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Biomolecules May 2024Chronic myeloid leukemia (CML) is an oncological myeloproliferative disorder that accounts for 15 to 20% of all adult leukemia cases. The molecular basis of this disease... (Review)
Review
Chronic myeloid leukemia (CML) is an oncological myeloproliferative disorder that accounts for 15 to 20% of all adult leukemia cases. The molecular basis of this disease lies in the formation of a chimeric oncogene BCR-ABL1. The protein product of this gene, p210 BCR-ABL1, exhibits abnormally high constitutive tyrosine kinase activity. Over recent decades, several targeted tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1 have been developed and introduced into clinical practice. These inhibitors suppress BCR-ABL1 activity through various mechanisms. Furthermore, the advent of RNA interference technology has enabled the highly specific inhibition of BCR-ABL1 transcript expression using small interfering RNA (siRNA). This experimental evidence opens avenues for the development of a novel therapeutic strategy for CML, termed siRNA therapy. The review delves into molecular genetic mechanisms underlying the pathogenesis of CML, challenges in CML therapy, potential molecular targets for drug development, and the latest results from the application of siRNAs in in vitro and in vivo CML models.
Topics: Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Humans; RNA, Small Interfering; Fusion Proteins, bcr-abl; Molecular Targeted Therapy; Animals; Protein Kinase Inhibitors; RNA Interference
PubMed: 38927048
DOI: 10.3390/biom14060644 -
Stem Cell Research & Therapy Jun 2024Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of...
BACKGROUND
Human induced pluripotent stem cells (hiPSCs) and their differentiated cell types have a great potential for tissue repair and regeneration. While the primary focus of using hiPSCs has historically been to regenerate damaged tissue, emerging studies have shown a more potent effect of hiPSC-derived paracrine factors on tissue regeneration. However, the precise contents of the transplanted hiPSC-derived cell secretome are ambiguous. This is mainly due to the lack of tools to distinguish cell-specific secretome from host-derived proteins in a complex tissue microenvironment in vivo.
METHODS
In this study, we present the generation and characterization of a novel hiPSC line, L274G-hiPSC, expressing the murine mutant methionyl-tRNA synthetase, L274GMmMetRS, which can be used for tracking the cell specific proteome via biorthogonal non-canonical amino acid tagging (BONCAT). We assessed the trilineage differentiation potential of the L274G-hiPSCs in vitro and in vivo. Furthermore, we assessed the cell-specific proteome labelling in the L274G-hiPSC derived cardiomyocytes (L274G-hiPSC-CMs) in vitro following co-culture with wild type human umbilical vein derived endothelial cells and in vivo post transplantation in murine hearts.
RESULTS
We demonstrated that the L274G-hiPSCs exhibit typical hiPSC characteristics and that we can efficiently track the cell-specific proteome in their differentiated progenies belonging to the three germ lineages, including L274G-hiPSC-CMs. Finally, we demonstrated cell-specific BONCAT in transplanted L274G-hiPSC-CMs.
CONCLUSION
The novel L274G-hiPSC line can be used to study the cell-specific proteome of hiPSCs in vitro and in vivo, to delineate mechanisms underlying hiPSC-based cell therapies for a variety of regenerative medicine applications.
Topics: Induced Pluripotent Stem Cells; Humans; Proteome; Animals; Mice; Cell Differentiation; Myocytes, Cardiac; Amino Acids; Human Umbilical Vein Endothelial Cells; Methionine-tRNA Ligase
PubMed: 38926849
DOI: 10.1186/s13287-024-03792-3 -
Molecular Cancer Jun 2024TFE3-rearranged renal cell carcinoma (TFE3-rRCC) is a rare but highly heterogeneous renal cell carcinoma (RCC) entity, of which the clinical treatment landscape is...
BACKGROUND
TFE3-rearranged renal cell carcinoma (TFE3-rRCC) is a rare but highly heterogeneous renal cell carcinoma (RCC) entity, of which the clinical treatment landscape is largely undefined. This study aims to evaluate and compare the efficacy of different systemic treatments and further explore the molecular correlates.
METHODS
Thirty-eight patients with metastatic TFE3-rRCC were enrolled. Main outcomes included progression-free survival (PFS), overall survival, objective response rate (ORR) and disease control rate. RNA sequencing was performed on 32 tumors.
