The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities. The Hsp70 chaperones regulate protein metabolism, including folding, unfolding, subcellular localization, aggregation/disaggregation and incorporation into protein complexes. Recent studies have revealed the mechanisms of functions of Hsp70s and their co-chaperones, highlighting new opportunities for modulating disease-related Hsp70 roles.

The Hsp70 chaperone network

The p53 protein is modified by as many as 50 individual posttranslational modifications. Many of these occur in response to genotoxic or nongenotoxic stresses and show interdependence, such that one or more modifications can nucleate subsequent events. This interdependent nature suggests a pathway that operates through multiple cooperative events as opposed to distinct functions for individual, isolated modifications. This concept, supported by recent investigations, which provide exquisite detail as to how various modifications mediate precise protein-protein interactions in a cooperative manner, may explain why knockin mice expressing p53 proteins substituted at one or just a few sites of modification typically show only subtle effects on p53 function. The present article focuses on recent, exciting progress and develops the idea that the impact of modification on p53 function is achieved through collective and integrated events.

https://cshperspectives.cshlp.org/content/1/6/a000950.full.pdf

Posttranslational Modification of p53: Cooperative Integrators of Function

Lactobionic acid: A high value-added lactose derivative for food and pharmaceutical applications

The p53 tumor suppressor is a critical component of the cellular response to stress. As it can inhibit cell growth, p53 is mutated or functionally inactivated in most tumors. A multitude of protein-protein interactions with transcriptional cofactors are central to p53-dependent responses. In its activated state, p53 is extensively modified in both the N- and C-terminal regions of the protein. These modifications, especially phosphorylation of serine and threonine residues in the N-terminal transactivation domain, affect p53 stability and activity by modulating the affinity of protein-protein interactions. Here, we review recent findings from in vitro and in vivo studies on the role of p53 N-terminal phosphorylation. These modifications can either positively or negatively affect p53 and add a second layer of complex regulation to the divergent interactions of the p53 transactivation domain.

/pdf/p53-n-terminal-phosphorylation-a-defining-layer-of-complex-564n7gf936.pdf

p53 N-terminal phosphorylation: a defining layer of complex regulation

http://www.jbc.org/content/289/37/25431.full.pdf

Inhibition of epithelial to mesenchymal transition by E-cadherin up-regulation via repression of slug transcription and inhibition of E-cadherin degradation: dual role of scaffold/matrix attachment region-binding protein 1 (SMAR1) in breast cancer cells.

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

Cellobiose dehydrogenase production by the mycelial culture of the mushroom Termitomyces clypeatus

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.

Conformational Adaptation in the E. coli Sigma 32 Protein in Response to Heat Shock

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 σ32 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, σF 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-...

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

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.

Srijata Mukherjee

Papers

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

Cellobiose dehydrogenase production by the mycelial culture of the mushroom Termitomyces clypeatus

Conformational Adaptation in the E. coli Sigma 32 Protein in Response to Heat Shock

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

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