Ginsenoside CK targeting KEAP1-DGR/Kelch domain disrupts the binding between KEAP1 and NRF2-DLG motif to ameliorate oxidative stress damage.

BACKGROUND: Panax ginseng and Panax notoginseng as traditional Chinese medicines, are widely used in the treatment of qi deficiency, viral or bacterial infection, inflammation and cancer. Ginsenoside CK, an active metabolite of protopanoxadiol among the ginseng saponins, has been shown in previous studies to improve the organism's oxidative balance by regulating the KEAP1-NRF2/ARE pathway, thus slowing the progression of diseases. However, the specific targets and mechanisms of CK in improving oxidative stress remain unclear. PURPOSE: The aim of this study was to determine the potential therapeutic targets and molecular mechanisms of CK in improving oxidative stress injury both in vitro and in vivo. METHODS: LPS was used to induce oxidative damage in RAW 264.7 cells to evaluate the regulatory effects of CK on the KEAP1-NRF2/ARE pathway. Drug affinity responsive target stability technology (DARTS) combined with proteomics was employed to identify CK's potential target proteins. CK functional probe were designed to analyze the target protein using click chemistry. Furthermore, small molecule and protein interaction technologies were used to verify the mechanism, and computer dynamic simulation technology was used to analyze the interaction sites between CK and the target protein. The pharmacological effects and mechanism of CK in improving oxidative damage were verified in vivo by LPS-induced acute injury in mice and physical mechanical injury in rat soft tissues. RESULTS: KEAP1 was identified as the target protein that CK regulates to improve oxidative damage through the KEAP1-NRF2/ARE pathway. CK competitively binds to the DGR/Kelch domain of KEAP1, disrupting the binding between DLG peptide in NRF2 and KEAP1, thereby inhibiting the occurrence of oxidative damage induced by LPS or physical mechanical stress. CONCLUSIONS: CK functions as a natural KEAP1-NRF2 inhibitor, disrupting the binding between KEAP1 and NRF2-DLG motifs by targeting the DGR/Kelch domain of KEAP1, activating the antioxidant transcriptional program of NRF2, and reducing oxidative stress damage.

A prognostic and immunological analysis of 7B-containing Kelch structural domain (KLHDC7B) in pan-cancer: a potential target for immunotherapy and survival.

PURPOSE: KLHDC7B is a member of Kelch family, with a Kelch domain in the C-terminal half, which plays a role in various cellular events, such as cytoskeletal arrangement, protein degradation, gene expression. Although there is increasing evidence supporting KLHDC7B's vital role in tumorigenesis, a systematic analysis of KLHDC7B in cancers remains lacking. Therefore, we intended to investigate the prognostic value for KLHDC7B across 33 cancer types and explore its potential immunological function. METHODS: GEO (Gene Expression Omnibus database) and TCGA (The Cancer Genome Atla) database were used to explore the role of KLHDC7B in 33 cancers. TIMER2, GEPIA2 and Kaplan-Meier plotter were utilized to explore the KLHDC7B expression level and prognostic value in different cancers. The pan cancer genetic variation and DNA methylation of KLHDC7B were analyzed by cBioPortal and MEXPRESS. TIMER2 was employed to investigate the correlation between KLHDC7B expression and immune infiltration. The relationship of KLHDC7B expression with TMB (tumor mutational burden) and MSI (microsatellite instability) were evaluated using Spearman correlation analysis. Finally, by GO and KEGG enrichment analysis, the underlying mechanisms of KLHDC7B in tumor pathophysiology were further investigated. RESULTS: KLHDC7B expression level was related to pathological stages, MSI, TMB, immune checkpoint and immune cell infiltration in most cancers. Especially, we found that the KLHDC7B expression was negatively correlated with the immune infiltration of Myeloid derived suppressor cells into TGCT and GBM. Additionally, survival analysis showed that the expression of KLHDC7B was connected with overall survival (OS) in 3 cancers and disease-free survival (DFS) in 5 cancers. Furthermore, the enrichment analysis revealed that the KLHDC7B collecting genes and binding proteins are related to the function of proteins and immune response. CONCLUSION: KLHDC7B demonstrates strong clinical utility as markers of prognostic and immune response in pan-cancer.

