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Nature Structural & Molecular Biology Jul 2024The mitochondrial chaperonin, mitochondrial heat shock protein 60 (mtHsp60), promotes the folding of newly imported and transiently misfolded proteins in the...
The mitochondrial chaperonin, mitochondrial heat shock protein 60 (mtHsp60), promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin progresses through its ATP-dependent client folding cycle are not clear. Here, we determined cryo-EM structures of a hyperstable disease-associated human mtHsp60 mutant, V72I. Client density is identified in three distinct states, revealing interactions with the mtHsp60 apical domains and C termini that coordinate client positioning in the folding chamber. We further identify an asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60-10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify distinct roles for the apical domains in coordinating client capture and progression through the chaperone cycle, supporting a conserved mechanism of group I chaperonin function.
PubMed: 38951622
DOI: 10.1038/s41594-024-01352-0 -
Methods in Molecular Biology (Clifton,... 2024Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The...
Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.
Topics: Gene Editing; Escherichia coli; Baculoviridae; Genetic Vectors; Chromosomes, Artificial, Bacterial; Genome, Viral; Genetic Engineering; Bacteriophage lambda; Homologous Recombination
PubMed: 38951330
DOI: 10.1007/978-1-0716-3961-0_8 -
Planta Jul 2024Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1...
Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.
Topics: Cicer; Seeds; Anthocyanins; Plant Proteins; Gene Expression Regulation, Plant; Proanthocyanidins; Transcription Factors; Plants, Genetically Modified; Arabidopsis; Flowers
PubMed: 38951258
DOI: 10.1007/s00425-024-04470-7 -
Current Microbiology Jul 2024Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1...
Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, HO-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and HO-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.
Topics: Polyphosphates; Myxococcus xanthus; Spores, Bacterial; Bacterial Proteins; Phosphotransferases (Phosphate Group Acceptor); Acid Anhydride Hydrolases; Culture Media
PubMed: 38951187
DOI: 10.1007/s00284-024-03778-7 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Apr 2024
Topics: Humans; Mutation; Oncogene Proteins, Fusion; Male; Transcription Factors; Child; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma
PubMed: 38951073
DOI: 10.3760/cma.j.121090-20231012-00195 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Apr 2024Twelve DEK-NUP214 fusion gene-positive patients with acute myeloid leukemia and on allo-HSCT treatment at the Hematology Hospital of the Chinese Academy of Medical...
Twelve DEK-NUP214 fusion gene-positive patients with acute myeloid leukemia and on allo-HSCT treatment at the Hematology Hospital of the Chinese Academy of Medical Sciences from November 2016 to August 2022 were included in the study, and their clinical data were retrospectively analyzed. The patients comprised five men and seven women with a median age of 34 (16-52) years. At the time of diagnosis, all the patients were positive for the DEK-NUP214 fusion gene. Chromosome karyotyping analysis showed t (6;9) (p23;q34) translocation in 10 patients (two patients did not undergo chromosome karyotyping analysis), FLT3-ITD mutation was detected in 11 patients, and high expression of WT1 was observed in 11 patients. Nine patients had their primary disease in the first complete remission state before transplantation, one patient had no disease remission, and two patients were in a recurrent state. All patients received myeloablative pretreatment, five patients received sibling allogeneic hematopoietic stem cell transplantation, and seven patients received haploid hematopoietic stem cell transplantation. The median number of mononuclear cells in the transplant was 10.87 (7.09-17.89) ×10(8)/kg, and the number of CD34(+) cells was 3.29 (2.53-6.10) ×10(6)/kg. All patients achieved blood reconstruction, with a median time of 14 (10-20) days for neutrophil implantation and 15 (9-27) days for platelet implantation. The 1 year transplant-related mortality rate after transplantation was 21.2%. The cumulative recurrence rates 1 and 3 years after transplantation were 25.0% and 50.0%, respectively. The leukemia free survival rates were (65.6±14.0) % and (65.6±14.0) %, respectively. The overall survival rates were (72.2±13.8) % and (72.2±13.8) %, respectively.
Topics: Humans; Male; Female; Adult; Hematopoietic Stem Cell Transplantation; Middle Aged; Leukemia, Myeloid, Acute; Adolescent; Retrospective Studies; Young Adult; Nuclear Pore Complex Proteins; Transplantation, Homologous; Chromosomal Proteins, Non-Histone; Poly-ADP-Ribose Binding Proteins; Oncogene Proteins, Fusion; Oncogene Proteins; Translocation, Genetic
PubMed: 38951067
DOI: 10.3760/cma.j.cn121090-20230913-00115 -
Journal of Managed Care & Specialty... Jul 2024Neurotrophic tyrosine receptor kinase () gene fusions are rare oncogenic drivers prevalent in 0.3% of solid tumors. They are most common in salivary gland cancer (2.6%),...
