal extracts obtained from endophytes: A. alternata, C. cladosporioides and A. brassicae upon purification and further identification of the bioactive compounds can be a fascinating source for novel pharmaceutical agents.