Suppression of cuticular wax biosynthesis mediated by rice LOV KELCH REPEAT PROTEIN 2 supports a negative role in drought stress tolerance.

Drought tolerance is important for grain crops, including rice (Oryza sativa); for example, rice cultivated under intermittent irrigation produces less methane gas compared to rice grown in anaerobic paddy field conditions, but these plants require greater drought tolerance. Moreover, the roles of rice circadian-clock genes in drought tolerance remain largely unknown. Here, we show that the mutation of LOV KELCH REPEAT PROTEIN 2 (OsLKP2) enhanced drought tolerance by increasing cuticular wax biosynthesis. Among ZEITLUPE family genes, OsLKP2 expression specifically increased under dehydration stress. OsLKP2 knockdown (oslkp2-1) and knockout (oslkp2-2) mutants exhibited enhanced drought tolerance. Cuticular waxes inhibit non-stomatal water loss. Under drought conditions, total wax loads on the leaf surface increased by approximately 10% in oslkp2-1 and oslkp2-2 compared to the wild type, and the transcript levels of cuticular wax biosynthesis genes were upregulated in the oslkp2 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays revealed that OsLKP2 interacts with GIGANTEA (OsGI) in the nucleus. The osgi mutants also showed enhanced tolerance to drought stress, with a high density of wax crystals on their leaf surface. These results demonstrate that the OsLKP2-OsGI interaction negatively regulates wax accumulation on leaf surfaces, thereby decreasing rice resilience to drought stress.

Kelch-like proteins in the gastrointestinal tumors.

Gastrointestinal tumors have become a worldwide health problem with high morbidity and poor clinical outcomes. Chemotherapy and surgery, the main treatment methods, are still far from meeting the treatment needs of patients, and targeted therapy is in urgent need of development. Recently, emerging evidence suggests that kelch-like (KLHL) proteins play essential roles in maintaining proteostasis and are involved in the progression of various cancers, functioning as adaptors in the E3 ligase complex and promoting the specific degradation of substrates. Therefore, KLHL proteins should be taken into consideration for targeted therapy strategy discovery. This review summarizes the current knowledge of KLHL proteins in gastrointestinal tumors and discusses the potential of KLHL proteins as potential drug targets and prognostic biomarkers.

A kelch motif-containing protein KLHDC2 regulates immune responses against Vibrio parahaemolyticus and white spot syndrome virus in Penaeus vannamei.

The kelch motif-containing proteins are widely present in organisms and known to be involved in various biological processes, but their roles in immunity remain unclear. In this study, a kelch motif-containing protein KLHDC2 was identified from Pacific white shrimp Penaeus vannamei and its immune function was investigated. The klhdc2 gene was widely expressed in shrimp tissues and its protein product was mainly present in the nucleus. Expression of klhdc2 was regulated by shrimp NF-κB family members Dorsal and Relish, and changed after immune stimulation. KLHDC2 could enhance the immune defense against Vibrio parahaemolyticus in shrimp but inhibit that against white spot syndrome virus (WSSV). Further analyses showed that KLHDC2 did not affect the phagocytosis of hemocytes but regulated the expression of a series of immune effector genes. KLHDC2 has a complex regulatory relationship with Dorsal and Relish, which may partly contribute to its positive role in antibacterial response by regulating humoral immunity. Moreover, the regulatory effect of KLHDC2 on WSSV ie1 gene contributed to its negative effect on antiviral response. Therefore, the current study enrichs the knowledge on the Kelch family and helps to learn more about the regulatory mechanism of shrimp immunity.

Generation and analysis of pseudohypoaldosteronism type II knock-in mice caused by a nonsense KLHL3 mutation in the Kelch domain.