RESULTS
Patients receiving first-line immune checkpoint inhibitor (ICI) based combination therapy achieved longer PFS than those treated without ICI (median PFS: 11.5 vs. 5.1 months, P = 0.098). After stratification of fusion partners, the superior efficacy of first-line ICI based combination therapy was predominantly observed in ASPSCR1-TFE3 rRCC (median PFS: not reached vs. 6.5 months, P = 0.01; ORR: 67.5% vs. 10.0%, P = 0.019), but almost not in non-ASPSCR1-TFE3 rRCC. Transcriptomic data revealed enrichment of ECM and collagen-related signaling in ASPSCR1-TFE3 rRCC, which might interfere with the potential efficacy of anti-angiogenic monotherapy. Whereas angiogenesis and immune activities were exclusively enriched in ASPSCR1-TFE3 rRCC and promised the better clinical outcomes with ICI plus tyrosine kinase inhibitor combination therapy.
CONCLUSIONS
The current study represents the largest cohort comparing treatment outcomes and investigating molecular correlates of metastatic TFE3-rRCC based on fusion partner stratification. ICI based combination therapy could serve as an effective first-line treatment option for metastatic ASPSCR1-TFE3 rRCC patients. Regarding with other fusion subtypes, further investigations should be performed to explore the molecular mechanisms to propose pointed therapeutic strategy accordingly.
Topics: Humans; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Renal Cell; Female; Male; Middle Aged; Kidney Neoplasms; Aged; Immune Checkpoint Inhibitors; Oncogene Proteins, Fusion; Adult; Antineoplastic Combined Chemotherapy Protocols; Gene Rearrangement; Biomarkers, Tumor; Treatment Outcome; Prognosis; Intracellular Signaling Peptides and Proteins
PubMed: 38926757
DOI: 10.1186/s12943-024-02044-5 -
Acta Neuropathologica Communications Jun 2024
Topics: Humans; Central Nervous System Neoplasms; Male; Child; Female; Oncogene Proteins, Fusion; Repressor Proteins
PubMed: 38926750
DOI: 10.1186/s40478-024-01824-w -
Microbial Cell Factories Jun 2024Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of...
BACKGROUND
Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.
RESULTS
In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.
CONCLUSIONS
We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.
Topics: Botrytis; Abscisic Acid; Fermentation; Metabolic Engineering; Fungal Proteins
PubMed: 38926702
DOI: 10.1186/s12934-024-02460-8 -
Scientific Reports Jun 2024Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations...
Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.
Topics: Humans; Homeodomain Proteins; Nerve Tissue Proteins; Mutation; HEK293 Cells; Autism Spectrum Disorder; Heart Diseases; Facies; Neurodevelopmental Disorders
PubMed: 38926592
DOI: 10.1038/s41598-024-65608-x -
Scientific Reports Jun 2024To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA...
To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA and protein levels via the use of recombinant carriers in vitro. Whole-exome sequencing (WES) and Sanger sequencing were used to analyse the pathogenic genes of the child and validate the mutations in the parents. pEGFP and phage vectors carrying wild-type and mutant OCA2 were constructed using the coding DNA sequence (CDS) of the whole gene-synthesized OCA2 as a template and transfected into HEK293T cells, after which expression analysis was performed. The child in this study was born with white skin, hair, eyelashes, and eyebrows and exhibited nystagmus. Genetic analysis indicated that the child carried two heterozygous mutations: c.1079C > T (p.Ser360Phe) of maternal origin and c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) of paternal origin, conforming to an autosomal recessive inheritance pattern. In vitro analysis showed that the expression of the c.1079C > T (p.Ser360Phe) mutant did not significantly change at the mRNA level but did increase at the protein level, suggesting that the mutation may lead to enhanced protein stability, and the c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) mutation resulted in the loss of three amino acids in exon 10, producing a truncated protein. In vitro expression analysis also revealed that the expression of the mutant gene was significantly downregulated at both the mRNA and protein levels, suggesting that the mutation can simultaneously produce truncated proteins and lead to protein degradation. This case study enriches the phenotypic spectrum of OCA2 gene disease. In vitro expression analysis confirmed that both mutations affect protein expression, providing a theoretical basis for analysing the pathogenicity of these two mutations.
Topics: Humans; HEK293 Cells; Mutation; Albinism, Oculocutaneous; Membrane Transport Proteins; Exome Sequencing; Female; Male; Pedigree; RNA, Messenger
PubMed: 38926510
DOI: 10.1038/s41598-024-64782-2 -
Nature Communications Jun 2024Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to...
