10 research outputs found
Crystallographic study of G178S mutant of human proliferating cell nuclear antigen
Crystallization and diffraction studies of human PCNA G178S mutant are reported
Initial crystallographic study of human PCNA in complex with a peptide containing the noncanonical PIP-box sequence of human DNA polymerase Ī¹
Crystallization and diffraction studies of a human PCNAāPolĪ¹ peptide complex are reported
Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1/PCTK1/Cdk16 Inhibits In Vivo Cancer Growth
PCTAIRE1/CDK16/PCTK1 plays critical roles in cancer cell proliferation and antiapoptosis. To advance our previously published in vitro results with PCTAIRE1 silencing, we examined the in vivo therapeutic potential of this approach by using small interfering RNA (siRNA) encapsulated by lipid nanoparticles. Therapy experiments of PCTAIRE1 siRNA were performed using human HCT116 colorectal cancer cells and human A2058 melanoma cells. A single dose of PCTAIRE1 siRNA-lipid nanoparticles was found to be highly effective in reducing in vivo PCTAIRE1 expression for up to 4 days as assayed by immunoblotting. Therapy experiments were started 4 days after subcutaneous injection of cancer cells. Treatment with PCTAIRE1 siRNA-lipid nanoparticles (0.5āmg/kg RNA, twice a week) reduced tumor volume and weight significantly compared with the scramble-control group. Histopathological analysis (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) showed increased apoptosis of tumor cells treated with PCTAIRE1-siRNA. Overall, our results demonstrate that siRNA treatment targeting PCTAIRE1 is effective in vivo, suggesting that PCTAIRE1 siRNA-lipid nanoparticles might be a novel therapeutic approach against cancer cells
Structural Basis for Novel Interactions between Human Translesion Synthesis Polymerases and Proliferating Cell Nuclear Antigen*Sā
Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows
continued DNA synthesis, even in the presence of damaged DNA templates.
Mammals have multiple DNA polymerases specialized for TLS, including PolĪ·,
PolĪ¹, and PolĪŗ. These enzymes show preferential bypass for
different lesions. Proliferating cell nuclear antigen (PCNA), which functions
as a sliding clamp for the replicative polymerase PolĪ“, also interacts
with the three TLS polymerases. Although many PCNA-binding proteins have a
highly conserved sequence termed the PCNA-interacting protein box (PIP-box),
PolĪ·, PolĪ¹, and PolĪŗ have a noncanonical PIP-box sequence. In
response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the
RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS.
Consistent with this, these three TLS polymerases have one or two ubiquitin
binding domains and are recruited to replication forks via interactions with
ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding
domain. However, it is unclear how these TLS polymerases interact with PCNA.
To address the structural basis for interactions between different TLS
polymerases and PCNA, we determined crystal structures of PCNA bound to
peptides containing the noncanonical PIP-box of these polymerases. We show
that the three PIP-box peptides interact with PCNA in different ways, both
from one another and from canonical PIP-box peptides. Especially, the PIP-box
of PolĪ¹ adopts a novel structure. Furthermore, these structures enable us
to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated
PCNA. Our results will provide clues to understanding the mechanism of
preferential recruitment of TLS polymerases to the stalled forks
Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins
B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy. We used a combination of NMR spectroscopy-based methods, mutagenesis, and functional studies to define the interaction site of a Nur77 peptide on anti-apoptotic Bcl-2 family proteins and reveal a novel interaction surface. Nur77 binds adjacent to the BH3 peptide-binding crevice, suggesting the possibility of cross-talk between these discrete binding sites. Mutagenesis of residues lining the identified interaction site on Bcl-B negated the interaction with Nur77 protein in cells and prevented Nur77-mediated modulation of apoptosis and autophagy. The findings establish a new protein interaction site with the potential to modulate the apoptosis and autophagy mechanisms governed by Bcl-2 family proteins