Mutations in the Kelch-like 3 (KLHL3) gene are the most common cause of inherited pseudohypoaldosteronism type II (PHAII) featuring thiazide-sensitive hypertension and hyperkalemic metabolic acidosis. Although Klhl3R528H/+ knock-in (KI) mice carrying a missense mutation in the Kelch repeat domain have been reported, nonsense KLHL3 mutations in the same domain that cause PHAII have not been fully investigated in vivo. We generated and analyzed Klhl3 KI mice harboring a nonsense W523X mutation (corresponding to the human KLHL3 W470X mutation). Both heterozygous and homozygous Klhl3W523X/+ KI mice exhibited typical PHAII with low-renin hypertension, hyperkalemia with reduced renal potassium excretion, and hyperchloremic metabolic acidosis. Their kidney tissues showed the presence of Klhl3 mRNA and increased Klhl3 protein levels along with enhanced downstream Wnk1/4-Spak/Osr1-N(k)cc phosphorylation. Increased protein expression of total Spak, phosphor(p-)Spak, total Ncc, and p-Ncc from urinary extracellular vesicles (uEVs) also confirmed the activation of the Wnk-mediated Ncc pathway. In vitro studies showed that the human KLHL3 W470X mutation resulted in increased KLHL3 protein stability and disrupted its binding affinity for WNK1/4, leading to the attenuated degradation and increased abundance of total WNKs. In conclusion, nonsense Klhl3W523X/+ mice recapitulating PHAII phenotypes exhibit Klhl3 protein stability, abrogating its binding to Wnks, with enhanced Ncc expression in the kidney tissue and even in uEVs. Activation of the WNK-mediated Na+ -Cl- co-transporter reiterated the in vivo pathogenic role of nonsense KLHL3 mutations in PHAII.

CRL2KLHDC3 mediates p14ARF N-terminal ubiquitylation degradation to promote non-small cell lung carcinoma progression.

Kelch superfamily involves a variety of proteins containing multiple kelch motif and is well characterized as substrate adaptors for CUL3 E3 ligases, which play critical roles in carcinogenesis. However, the role of kelch proteins in lung cancer remains largely unknown. In this study, the non-small cell lung cancer (NSCLC) patients with higher expression of a kelch protein, kelch domain containing 3 (KLHDC3), showed worse overall survival. KLHDC3 deficiency affected NSCLC cell lines proliferation in vitro and in vivo. Further study indicated that KLHDC3 mediated CUL2 E3 ligase and tumor suppressor p14ARF interaction, facilitating the N-terminal ubiquitylation and subsequent degradation of p14ARF. Interestingly, Gefitinib-resistant NSCLC cell lines displayed higher KLHDC3 protein levels. Gefitinib and Osimertinib medications were capable of upregulating KLHDC3 expression to promote p14ARF degradation in the NSCLC cell lines. KLHDC3 shortage significantly increased the sensitivity of lung cancer cells to epidermal growth factor receptor (EGFR)-targeted drugs, providing an alternative explanation for the development of Gefitinib and Osimertinib resistance in NSCLC therapy. Our works suggest that CRL2KLHDC3 could be a valuable target to regulate the abundance of p14ARF and postpone the occurrence of EGFR-targeted drugs resistance.

A kelch domain cell end protein, PoTea1, mediates cell polarization during appressorium morphogenesis in Pyricularia oryzae.

The rice blast fungus Pyricularia oryzae differentiates into an infection structure, called an appressorium, for plant penetration. The process of appressorium formation requires the transformation of polarized growth to isotropic growth, while penetration requires the opposite growth transformation from isotropic to polarized. Polarized growth requires coordinated organization of cytoskeletal elements, such as microtubule and actin. We identified PoTea1, a homolog of Tea1 from Schizosaccharomyces pombe, and characterized its roles in P. oryzae. After PoTEA1 deletion, ∆Potea1 displayed slowed hyphal growth, decreased sporulation, increased hyphal branches, abnormal two-celled spores, and reduced plant penetration and virulence. During appressorium formation, ∆Potea1 developed a long germ tube with a small appressorium, leading to delayed appressorium differentiation and reduced glycogen and lipid droplet degradation. ∆Potea1 is defective in cAMP-PKA and Pmk1 MAPK pathways. PoTea1 localized at hyphal tips and appressoria as bright dots and was highly dynamic during appressorium formation. PoTea1 formed a complex with itself by self-assembly that was highly dependent on its kelch motif. The coiled-coil motif C2 of PoTea1 is involved in self polymerization and appressorium formation. Benomyl and latrunculin A, two cytoskeleton inhibitors, disturbed the stable localization of PoTea1 at vegetative hyphal tips. We speculate that PoTea1 functions in appressorium formation and virulence by mediating cell polarity in P. oryzae.

