Biology subjects

Lee, K. J.

Publications and source records attributed to Lee, K. J..

24 records · Page 2Linked to original sources

bioRxiv2021

Pectin remodeling belongs to a homeostatic system and triggers transcriptomic and hormonal modulations

O_LIHere, we focused on the biological modifications arisen from a strong and transient variation of the pectin methylesterification status during the seed-to-seedling transition. C_LIO_LIA reverse genetic approach was used to trigger specific reduction of pectin de-methylesterification during the seed maturation stage and the related physiological effects were assessed using a combination of biochemical, transcriptomic and microscopic analyses. C_LIO_LIArabidopsis PME36 is required to implement the characteristic pattern of de-methylesterified pectin in the mature seed. While this pattern is strongly impaired in pme36-1 and pme36-2 mature seed, no phenotypical effect is observed in the knockout mutant during seed germination. By analyzing hormone homeostasis and gene expression regulation, we show a strong and dynamic physiological disorder in the mutant, which reveals the existence of a complex compensatory mechanism overcoming the defect in pectin de-methylesterification. C_LIO_LIOur results reveal that pectin methylesterification status acts as upstream modulator involved in an undescribed homeostatic system in which pectin remodeling, hormone signaling and transcriptomic regulations interact to ensure the maintenance of a normal seed-to-seedling developmental program. C_LI

plant biology↗

bioRxiv2021

In vitro assessment of an engineered tBID-based safety switch system in human T lymphocytes

Cell therapy as a promising therapeutic modality to treat cancer has been intensively studied for decades. However, the clinical trials have indicated that patients under T cell therapy may develop severe cytokine release syndrome resulting in hospitalization or even death. Furthermore, genetic modifications to promote proliferation and persistence of T cells could result in high numbers of long-lived engineered cells in patients after treatment. In this study we developed a novel tBID-based safety switch regulated through a small molecule inducible on switch to control undesired toxicity or ablate engineered cells as needed. We compared several tBID-based safety switch constructs with the clinically validated safety switches, HSV-TK (human herpes simplex virus thymidine kinase) and iCasp9 (inducible Caspase 9), few tBID-based safety switch constructs were systematically tested and investigated in vitro in Jurkat or human primary T cells. We demonstrated that our novel tBID based safety switch, with optimization, was able to eliminate up to ~90% of transduced human primary T cells within 72hr after activation, providing an alternative switch system to manage safety concerns for cell therapy.

molecular biology↗

bioRxiv2020

Active synthesis of collagen (I) homotrimer and matrisomal proteins in Dupuytren's fibrosis

Dupuytrens disease is a common fibroproliferative disease of the palmar fascia of the hand with advanced cases treated surgically. Anti-tumour necrosis factor (TNF) injection has undergone phase 2 trials and may be effective in slowing early-stage disease progression. Here we sought to determine how new synthesis of type I collagen in Dupuytrens differs from normal palmar fascia samples and to analyse the role of TNF in aberrant collagen synthesis. Model non-fibrotic, but fibrous connective tissues, were used to analyse active type I collagen protein synthesis in development, ageing and degenerative disease, where it was restricted to early development and ruptured tissue. Dupuytrens tissue was shown to actively synthesise type I collagen, including abnormal type I collagen homotrimer. TNF- reduced COL1A2 gene expression only in the presence of serum in 2D cell culture and had opposing effects on collagen protein production in the presence or absence of serum. TNF- had only limited effects in 3D tendon-like constructs. Anti-TNF did not reduce type I collagen synthesis in 3D tendon-like constructs or prevent type I collagen homotrimer synthesis in Dupuytrens tissue. Hence, modulation of the TNF- pathway in Dupuytrens disease is unlikely to prevent the pathological collagen accumulation that is characteristic of fibrosis.

cell biology↗

bioRxiv2020

Collagen (I) homotrimer does not cause bone fragility but potentiates the osteogenesis imperfecta (oim) mutant allele

