A small HDM2 antagonist peptide and a USP7 inhibitor synergistically inhibit the p53-HDM2-USP7 circuit.

HDM2, an E3 ubiquitin ligase, is a crucial regulator of many proliferation-related pathways. It is also one of the primary regulators of p53. USP7, a deubiquitinase, also plays a key role in the regulation of both p53 and HDM2, thus forming a small regulatory network with them. This network has emerged as an important drug target. Development of a synergistic combination targeting both proteins is desirable and important for regulating this module. We have developed a small helically constrained peptide that potently inhibited p53-HDM2 interaction and exerted anti-proliferative effects on p53+/+ cells. A combination of this peptide-when attached to cell entry and nuclear localization tags-and a USP7 inhibitor showed synergistic anti-proliferative effects against cells harboring wild-type alleles of p53. Synergistic inhibition of two important drug targets may lead to novel therapeutic strategies.

Conformational adaptation in the E. coli sigma 32 protein in response to heat shock.

E. coli, like other organisms, responds to heat shock by rapidly up-regulating several proteins, including chaperones. The heat-shock sigma factor, sigma 32 (σ(32)), a transcription factor, plays a pivotal role in this response. The level of σ(32) is normally kept low through a DnaK/J mediated degradation. Elevated temperature rapidly increases the σ(32) level and initiates a heat-shock response. A plausible way for the up-regulation of free σ(32) levels would be to destabilize the σ(32):DnaK:DnaJ complex initiated via a conformational change in σ(32) structure at elevated temperatures. In this study, we have modeled the E. coli σ(32) structure by homology modeling and conducted extensive molecular dynamics (MD) simulations at non-heat-shock (30 °C) and heat-shock (42 °C) temperatures. Substantial structural rearrangements at 42 °C were observed around the N-terminus (residues 11-60, which cover the DnaJ binding region) and the region spanning residues 190-210 (covering the DnaK binding site, residues 198-201). At 42 °C, a large amount of helix melting and structural destabilization was observed around residues 11-60, while regions 91-101 and 216-221 of σ(32) undergo conformational change, leading to formation of a lid-like structure over region 198-VLYL-201 resulting in reduced accessibility of the DnaK binding sites. These temperature induced melting and fluctuations observed around the DnaJ and/or DnaK binding regions suggest reduction of DnaK/DnaJ affinity for σ(32) at 42 °C, which is further supported by our molecular docking analysis. Emission maxima of environment sensitive fluorescence probes inserted at several cysteine mutants of σ(32) protein at 30 and 42 °C are also supportive of the structural changes observed in the molecular dynamics study.

Alternative sigma factors in the free state are equilibrium mixtures of open and compact conformations.

Conformational switching upon core RNA polymerase binding is an integral part of functioning of bacterial sigma factors. Here, we have studied dynamical features of two alternative sigma factors. A study of fluorescence resonance energy transfer and hydrodynamic measurements in Escherichia coli σ(32) suggest a compact shape like those found in complex with anti-sigma factors. On the other hand, the fluorescence anisotropy of probes attached to different regions of the protein and previous hydrogen exchange measurements suggest significant internal flexibility, particularly in the C-terminal half and region 1. In a homologous sigma factor, σ(F) of Mycobacterium tuberculosis, emission spectra and fluorescence resonance energy transfer between the single tryptophan (W112) and probes placed in different regions suggest a compact conformation for a major part of the N-terminal half encompassing region 2 and the flexible C-terminal half. Fluorescence anisotropy measurements suggest significant flexibility in the C-terminal half and region 1, as well. Thus, free alternative sigma factors may be in equilibrium between two conformations: a compact one in which the promoter interacting motifs are trapped in the wrong conformation and another less abundant one with a more open and flexible conformation. Such flexibility may be important for promoter recognition and interaction with many partner proteins.

SMAR1 forms a ternary complex with p53-MDM2 and negatively regulates p53-mediated transcription.

The intra-cellular level of tumor suppressor protein p53 is tightly controlled by an autoregulatory feedback loop between the protein and its negative regulator MDM2. The role of MDM2 in down-regulating the p53 response in unstressed conditions and in the post-stress recovery phase is well documented. However, interplay between the N-terminal phosphorylations and C-terminal acetylations of p53 in this context remains unclear. Here, we show that an MAR binding protein SMAR1 interacts with MDM2 and the Ser15 phosphorylated form of p53, forming a ternary complex in the post stress-recovery phase. This triple complex formation between p53, MDM2 and SMAR1 results in recruitment of HDAC1 to deacetylate p53. The deacetylated p53 binds poorly to the target promoter (p21), which results in switching off the p53 response, essential for re-entry into the cell cycle. Interestingly, the knock-down of SMAR1 using siRNA leads to a prolonged cell-cycle arrest in the post stress recovery phase due to ablation of p53-MDM2-HDAC1 interaction. Thus, the results presented here for the first time highlight the role of SMAR1 in masking the active phosphorylation site of p53, enabling the deacetylation of p53 by HDAC1-MDM2 complex, thereby regulating the p53 transcriptional response during stress rescue.