Absence of Plasmodium falciparum artemisinin resistance gene mutations eleven years after the adoption of artemisinin-based combination therapy in Nigeria.

BACKGROUND: The occurrence of artemisinin resistance (ART)-associated polymorphism of Plasmodium falciparum K13-propeller (pfk13) gene before and after the introduction of artemisinin-based combination therapy (ACT) in two regions of Nigeria was investigated in this study. Regular surveillance is necessary to make a definite conclusion on the emergence and pattern of possible resistance to ART. METHODS: This cross-sectional study was carried out in the Southwestern and Southeastern geopolitical zones of Nigeria. A total of 150, 217, and 475 participants were enrolled for the study in the Southwest (2004_Group A), Southwest (2015_Group B), and southeast (2015_Group C), respectively. Blood samples were collected from the study participants for DNA extraction and a nested PCR for P. falciparum identification. Samples that were positive for P. falciparum were genotyped for the pfk13 gene using the Sanger sequencing method. The single nucleotide polymorphisms were analysed using the Bioedit software. RESULTS: A total of 116, 125, and 83 samples were positive for P. falciparum, respectively for the samples collected from the Southwest (2004 and 2015) and southeast (2015). Parasite DNA samples collected from febrile children in 2004 (Group A; n = 71) and 2015 (Group B; n = 73) in Osogbo Western Nigeria and 2015_Group C (n = 36) in southeast Nigeria were sequenced successfully. This study did not observe mutations associated with the in vitro resistance in southeast Asia, such as Y493H, R539T, I543T, and C580Y. Two new polymorphisms V520A and V581I were observed in two samples collected in Osogbo, Southwest Nigeria. These two mutations occurred in the year 2004 (Group A) before the introduction of ACT. Six mutations were identified in 17% of the samples collected in southeast Nigeria. One of these mutations (D547G) was non-synonymous, while the remaining (V510V, R515R, Q613Q, E688E, and N458N) were synonymous. Also, one (2%) heterozygote allele was identified at codon 458 in the 2015 (Group C) samples. CONCLUSIONS: None of the mutations observed in this study were previously validated to be associated with ART resistance. These results, therefore, suggest that artemisinin is likely to remain highly effective in treating malaria in the study areas that are malarious zone.

Increase in the proportion of Plasmodium falciparum with kelch13 C580Y mutation and decline in pfcrt and pfmdr1 mutant alleles in Papua New Guinea.

BACKGROUND: The C580Y mutation in the Plasmodium falciparum kelch13 gene is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identified outside the GMS, except for Guyana/Amazonia. In 2017, three parasites carrying the C580Y mutation were identified in Papua New Guinea (PNG). As the C580Y allele rapidly spread in the GMS, there is concern that this mutant is now spreading in PNG. METHODS: In 2020, a cross-sectional survey was conducted at two clinics in Wewak, PNG. Symptomatic patients infected with P. falciparum were treated with artemether plus lumefantrine following a national treatment policy. Blood samples were obtained before treatment, and polymorphisms in kelch13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3. The results were compared with those of previous studies conducted in 2002, 2003, and 2016-2018. RESULTS: A total of 94 patients were included in this analysis. The proportion of C580Y was significantly increased (2.2% in 2017, 5.7% in 2018, and 6.4% in 2020; p = 4.2 × 10-3). A significant upward trend was observed in the wild-type proportion for pfcrt (1.9% in 2016 to 46.7% in 2020; p = 8.9 × 10-16) and pfmdr1 (59.5% in 2016 to 91.4% in 2020; p = 2.3 × 10-6). Among 27 patients successfully followed on day 3, including three with C580Y infections, none showed positive parasitaemia. CONCLUSIONS: Under the conditions of significant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in kelch13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used first-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effects are important.

