Potential for targeting small heat shock protein modifications

Abstract

Small heat shock proteins (sHSPs) play key roles in cellular stress and several human diseases. The direct effects of some post-translational modifications (PTMs) on certain sHSPs have been characterized, raising the possibility that small molecules could be used to modulate these modifications and indirectly up- or downregulate sHSP activity.

sHSPs are oligomeric chaperones that prevent protein aggregation

sHSPs are ATP-independent protein chaperones that bind to destabilized client proteins to clear unfolded or misfolded proteins in response to different stress conditions [1,2]. There are ten sHSPs in humans, with gene names HSPB1–HSBP10. The corresponding proteins show different expression patterns, with several being ubiquitous, while others are confined to specific tissues or structures. The general structure of sHSPs consists of a disordered hydrophobic N-terminal region (NTR), a structured α-crystallin domain (ACD), and a polar C-terminal domain (CTD) (Figure 1A). The ACD has an IgG-like β-sandwich fold structure that enables dimerization via an antiparallel alignment resulting in the formation of hydrophobic cleft in the ACD. This cleft is the major site of client-protein binding and is required for most of the sHSP chaperone functions. The conserved sHSPs are observed as oligomers mediated by interactions in the NTR and CTD and regulated in response to different environmental conditions to control the chaperone activity (Figure 1A). One well-studied example is the interaction between the ACD cleft and a three-residue IXI motif (isoleucine/valine – any amino acid – isoleucine/valine) found in either the NTR or CTD of some sHSPs. The IXI motif can dynamically bind to the ACD cleft [3], allowing sHSPs to switch between ACD-bound and unbound states, regulating their activity and oligomerization (Figure 1B). This and other interactions between different dimeric building blocks allow sHSPs to grow into heterogeneous complexes which contain 12 or more subunits. Here, we highlight the general areas of human health and disease where modulation of sHSP activity could be therapeutically beneficial. We then describe PTMs that have been characterized to directly change sHSP activity as potential, targetable mechanisms for therapeutic development.

Figure 1. Small heat shock protein (sHSP) structure.

(A) sHSPs contain a central α-crystallin domain (ACD) that binds client proteins in a hydrophobic cleft. Additional interactions result in oligomerization of the sHSP. Illustrated by HSP27 (PDBs: 4MJH, 6DV5). (B) Some sHSPs have an IXI motif that can bind to the ACD cleft and dynamically regulate substrate binding.

sHSPs play important roles in human diseases but are difficult to directly target

As indicated within their names, sHSPs are upregulated by increases in temperature change, and they are sensitive to other environmental changes to adjust their activities. The largest function of sHSPs appears to be the direct interaction with misfolded or unfolded proteins to inhibit protein aggregation in response to various cellular stressors. Additionally, they can bind to exposed portions of folded proteins to regulate different cell signaling pathways. This family of proteins plays an important role in many diseases. Mutations in certain sHSPs can cause degenerative diseases, such as Charcot-Marie-Tooth neuropathy (CMT2) and/or distal hereditary motor neuropathy (dHMN). They can also contribute to cardiomyopathies and reduced cardiac function [4]. sHSPs can also regulate amyloid fibril aggregation associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Some sHSPs are upregulated in Alzheimer’s disease brains and colocalize with protein aggregates [5]. sHSPs can also inhibit multiple steps of amyloid fibril formation in vitro, suggesting a protective function for these proteins that is ultimately in a losing battle. Increased expression of sHSPs is closely associated with the progression of cancers, where their expression levels are upregulated in various types of tumors [6]. sHSPs can inactivate crucial cell-death regulators, such as caspases or Bcl-2 proteins, enabling cancer cells to escape from apoptosis more readily. sHSPs also play essential roles in the formation and upkeep of cytoskeletal structure, such as those found in the eye lens and muscle tissues. Notably, two sHSPs, αA-crystallin (HSPB4) and αB-crystallin (HSPB5), make up 40% of the total protein content in the lens. Finally, high levels of sHSPs are expressed in muscle tissues due to the high metabolic demand and downstream oxidative stress.

These disease associations and mechanisms of action make sHSPs potentially attractive targets for drugs that either increase or decrease their chaperone activities [7]. However, potentially due to their heterogeneous structure, this overall goal has not yet been achieved. For example, one HSP27 inhibitor in clinical development (Apatorsen, OGX-427) is an antisense oligonucleotide for depleting HSP27 levels, and not a direct modulator.

PTMs can directly up- or downregulate sHSP activity

PTMs are covalent, chemical modifications of proteins that have the potential to alter underlying biophysical properties and biochemistry. Several sHSPs have been found to bear PTMs that alter their chaperone activity. Here, we highlight those PTMs where direct biochemical experiments have determined their effects on sHSP activity and structure, including phosphorylation, O-GlcNAc modification, and chemical glycation.

