Heat shock protein, protein folding

The yeast, Saccharomyces cerevisiae, the model eukaryote, has provided much understanding of molecular and cellular biology, as well as insights into many human diseases. In this paper we review how yeast studies are contributing to knowledge about the role of oxidative damage to cell health, and how one of the key players in Alzheimer's disease, amyloid beta (Aβ) is linked to the reactive oxygen species response involving AHP1, which encodes an alkyl hydroperoxidase, Ahp1p, a protein involved in protection from lipid peroxidation.

Living organisms are composed of biopolymers (proteins, nucleic acids, carbohydrates and lipid polymers) that are used to keep or transmit information relevant to the state of these organisms at any given time. In these processes, proteins play a central role by displaying different activities required to keep or transmit this information. In this review, I present the current knowledge about the protein sequence–structure–activity relationship and the basis for modeling this relationship. Three representative predictors relevant to the modeling of this relationship are summarized to highlight areas that require further improvement and development. I will describe how a basic understanding of this relationship is fundamental in the development of new methods to design proteins, which represents an area of multiple applications in the areas of health and biotechnology.

Research into the role of bacterial lipopolysaccharides (LPS) has escalated with the improved understanding of the LPS effects on astrocytes and neurons with relevance to brain neuroinflammation [1,2] that is now of major concern in neurodegenerative diseases. LPS activates the toll-like receptor 4 (TLR-4) in mouse and human astrocytes with TLR-4 linked to neuroinflammation and neuron death . LPS related toxicity may influence neuron membrane cholesterol by binding to cell membranes that promote amyloid beta (Aβ) aggregation and fibril formation [7] to mediate accelerated neuron death. LPS are endotoxins and essential components of the outer membrane of gram negative bacteria and consist of covalently linked segments, surface carbohydrate polymer, core oligosaccharide and acylated glycolipid that can bind to cell membranes to alter membrane interactions . LPS regulate plasma acute phase proteins (gelsolin, serum amyloid protein A, serum amyloid protein, C-reactive protein, clusterin, transthyretin) [7] and various other acute phase proteins (APP) involved in Aβ aggregation (transferrin, albumin, phospholipid transfer protein, LPS binding protein (LBP), albumin).

Around two thirds of genome sequenced bacteria encode one chaperonin
60 protein with the other third encoding between two and eight chaperonin 60 paralogues.
A surprising fi nding is that these bacterial proteins have a wide, and growing,
range of additional functions both within the bacterium, but principally when the
Cpn60 protein exits the cell and exists on the bacterial cell wall or in the bacterium’s
external milieu. These fi ndings have occurred at the same time that it has been
realised that bacterial Cpn60 proteins can assume lower oligomeric forms than that
of the prototypic tetradecameric E. coli GroEL. It is possible that lower oligomeric
forms of Cpn60 may more readily be secreted and interact with biopolymers in a
distinct manner to that of the tetradecameric homologues and paralogues. How the
Cpn60 moonlighting functions evolved is a key question to be addressed. To address
this question we postulate that the chaperonin genes have been subject to different
selective constraints over evolutionary time. Gene duplication, followed by sequence
divergence, resulted in the evolution of paralogous Cpn60 proteins that have distinct
moonlighting activities. Moreover, these functional variations might be acquired by
incorporating chemically dissimilar substitutions at functionally important residues.

Chaperonins are a group of molecular chaperones
that form large multi subunit structures and are
found in all forms of life. Encoded by the groEL and
groES genes, bacterial chaperonins are required for
appropriate folding of many cellular proteins. A
significant number of bacterial species are known to
express multiple copies of chaperonin genes, possibly
to confer redundancy of GroEL function in these species.
It is also likely that the paralogous GroELs might
be undergoing diversification of function as a consequence
of gene duplication. We argue in this article
that different chaperonins in an organism might be
involved in distinct biochemical functions that remain
to be discovered, some of which might be modulated
by different oligomeric states of the chaperonins.

Heat shock proteins (Hsps) play an important role in the development and pathogenicity of
malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of
other proteins. Hsps are thought to play a crucial role when malaria parasites invade their
host cells and during their subsequent development in hepatocytes and red blood cells. It
is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria
parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is
capable of independent folding of some proteins, its functional cooperation with heat shock
protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone
receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs
through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously
characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the
protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the
blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a
stress-inducible protein. We further explored the direct interaction between PfHop and
PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction
of the two proteins was further validated by co-immunoprecipitation studies. We
observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP
or ADP. However, ADP appears to promote the association of the two proteins better than
ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains
and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of
PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted
with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally
eluted as a protein species of twice its predicted size, suggesting that it may occur as a
dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association
in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a
stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition,
the protein is capable of self-association. The findings suggest that PfHop serves as a module
that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional
complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this
study are important towards the development of possible antimalarial inhibitors targeting
the cooperation of these two chaperones.

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous a-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.

The 42 amino acid Alzheimer’s Ab peptide is involved in the progression of Alzheimer’s disease. Here we describe the effects of intracellular Ab, produced through its attachment to either end of a green fluorescent protein, in yeast. Cells producing Ab exhibited a lower growth yield and a heat shock response, showing that Ab fusions promote stress in cells and supporting the notion that intracellular Ab is a toxic molecule. These studies have relevance in understanding the role of Ab in the death of neuronal cells, and indicate that yeast may be a new tractable model system for
the screening for inhibitors of the stress caused by Ab.