Kelch13 mutations in Plasmodium falciparum and risk of spreading in Amazon basin countries.

BACKGROUND: The first potential focus for artemisinin resistance in South America was recently confirmed with the presence of the C580Y mutation in the Plasmodium falciparum kelch 13 gene (pfk13) in Guyana. OBJECTIVES: This study aimed to strengthen pfk13 monitoring in the Amazon basin countries, to compile the available data and to evaluate the risk of spreading of mutations. METHODS: Sanger sequencing was done on 862 samples collected between 1998 and 2019, and a global map of pfk13 genotypes available for this region was constructed. Then, the risk of spreading of mutations based on P. falciparum case importation between 2015 and 2018 within countries of the Amazon basin was evaluated. RESULTS: No additional pfk13 C580Y foci were identified. Few mutations (0.5%, 95% CI = 0.3%-0.8%) in the propeller domain were observed in the general parasite population of this region despite a high proportion of K189T mutations (49.1%, 95% CI = 46.2%-52.0%) in the non-propeller domain. Case information revealed two patterns of intense human migration: Venezuela, Guyana and the Roraima State in Brazil; and French Guiana, Suriname and the Amapá State in Brazil. CONCLUSIONS: There are few pfk13 mutant foci, but a high risk of dispersion in the Amazon basin, mainly from the Guiana Shield, proportionate to mining activities. Therefore, access to prompt diagnosis and treatment, and continuous molecular monitoring is essential in these geographical areas.

Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat.

F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) genes, unevenly distributed across 21 chromosomes and encoding 74 proteins, were identified in EnsemblPlants. Protein sequences were compared with those of Arabidopsis and three cereal species by phylogenetic and domain analyses, where the wheat sequences were resolved into 6 clades. In silico analysis of a digital PCR dataset revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homolog of the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. Reverse-transcription quantitative real-time PCR (RT-qPCR) was carried out to determine tissue-specific, hormone and stress (abiotic/biotic) responsive expression patterns. Of interest, TaAFR was expressed most abundantly in the leaves, and its expression in response to leaf rust variants suggests a potential role in compatible vs incompatible rust responses. The protein was predicted to localize in cytosol, but it was shown experimentally to localize in both the cytosol and the nucleus of tobacco. A series of protein interaction studies, starting with a yeast-2-hybrid (Y2H) library screen (wheat leaf infected with incompatible leaf rust pathogens), led to the identification of three TaAFR interacting proteins. Skp1/ASK1-like protein (Skp1) was found to interact with the F-box domain of TaAFR, while ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL) were shown to interact with its Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.

Artemisinin combination therapy fails even in the absence of Plasmodium falciparum kelch13 gene polymorphism in Central India.

Artemisinin is the frontline fast-acting anti-malarial against P. falciparum. Emergence and spread of resistant parasite in eastern-India poses a threat to national malaria control programs. Therefore, the objective of our study is to evaluate the artesunate-sulfadoxine-pyrimethamine efficacy in Central India. 180 monoclonal P. falciparum-infected patients received standard ASSP therapy during August 2015-January 2017, soon after diagnosis and monitored over next 42-days. Artemisinin-resistance was assessed through in-vivo parasite clearance half-life (PC1/2), ex-vivo ring-stage survivability (RSA), and genome analysis of kelch13 and other candidate gene (pfcrt, pfmdr1, pfatpase 6, pfdhfr and pfdhps). Of 180 P. falciparum positive patients, 9.5% showed increased PC1/2 (> 5.5 h), among them eleven isolates (6.1%) showed reduced sensitivity to RSA. In 4.4% of cases, parasites were not cleared by 72 h and showed prolonged PC1/2(5.6 h) (P < 0.005) along with significantly higher RSA (2.2%) than cured patients (0.4%). None of day-3 positive isolates contained the pfkelch13 mutation implicated in artemisinin resistance. Parasite recrudescence was observed in 5.6% patients, which was associated with triple dhfr-dhps (A16I51R59N108I164-S436G437K540G581T613) combination mutation. Emergence of reduced sensitivity to artesunate-sulfadoxine-pyrimethamine, in central India highlighted the risk toward spread of resistant parasite across different parts of India. Day-3 positive parasite, featuring the phenotype of artemisinin-resistance without pfkelch13 mutation, suggested kelch13-independent artemisinin-resistance.