Anaerobic, acetogenic bacteria are well known for their ability to convert various one-carbon compounds, promising feedstocks for a future, sustainable biotechnology, to products such as acetate and biofuels. The model acetogen Acetobacterium woodii can grow on CO, formate or methanol, but not on carbon monoxide, an important industrial waste product. Since hydrogenases are targets of CO inhibition, here, we genetically delete the two [FeFe] hydrogenases HydA2 and HydBA in A. woodii. We show that the ∆hydBA/hydA2 mutant indeed grows on CO and produces acetate, but only after a long adaptation period. SNP analyzes of CO-adapted cells reveal a mutation in the HycB2 subunit of the HydA2/HydB2/HydB3/Fdh-containing hydrogen-dependent CO reductase (HDCR). We observe an increase in ferredoxin-dependent CO reduction and vice versa by the HDCR in the absence of the HydA2 module and speculate that this is caused by the mutation in HycB2. In addition, the CO-adapted ∆hydBA/hydA2 mutant growing on formate has a final biomass twice of that of the wild type.
Topics: Acetobacterium; Formates; Carbon Monoxide; Bacterial Proteins; Hydrogenase; Mutation; Carbon Dioxide; Electron Transport; Biomass; Acetates; Polymorphism, Single Nucleotide
PubMed: 38926344
DOI: 10.1038/s41467-024-49680-5 -
Cancer Medicine Jun 2024Members of the neurotrophic tropomyosin receptor kinase (NTRK) gene family, NTRK1, NTRK2, and NTRK3 encode TRK receptor tyrosine kinases. Intra- or inter-chromosomal...
BACKGROUND
Members of the neurotrophic tropomyosin receptor kinase (NTRK) gene family, NTRK1, NTRK2, and NTRK3 encode TRK receptor tyrosine kinases. Intra- or inter-chromosomal gene rearrangements produce NTRK gene fusions encoding fusion proteins which are oncogenic drivers in various solid tumors.
METHODS
This study investigated the prevalence of NTRK fusion genes and identified fusion partners in Japanese patients with solid tumors recorded in the Center for Cancer Genomics and Advanced Therapeutics database of comprehensive genomic profiling test.
RESULTS
In the analysis population (n = 46,621), NTRK fusion genes were detected in 91 patients (0.20%). The rate was higher in pediatric cases (<18 years; 1.69%) than in adults (0.16%). NTRK gene fusions were identified in 21 different solid tumor types involving 38 different partner genes including 22 (57.9%) previously unreported NTRK gene fusions. The highest frequency of NTRK gene fusions was head and neck cancer (1.31%) and thyroid cancer (1.31%), followed by soft tissue sarcoma (STS; 0.91%). A total of 97 NTRK fusion gene partners were analyzed involving mainly NTRK1 (49.5%) or NTRK3 (44.2%) gene fusions. The only fusion gene detected in head and neck cancer was ETV6::NTRK3 (n = 22); in STS, ETV6::NTRK3 (n = 7) and LMNA::NTRK1 (n = 5) were common. Statistically significant mutual exclusivity of NTRK fusions with alterations was confirmed in TP53, KRAS, and APC. NTRK gene fusion was detected from 11 STS cases: seven unclassified sarcoma, three sarcoma NOS, and one Ewing sarcoma.
CONCLUSIONS
NTRK gene fusion identification in solid tumors enables accurate diagnosis and potential TRK inhibitor therapy.
Topics: Humans; Japan; Oncogene Proteins, Fusion; Receptor, trkA; Male; Neoplasms; Female; Child; Adult; Receptor, trkC; Adolescent; Receptor, trkB; Prevalence; Young Adult; Middle Aged; Child, Preschool; Aged; Membrane Glycoproteins
PubMed: 38925616
DOI: 10.1002/cam4.7351 -
The Journal of Biological Chemistry Jun 2024The human AlkB homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct...
The human AlkB homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair (DRR) enzymes that remove 1meA and 3meC lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1ALKBH3 cells showed enhanced resistance towards S1- and S2-type methylating agents, whereas MLH1ALKBH2 cells were only resistant to S1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH23) rendered cells sensitive to S1- and S2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH23 cells have an ATR-dependent arrest at the G/M checkpoint, increased apoptotic signalling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1ALKBH23 cells. Finally, in MLH1ALKBH23 cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1.
PubMed: 38925328
DOI: 10.1016/j.jbc.2024.107492