HOP stabilizes the HSFA1a and plays a main role in the onset of thermomorphogenesis.

Living organisms have the capacity to respond to environmental stimuli, including warm conditions. Upon sensing mild temperature, plants launch a transcriptional response that promotes morphological changes, globally known as thermomorphogenesis. This response is orchestrated by different hormonal networks and by the activity of different transcription factors, including the heat shock factor A1 (HSFA1) family. Members of this family interact with heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90); however, the effect of this binding on the regulation of HSFA1 activity or of the role of cochaperones, such as the HSP70-HSP90 organizing protein (HOP) on HSFA1 regulation, remains unknown. Here, we show that AtHOPs are involved in the folding and stabilization of the HSFA1a and are required for the onset of the transcriptional response associated to thermomorphogenesis. Our results demonstrate that the three members of the AtHOP family bind in vivo to the HSFA1a and that the expression of multiple HSFA1a-responsive-responsive genes is altered in the hop1 hop2 hop3 mutant under warm temperature. Interestingly, HSFA1a is accumulated at lower levels in the hop1 hop2 hop3 mutant, while control levels are recovered in the presence of the proteasome inhibitor MG132 or the synthetic chaperone tauroursodeoxycholic acid (TUDCA). This uncovers the HSFA1a as a client of HOP complexes in plants and reveals the participation of HOPs in HSFA1a stability.

The role of the co-chaperone HOP in plant homeostasis during development and stress.

Proteins need to acquire their native structure in order to become fully functional. In specific cases, the active conformation is obtained spontaneously; nevertheless, many proteins need the assistance of chaperones and co-chaperones to be properly folded. These proteins help to maintain protein homeostasis under control conditions and under different stresses. HOP (HSP70-HSP90 organizing protein) is a highly conserved family of co-chaperones that assist HSP70 and HSP90 in the folding of specific proteins. In the last few years, findings in mammals and yeast have revealed novel functions of HOP and re-defined the role of HOP in protein folding. Here, we provide an overview of the most important aspects of HOP regulation and function in other eukaryotes and analyse whether these aspects are conserved in plants. In addition, we highlight the HOP clients described in plants and the role of HOP in plant development and stress response.

Native Mass Spectrometry-Guided Screening Identifies Hit Fragments for HOP-HSP90 PPI Inhibition.

Contemporary medicinal chemistry considers fragment-based drug discovery (FBDD) and inhibition of protein-protein interactions (PPI) as important means of expanding the volume of druggable chemical space. However, the ability to robustly identify valid fragments and PPI inhibitors is an enormous challenge, requiring the application of sensitive biophysical methodology. Accordingly, in this study, we exploited the speed and sensitivity of nanoelectrospray (nano-ESI) native mass spectrometry to identify a small collection of fragments which bind to the TPR2AB domain of HOP. Follow-up biophysical assessment of a small selection of binding fragments confirmed binding to the single TPR2A domain, and that this binding translated into PPI inhibitory activity between TPR2A and the HSP90 C-terminal domain. An in-silico assessment of binding fragments at the PPI interfacial region, provided valuable structural insight for future fragment elaboration strategies, including the identification of losartan as a weak, albeit dose-dependent inhibitor of the target PPI.

The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70-HSP90 organising protein) is a conserved family of co-chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin-related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co-chaperone) and these co-chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co-chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

HOP family plays a major role in long-term acquired thermotolerance in Arabidopsis.

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods.

Augmentation of CFTR maturation by S-nitrosoglutathione reductase.

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

Plasmodium falciparum Hop: detailed analysis on complex formation with Hsp70 and Hsp90.

The heat shock organizing protein (Hop) is important in modulating the activity and co-interaction of two chaperones: heat shock protein 70 and 90 (Hsp70 and Hsp90). Recent research suggested that Plasmodium falciparum Hop (PfHop), PfHsp70 and PfHsp90 form a complex in the trophozoite infective stage. However, there has been little computational research on the malarial Hop protein in complex with other malarial Hsps. Using in silico characterization of the protein, this work showed that individual domains of Hop are evolving at different rates within the protein. Differences between human Hop (HsHop) and PfHop were identified by motif analysis. Homology modeling of PfHop and HsHop in complex with their own cytosolic Hsp90 and Hsp70 C-terminal peptide partners indicated excellent conservation of the Hop concave TPR sites bound to the C-terminal motifs of partner proteins. Further, we analyzed additional binding sites between Hop and Hsp90, and showed, for the first time, that they are distinctly less conserved between human and malaria parasite. These sites are located on the convex surface of Hop TPR2, and involved in interactions with the Hsp90 middle domain. Since the convex sites are less conserved than the concave sites, it makes their potential for malarial inhibitor design extremely attractive (as opposed to the concave sites which have been the focus of previous efforts).