Targeting Cul3-scaffold E3 ligase complex via KLHL substrate adaptors for cancer therapy.

Targeted therapy has become increasingly important and indispensable in cancer therapy. Cullin3-RING ligases (CRL3) serve as essential executors for regulating protein homeostasis in cancer development, highlighting that CRL3 might be promising targets in various cancer treatment. However, how to design new targeted therapies by disrupting the function of CRL3 is poorly understood. Here, we focus on the substrate adaptors of CRL3, and carry out a systematical research on the function of Kelch-like (KLHL) family proteins. We have identified twenty-four KLHL proteins with function of tumor promotion and thirteen KLHL proteins with high clinical significance on cancer therapy. Furthermore, we have clarified the novel biological function of KLHL13 as a vital factor that contributes to malignant progression in lung cancer. Taken together, our findings reveal multiple potential therapeutical targets and provide evidence for targeting CRL3 via KLHL substrate adaptors for cancer therapy.

HPV E7 affects the function of cervical cancer cells via the TAL1/lnc­EBIC/KLHDC7B axis.

High­risk human papillomavirus (HPV)16 and 18 are the primary cause of cervical cancer (CC) and long non­coding RNAs (lncRNAs/lncs) are often abnormally expressed in patients with CC. The authors' previous study indicated that oncogenic enhancer of zeste homolog 2 (EZH2)­binding lncRNA in cervical cancer (lnc­EBIC) serves a role in the tumorigenic activity of the HPV E6 protein in patients with CC. However, whether HPV E7 affects the development of CC through lnc­EBIC, and the potential mechanisms underlying this remains unclear. Therefore, the present study investigated the effects of lnc­EBIC and HPV E7 in cervical cancer cell lines HeLa, CaSki and C33A in vitro. CCK­8, EdU and DAPI staining assays, flow cytometry, RT­qPCR, western blotting and Transwell assay were performed on these cell lines. The results revealed that exogenous expression of HPV16/18 E7 significantly promoted lnc­EBIC expression, and conversely, lnc­EBIC was downregulated by silencing endogenous HPV16/18 E7 expression in corresponding CaSki and HeLa cells. Overexpression of lnc­EBIC significantly increased cellular proliferation, migration and invasion, and inhibited apoptosis in HPV­ C33A cells. The tumorigenic effects of HPV16/18 E7 in corresponding CaSki and HeLa cells were significantly blocked by the silencing of lnc­EBIC expression. Molecular analysis revealed that HPV16/18 E7 depended on TAL BHLH transcription factor 1, erythroid differentiation factor inhibition to promote lnc­EBIC expression, which also resulted in the upregulation of oncogenic Kelch domain­containing 7B (KLHDC7B) in corresponding CaSki and HeLa cells. Additionally, KLHDC7B knockdown blocked the tumor­promotive effects of lnc­EBIC in HPV­ C33A cells. Collectively, the results of the present study indicated that lnc­EBIC acts as an oncogenic lncRNA by enhancing KLHDC7B expression in HPV+ and HPV­ CC cells, and can be exploited by HPV16/18 E7 to accelerate tumorigenic activity in CC. These results further revealed that the lnc­EBIC/KLHDC7B axis represents a novel molecular mechanism and potential therapeutic target for CC.

Functional domain studies uncover novel roles for the ZTL Kelch repeat domain in clock function.

