Mutations inisocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1-5. Mutant IDH proteins in IHCC ...and other malignancies acquire an abnormal enzymatic activity allowing them to convert a-ketoglutarate (aKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple aKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extra cellular matrix maturation6-10.However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4a, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4a silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs4,5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in Isocitrate dehydrogenase 1 (
IDH1
) and
IDH2
are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer
1
–
5
. Mutant IDH proteins in ...IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert alpha-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation
6
–
10
. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models (GEMMs) expressing mutant IDH in the adult liver show aberrant response to hepatic injury, characterized by HNF4α silencing, impaired hepatocyte differentiation and markedly elevated levels of cell proliferation. Moreover, mutant IDH and activated Kras, genetic alterations that co-exist in a subset of human IHCCs
4
,
5
, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel GEMM of IDH-driven malignancy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ionic liquids (ILs) are known to stabilize protein conformations in aqueous medium. Importantly, ILs can also act as refolding additives in urea-driven denaturation of proteins. However, despite the ...importance of the problem, detailed microscopic understanding of the counteraction effects of ILs on urea-induced protein denaturation remains elusive. In this work, atomistic molecular dynamics (MD) simulations of the protein α-lactalbumin have been carried out in pure aqueous medium, in 8 M binary urea–water solution and in ternary urea–IL–water solutions containing ammonium-based ethyl ammonium acetate (EAA) as the IL at different concentrations (1–4 M). Attempts have been made to quantify detailed molecular-level understanding of the origin behind the counteraction effects of the IL on urea-induced partial unfolding of the protein. The calculations revealed significant conformational changes of the protein with multiple free energy minima due to its partial unfolding in binary urea–water solution. The counteraction effect of the IL was evident from the enhanced structural rigidity of the protein with propensity to transform into a single native free energy minimum state in ternary urea–IL–water solutions. Such an effect has been found to be associated with preferential direct binding of the IL components with the protein and simultaneous expulsion of urea from the interface, thereby providing additional stabilization of the protein in ternary solutions. Most importantly, modified rearrangement of the hydrogen bond network at the interface due to the formation of stronger protein–cation (PC) and protein–anion (PA) hydrogen bonds by breaking relatively weaker protein–urea (PU) and protein–water (PW) hydrogen bonds has been recognized as the microscopic origin behind the counteraction effects of EAA on urea-induced partial unfolding of the protein.
CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision ...editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms.
In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing.
Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.
Room temperature molecular dynamics (MD) simulations of the globular protein α-lactalbumin in aqueous solutions containing BMIM (1-butyl-3-methylimidazolium) based ionic liquids (ILs) with a series ...of Hofmeister anions have been carried out. In particular, effects of anions of different shapes/sizes and hydrophobic/hydrophilic characters, namely, thiocyanate (SCN–), dicyanamide (DCA–), methyl sulfate (MS–), triflate (TFO–), and bis(trifluoromethane) sulfonimide (TF2N–) on the heterogeneous dynamic environment at the interface around the protein have been explored. The calculations revealed exchange of population between water and IL cation–anion components beyond the first layer of bound water molecules at the protein surface. Further, increasingly restricted diffusivity of the IL components and water around the protein has been found to be associated with a longer time scale for the onset of dynamic heterogeneity at the interface. Restricted diffusivity of water molecules at the interface in the presence of the ILs has been found to be correlated with the longer time scale of structural relaxations of protein–water hydrogen bonds at the interface. More importantly, the time scale associated with the reorientations of the anions has been found to be anticorrelated with their translational diffusivity, with the effect being more at the interface as compared to the bulk IL solutions. It is demonstrated that the nonuniform ability of the anions to form hydrogen bonds with water due to their differential shapes and hydrophilic characters is the origin of such anticorrelation.