HOP co-chaperones contribute to GA signaling by promoting the accumulation of the F-box protein SNE in Arabidopsis.

Gibberellins (GAs) play important roles in multiple developmental processes and in plant response to the environment. Within the GA pathway, a central regulatory step relies on GA-dependent degradation of the DELLA transcriptional regulators. Nevertheless, the relevance of the stability of other key proteins in this pathway, such as SLY1 and SNE (the F-box proteins involved in DELLA degradation), remains unknown. Here, we take advantage of mutants in the HSP70-HSP90 organizing protein (HOP) co-chaperones and reveal that these proteins contribute to the accumulation of SNE in Arabidopsis. Indeed, HOP proteins, along with HSP90 and HSP70, interact in vivo with SNE, and SNE accumulation is significantly reduced in the hop mutants. Concomitantly, greater accumulation of the DELLA protein RGA is observed in these plants. In agreement with these molecular phenotypes, hop mutants show a hypersensitive response to the GA inhibitor paclobutrazol and display a partial response to the ectopic addition of GA when GA-regulated processes are assayed. These mutants also display different phenotypes associated with alterations in the GA pathway, such as reduced germination rate, delayed bolting, and reduced hypocotyl elongation in response to warm temperatures. Remarkably, ectopic overexpression of SNE reverts the delay in germination and the thermally dependent hypocotyl elongation defect of the hop1 hop2 hop3 mutant, revealing that SNE accumulation is the key aspect of the hop mutant phenotypes. Together, these data reveal a pivotal role for HOP in SNE accumulation and GA signaling.

HOP3 a new regulator of the ER stress response in Arabidopsis with possible implications in plant development and response to biotic and abiotic stresses.

HOPs (heat shock protein 70 (HSP70)-heat shock protein 90 (HSP90) organizing proteins) are a highly conserved family of cytosolic cochaperones. In a recent study we showed that HOP3, a member of the HOP family in Arabidopsis, plays an essential role during endoplasmic reticulum (ER) stress in plants. Interestingly, we also demonstrated that AtHOP3 interacts with binding immunoglobulin protein (BiP), a major ER-resident chaperone. All these data suggest that HOP3 could assist BiP in protein folding in the ER. These findings open the exciting possibility that HOP3, through its role in the alleviation of ER stress, could play an important function during different developmental processes and in response to different biotic and abiotic stresses.

The role of heat stress on the age related protein carbonylation.

Since the proteins are involved in many physiological processes in the organisms, modifications of proteins have important outcomes. Protein modifications are classified in several ways and oxidative stress related ones take a wide place. Aging is characterized by the accumulation of oxidized proteins and decreased degradation of these proteins. On the other hand protein turnover is an important regulatory mechanism for the control of protein homeostasis. Heat shock proteins are a highly conserved family of proteins in the various cells and organisms whose expressions are highly inducible during stress conditions. These proteins participate in protein assembly, trafficking, degradation and therefore play important role in protein turnover. Although the entire functions of each heat shock protein are still not completely investigated, these proteins have been implicated in the processes of protection and repair of stress-induced protein damage. This study has focused on the heat stress related carbonylated proteins, as a marker of oxidative protein modification, in young and senescent fibroblasts. The results are discussed with reference to potential involvement of induced heat shock proteins. This article is part of a Special Issue entitled: Protein Modifications. BIOLOGICAL SIGNIFICANCE: Age-related protein modifications, especially protein carbonylation take a wide place in the literature. In this direction, to highlight the role of heat shock proteins in the oxidative modifications may bring a new aspect to the literature. On the other hand, identified carbonylated proteins in this study confirm the importance of folding process in the mitochondria which will be further analyzed in detail.