N-terminal phosphorylation increases HSPB1 activity

sHSPs are phosphorylated [8] and the consequences of some of these modifications have been characterized. For example, HSP27 (HSPB1) is modified at several serine residues within NTR, including characterized phosphorylation at Ser15, Ser78, and Ser82. Size exclusion chromatography and electron microscopy have shown that phosphorylation of Ser15, Ser78, and Ser82 decreases the size of HSP27 oligomers [9]. To analyze the effect of phosphorylation within cells, the modified serine residues were substituted with the phosphoserine isosteres aspartate or glutamate. Consistent with the bona fide phosphorylation, the combination of all three mutants (S15D, S78D, and S82D) resulted in the formation of small HSPB1 oligomers. However, the individual mutants still formed large oligomers. Subsequent combinations of mutants demonstrated that phosphorylation of Ser15 and Ser82 is most likely responsible for the formation of small oligomers. Notably, HSP27 bearing triple phosphorylation or all three S to D mutations displayed increased chaperone activity with insulin as a model client in vitro [10]. In a separate study, mutation of Ser82 to alanine, thereby blocking phosphorylation, decreased the protection afforded to heat-shocked NIH3T3 cells overexpressing different HSP27 proteins [11], further suggesting an important role for increasing chaperone activity. Biochemical and cell-based experiments have indicated that phosphorylation of HSP27 in the NTR increases chaperone activity and may represent a dynamic mechanism to control HSP27 in response to cellular stimuli. Therefore, identification of the phosphatase that removes these modifications might be targeted to increase the levels of HSP27 phosphorylation and activity in human diseases associated with protein aggregation. Alternatively, kinase inhibitors might be exploited to lower levels of phosphorylation and reduce chaperone activity in cancer.

O-GlcNAc modification increases the activity of some sHSPs

Another representative PTM of sHSPs is O-GlcNAc modification. This type of glycosylation is a dynamic modification of serine and threonine residues of intracellular proteins with the monosaccharide N-acetylglucosamine. Notably, HSP27, αA-crystallin, and αB-crystallin have been long known to be O-GlcNAc modified, and proteomic analysis of cells and tissues has localized endogenous O-GlcNAc modifications to residues near their respective IXI motifs in the CTDs. We used synthetic protein chemistry centered around the technique of expressed protein ligation (EPL) to prepare each of these sHSPs bearing site-specific O-GlcNAc modifications at each of the identified sites. Subsequent analysis demonstrated that O-GlcNAc blocked the physical interaction between the ACD chaperone cleft and the IXI-motif. For all three sHSPs this resulted in increased chaperone activity against the amyloidforming client proteins α-synuclein and Aβ1-42 [12]. The effect of O-GlcNAc on HSPB1 was also sub-stoichiometric, demonstrating that not 100% of the chaperone needs to be modified to have increased antiaggregation activity. Small-molecule inhibitors that increase O-GlcNAc in the brain are currently in clinical trials for Alzheimer’s disease and sHSP O-GlcNAc modification may be a contributing factor to their protective effects. We speculate that these inhibitors may also show therapeutic benefit in other human diseases involving protein aggregation.

Glycation has site-specific effects on HSPB1 activity

sHSPs are also subject to nonenzymatic modifications such as glycation. Methylglyoxal (MG; 2-oxopropanal) modification has been identified within several sHSPs such as HSP27, αA-crystallin, and αB-crystallin. Nonspecific chemical glycation of αA-crystallin with glucose or fructose decreased its chaperone activity against model proteins, while modification with MG resulted in increased activity [13], suggesting that modification type and location might play important roles in chaperone function. The MG modification was localized to arginine residues in the NTR, but subsequent mutational studies yielded proteins that displayed both increased and decreased chaperone activity, making a definitive conclusion about the effect of glycation in this system elusive. Similar to the synthetic approach mentioned above for O-GlcNAc, HSP27 containing the advanced glycation end product argpyrimidine [(2S)-2-amino-5-[(5-hydroxy-4,6-dimethylpyrimidin-2-yl)amino] pentanoic acid] at residue 188 in the CTD was generated. Biochemical analysis showed that this modification impaired the chaperone activity in vitro [14]. The same group subsequently synthesized HSP27 with argpyrimidine at several residues in the NTR. In contrast to the CTD glycation, these modifications generally increased the chaperone activity and displayed an additive effect when all five argpyrimidines were incorporated [15]. Unlike phosphorylation and O-GlcNAc modification that appear to generally activate sHSPs, the effects of glycation are clearly more complicated and require additional investigation. These site-specific and conflicting effects and the direct chemical nature of glycation make the pharmacological targeting of this modification more difficult than others. However, we speculate that diseases that increase glycation, like uncontrolled hyperglycemia in diabetes, may disrupt normal sHSP activity with detrimental effects.

Concluding remarks

Recent studies have clearly demonstrated that PTMs can have a direct impact on the formation and function of sHSPs, as discussed above. As highlighted by the case of phosphorylation and O-GlcNAc, we believe that these PTMs may be targeted therapeutically to treat protein aggregation diseases. Additionally, it is interesting to consider whether a proteoform with a specific PTM or pattern of PTMs might be selectively targeted due to changes in ligandability. We believe that direct analysis of other PTMs and additional sHSPs has the potential to uncover other modifications, where the enzymes that add and remove those PTMs might be new targets for the development of therapeutics. Challenges in this area moving forward, include the preparation of homogenously modified sHSPs for biochemical studies. We recommend that simple loss-of-modification studies involving mutations that block PTMs should be judged carefully, as these types of mutations may have their own effects outside of the PTM of interest. Additionally, identification of the specific enzymes that contribute to a certain PTM is critical for inhibitor development. Finally, ATP-dependent chaperones like HSP90 are also regulated by a variety of PTMs, in a combinatorial fashion that has been termed the chaperone code. We believe that modifications of PTMs fit into this overall theme and the overall system should be considered to understand the global consequences of altering the levels of one PTM or another pharmacologically [16].