BACKGROUND
Neurotrophic tyrosine receptor kinase () gene fusions are rare oncogenic drivers prevalent in 0.3% of solid tumors. They are most common in salivary gland cancer (2.6%), thyroid cancer (1.6%), and soft-tissue sarcoma (1.5%). Currently, there are 2 US Food and Drug Administration-approved targeted therapies for gene fusions: larotrectinib, approved in 2018, and entrectinib, approved in 2019. To date, the real-world uptake of tyrosine receptor kinase inhibitor (TRKi) use for -positive solid tumors in academic cancer centers remains largely unknown.
OBJECTIVE
To describe the demographics, clinical and genomic characteristics, and testing and treatment patterns of patients with -positive solid tumors treated at US academic cancer centers.
METHODS
This was a retrospective chart review study conducted in academic cancer centers in the United States. All patients diagnosed with an fusion-positive (1, 2, 3) solid tumor (any stage) and who received cancer treatment at participating sites between January 1, 2012, and July 1, 2023, were included in this study. Patient demographics, clinical characteristics, genomic characteristics, testing data, and treatment patterns were collected from electronic medical records and analyzed using descriptive statistics as appropriate.
RESULTS
In total, 6 centers contributed data for 55 patients with -positive tumors. The mean age was 49.3 (SD = 20.5) years, 51% patients were female, and the majority were White (78%). The median duration of time from cancer diagnosis to testing was 85 days (IQR = 44-978). At the time of testing, 64% of patients had stage IV disease, compared with 33% at cancer diagnosis. Prevalent cancer types in the overall cohort included head and neck (15%), thyroid (15%), brain (13%), lung (13%), and colorectal (11%). 1 fusions were most common (45%), followed by 3 (40%) and 2 (15%). Across all lines of therapy, 51% of patients (n = 28) received a TRKi. Among TRKi-treated patients, 71% had stage IV disease at TRKi initiation. The median time from positive test to initiation of TRKi was 48 days (IQR = 9-207). TRKis were commonly given as first-line (30%) or second-line (48%) therapies. Median duration of therapy was 610 (IQR = 182-764) days for TRKi use and 207.5 (IQR = 42-539) days for all other first-line therapies.
CONCLUSIONS
This study reports on contemporary real-world testing patterns and use of TRKis in solid tumors, including time between testing and initiation of TRKi therapy and duration of TRKi therapy.
Topics: Humans; Female; Male; Retrospective Studies; Middle Aged; United States; Neoplasms; Receptor, trkC; Aged; Receptor, trkA; Adult; Protein Kinase Inhibitors; Receptor, trkB; Academic Medical Centers; Membrane Glycoproteins; Oncogene Proteins, Fusion; Cohort Studies; Pyrimidines; Pyrazoles; Benzamides; Young Adult; Indazoles
PubMed: 38950155
DOI: 10.18553/jmcp.2024.30.7.672 -
Cancer Research Communications Jul 2024Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by...
Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases. Myristoylation is emerging as a promising cancer therapeutic target, however the molecular determinants of sensitivity to N-myristoyltransferase inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that N-myristoyltransferases are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter Translocase of Inner Mitochondrial Membrane 17 homologue A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis N-myristoyltransferase-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.
PubMed: 38949950
DOI: 10.1158/2767-9764.CRC-23-0428 -
Journal of Agricultural and Food... Jul 2024Multiprotein bridging factor 1 (MBF1) is a very important transcription factor (TF) in plants, whose members influence numerous defense responses. Our study found that...
Multiprotein bridging factor 1 (MBF1) is a very important transcription factor (TF) in plants, whose members influence numerous defense responses. Our study found that MBF1c in Cucurbitaceae was highly conserved. expression was induced by temperature, salt stress, and abscisic acid (ABA) in cucumber. Overexpressed CsMBF1c enhanced the heat resistance of a cucumber, and the mutant showed decreased resistance to high temperatures (HTs). CsMBF1c played an important role in stabilizing the photosynthetic system of cucumber under HT, and its expression was significantly associated with heat-related TFs and genes related to protein processing in the endoplasmic reticulum (ER). Protein interaction showed that CsMBF1c interacted with dehydration-responsive element binding protein 2 (CsDREB2) and nuclear factor Y A1 (CsNFYA1). Overexpression of CsNFYA1 in improved the heat resistance. Transcriptional activation of CsNFYA1 was elevated by CsMBF1c. Therefore, plays an important regulatory role in cucumber's resistance to high temperatures.
PubMed: 38949485
DOI: 10.1021/acs.jafc.4c02398 -
Angewandte Chemie (International Ed. in... Jul 2024Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with...
Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provide a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug.
PubMed: 38949226
DOI: 10.1002/anie.202410791