The small LOV/F-box/Kelch family of E3 ubiquitin ligases plays an essential role in the regulation of plant circadian clocks and flowering time by sensing dusk. The family consists of three members, ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2), and FLAVIN-BINDING KELCH REPEAT F-BOX PROTEIN 1 (FKF1), which share a unique protein domain architecture allowing them to act as photoreceptors that transduce light signals via altering stability of target proteins. Despite intensive study of this protein family we still lack important knowledge about the biochemical and functional roles of the protein domains that comprise these unique photoreceptors. Here, we perform comparative analyses of transgenic lines constitutively expressing the photoreceptor LOV domain or the Kelch repeat protein-protein interaction domains of ZTL, FKF1, and LKP2. Expression of each domain alone is sufficient to disrupt circadian rhythms and flowering time, but each domain differs in the magnitude of effect. Immunoprecipitation followed by mass spectrometry with the ZTL Kelch repeat domain identified a suite of potential interacting partners. Furthermore, the ZTL Kelch repeat domain can interact with the ZTL homologs, LKP2 and FKF1, and the LOV domain of ZTL itself. This suggests a hypothesis that the Kelch repeat domain of ZTL may mediate inter- and intra-molecular interactions of the three LOV/F-box/Kelch proteins and provides added insight into the composition of the protein complexes and an additional role for the Kelch repeat domain.

Ginnalin A Binds to the Subpockets of Keap1 Kelch Domain To Activate the Nrf2-Regulated Antioxidant Defense System in SH-SY5Y Cells.

Ginnalin A (GA), a polyphenol from the red maple, was reported to be a potential ROS scavenger or an activator of nuclear factor erythroid-2 related factor 2 (Nrf2) in cancer cells. However, whether GA could activate Nrf2 in neuronal cells and the exact mode of action are unknown. We performed molecular docking calculations, which revealed that GA fits well into the five subpockets of the Kelch-like ECH-associated protein1 (Keap1) Kelch domain via hydrogen bonding and hydrophobic interaction. Our cytotoxicity assays demonstrate that pretreating SH-SY5Y cells with 20 µM GA effectively prevents cells from oxidative assault by 6-hydroxydopamine (6-OHDA). Fluorescence imaging indicates that upon the GA pretreatment, Nrf2 dissociates from the Keap1-Nrf2 complex and translocates into nucleus to activate the cellular antixodant system. Real-time qPCR quantification and Western blotting verified that the GA pretreatment elevates NAD(P)H quinone oxidoreductase-1 (NQO1) by more than 4.6-fold, heme oxygenase (HO-1) by about 1.2-fold, and the glutamate-cysteine ligase catalytic (GCLC) subunit by 0.7-fold. The higher antixidant protein levels, along with increased glutathione concentration, decrease intracellular reactive oxygen species and alleviate the 6-OHDA-induced oxidative damage. Silence of Nrf2 abrogates the cytoprotection of the GA pretreatment, confirming that the Keap1/Nrf2-ARE (antioxidant response element) pathway is solely responsible for the GA's biological effects. GA is a promising natural compound for sensitizing neuronal cells' antioxidative defense system to offset oxidative stress, a condition closely linked to the pathogenesis of Parkinson's disease.

Arabidopsis SnRK1 negatively regulates phenylpropanoid metabolism via Kelch domain-containing F-box proteins.

Phenylpropanoid metabolism represents a substantial metabolic sink for photosynthetically fixed carbon. The evolutionarily conserved Sucrose Non-Fermenting Related Kinase 1 (SnRK1) is a major metabolic sensor that reprograms metabolism upon carbon deprivation. However, it is not clear if and how the SnRK1-mediated sugar signaling pathway controls phenylpropanoid metabolism. Here, we show that Arabidopsis SnRK1 negatively regulates phenylpropanoid biosynthesis via a group of Kelch domain-containing F-box (KFB) proteins that are responsible for the ubiquitination and degradation of phenylalanine ammonia lyase (PAL). Downregulation of AtSnRK1 significantly promoted the accumulation of soluble phenolics and lignin polymers and drastically increased PAL cellular accumulation but only slightly altered its transcription level. Co-expression of SnRK1α with PAL in Nicotiana benthamiana leaves resulted in the severe attenuation of the latter's protein level, but protein interaction assays suggested PAL is not a direct substrate of SnRK1. Furthermore, up or downregulation of AtSnRK1 positively affected KFBPALs gene expression, and energy starvation upregulated KFBPAL expression, which partially depends on AtSnRK1. Collectively, our study reveals that SnRK1 negatively regulates phenylpropanoid biosynthesis, and KFBPALs act as regulatory components of the SnRK1 signaling network, transcriptionally regulated by SnRK1 and subsequently mediate proteasomal degradation of PAL in response to the cellular carbon availability.

Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum.

Malaria continues to impose a significant health burden in the continent of Africa with 213 million cases in 2018 alone, representing 93% of cases worldwide. Because of high transmission of malaria within the continent, the selection pressures to develop drug resistance in African parasites are distinct compared to the rest of the world. In light of the spread of resistance to artemisinin conferred by the C580Y mutation in the PfKelch13 propeller domain in Southeast Asia, and its independent emergence in South America, it is important to study genetic determinants of resistance in the African context using African parasites. Through in vitro evolution of Senegalese parasites, we had previously generated the artemisinin-resistant parasites Pikine_R and Thiès_R and established pfcoronin mutations to be sufficient to confer artemisinin resistance in the standard ring-stage survival assay (RSA). In the current study, we used genetic analysis of revertants to demonstrate pfcoronin to be the major driver of elevated RSA in the artemisinin-resistant parasites Pikine_R and Thiès_R evolved in vitro. We interrogated the role of a second gene PF3D7_1433800, which also had mutations in both the Pikine_R and Thiès_R selected lines, but found no evidence of a contribution to reduced susceptibility in the RSA survival assay. Nevertheless, our genetic analysis demonstrates that parasite genetic background is important in the level of pfcoronin mediated RSA survival, and therefore we cannot rule out a role for PF3D7_1433800 in other genetic backgrounds. Finally, we tested the potential synergy between the mutations of pfcoronin and pfkelch13 through the generation of single and double mutants in the Pikine genetic background and found that the contribution of pfcoronin to reduced susceptibility is masked by the presence of pfkelch13. This phenomenon was also observed in the 3D7 background, suggesting that pfcoronin may mediate its effects via the same pathway as pfkelch13. Investigating the biology of proteins containing the beta-propeller domain could further elucidate the different pathways that the parasite could use to attain resistance.

Case Report: The First Case of Genotypically Confirmed Plasmodium falciparum Kelch 13 Propeller Mutation in Sri Lanka and Its Implications on the Elimination Status of Malaria.

This case report discusses recrudescence of imported Plasmodium falciparum malaria, in the presence of P. falciparum Kelch13 (PfK13) propeller mutation, in a patient diagnosed and fully treated with artemether-lumefantrine under direct observation in Sri Lanka. This patient presented with a history of 5 days of fever following his arrival from the Democratic Republic of Congo (DRC). He had visited Rwanda 1 week before arrival to Sri Lanka. Treatment was commenced with artemisinin-based combination therapy, artemether-lumefantrine, which is the first-line drug recommended for uncomplicated falciparum malaria. Blood smears were negative for parasites by the third day of treatment. Approximately 2 weeks later, he developed fever again and was diagnosed as having a recrudescence of falciparum malaria. He was treated and responded to the second-line antimalarial dihydroartemisinin-piperaquine. Molecular testing of blood taken from the first infection revealed the presence of amino acid substitutions K189T and R561H within the PfK13 gene. R561H mutation is associated with delayed parasite clearance in Southeast Asia. Although seldom reported from DRC, an emergence and clonal expansion of parasites harboring R561H allele has been reported from Rwanda recently; thus, it is likely that this patient may have got the infection from Rwanda. Sri Lanka eliminated malaria in 2016. However, in the backdrop of continuing imported malaria cases, early diagnosis and prompt treatment is essential to prevent the re-establishment of the disease.