Type I collagen is the major structural component of bone where it exists as an (1)2(2)1 heterotrimer in all vertebrates. The osteogenesis imperfecta (oim) mouse model comprising solely homotrimeric (1)3 type I collagen, due to a dysfunctional 2 chain, has a brittle bone phenotype implying that the heterotrimeric form is required for physiological bone function. However, humans with rare null alleles preventing synthesis of the 2 chain have connective tissue and cardiovascular abnormalities (cardiac valvular Ehlers Danlos Syndrome), without evident bone fragility. Conversely a prevalent human single nucleotide polymorphism leading to increased homotrimer synthesis is associated with osteoporosis. Whilst the oim line is well-studied, whether homotrimeric type I collagen is functionally equivalent to the heterotrimeric form in bone has not been demonstrated. Col1a2 null and oim mouse lines were used in this study and bones analysed by microCT and 3-point bending. RNA was also extracted from heterozygote tissues and allelic discrimination analyses performed using qRT-PCR. Here we comprehensively show for the first time that mice lacking the 2(I) chain do not have impaired bone biomechanical or structural properties, unlike oim homozygous mice. However Mendelian inheritance was affected in male mice of both lines and male mice null for the 2 chain exhibited age-related loss of condition. The brittle bone phenotype of oim homozygotes could result from detrimental effects of the oim mutant allele, however, the phenotype of oim heterozygotes is known to be less severe. We used allelic discrimination to show that the oim mutant allele is not downregulated in heterozygotes. We then tested whether gene dosage was responsible for the less severe phenotype of oim heterozygotes by generating compound heterozygotes. Data showed that compound heterozygotes had impaired bone structural properties as compared to oim heterozygotes, albeit to a lesser extent than oim homozygotes. Hence, we concluded that the presence of heterotrimeric collagen-1 in oim heterozygotes alleviates the effect of the oim mutant allele but a genetic interaction between homotrimeric collagen-1 and the oim mutant allele leads to bone fragility.

physiology↗

bioRxiv2019

Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer

DNA repair defects have been increasingly focused on as therapeutic targets. In hormone positive breast cancer, XRCC1-deficient tumors have been identified and proposed as targets for combination therapies that damage DNA and inhibit DNA repair pathways. XRCC1 is a scaffold protein that functions in base excision repair (BER) by mediating essential interactions between DNA glycosylases, AP endonuclease, poly(ADP-ribose) polymerase 1, DNA polymerase {beta} (POL {beta}), and DNA ligases. Loss of XRCC1 confers BER defects and hypersensitivity to DNA damaging agents. BER defects have not been evaluated in triple negative breast cancer (TNBC), for which new therapeutic targets and therapies are needed. To evaluate the potential of XRCC1 as an indicator of BER defects in TNBC, we examined XRCC1 expression and localization in the TCGA database and in TNBC cell lines. High XRCC1 expression was observed for TNBC tumors in the TCGA database and expression of XRCC1 varied between TNBC cell lines. We also observed changes in XRCC1 subcellular localization in TNBCs that alter the ability to repair base lesions and single-strand breaks. Subcellular localization changes were also observed for POL {beta} that did not correlate with XRCC1 localization. Basal levels of DNA damage were also measured in the TNBC cell lines, and damage levels correlated with observed changes in XRCC1 expression, localization, and repair functions. The results confirmed that XRCC1 expression changes may indicate DNA repair capacity changes but emphasize that basal DNA damage levels along with expression and localization are better indicators of DNA repair defects. Given the observed over-expression of XRCC1 in TNBC preclinical models and the TCGA database, XRCC1 expression levels should be considered when evaluating treatment responses of TNBC preclinical model cells.

cancer biology↗

bioRxiv2018

PTK2 regulates the UPS impairment via p62 phosphorylation in TDP-43 proteinopathy

TDP-43 proteinopathy is a common feature in a variety of neurodegenerative disorders including Amyotrophic lateral sclerosis (ALS) cases, Frontotemporal lobar degeneration (FTLD), and Alzheimers disease. However, the molecular mechanisms underlying TDP-43-induced neurotoxicity are largely unknown. In this study, we demonstrated that TDP-43 proteinopathy induces impairment in ubiquitin-proteasome system (UPS) evidenced by an accumulation of ubiquitinated proteins and reduction of proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2 as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduces ubiquitin aggregates and attenuated TDP-43-induced cytotoxicity in Drosophila model of TDP-43 proteinopathy. We further identified that phosphorylation of p62 at serine 403 (p-p62S403), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TDP-43 overexpression and dependent on activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of p62 (p62S403A) significantly represses accumulation of polyubiquitinated proteins and neurotoxicity induced by TDP-43 overexpression in neuronal cells. In addition, inhibition of TBK1, a kinase which phosphorylates S403 of p62, ameliorates neurotoxicity upon UPS impairment in neuronal cells. Taken together, our data suggest that activation of PTK2-TBK1-p62 axis plays a critical role in the pathogenesis of TDP-43 by regulating neurotoxicity induced by UPS impairment. Therefore, targeting PTK2-TBK1-p62 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TDP-43 proteinopathy.

neuroscience↗