**1. Introduction**

The heat shock response is a highly conserved response of a cell to challenging environmental stress conditions [1], and the conserved set of proteins termed heat shock proteins (HSPs) is critical to the maintenance of cellular integrity under stress conditions. HSPs are primarily responsible for sustaining cellular protein homeostasis via their chaperone activity, by aiding the assembly and folding of proteins, and by inducing their degradation after irreversible damage [2].

Aside from these well-characterized functions, HSPs were also found to interact with biological membranes through binding to membrane lipids [3]. Based on previous observations, the interaction between HSPs and lipids could alleviate the deleterious consequences of stress on membranes and their integral proteins by stabilizing membrane structure [4]. Experimental evidence accumulating over the past several decades suggests that among HSPs, the family of small heat shock proteins (sHSPs) could specifically associate with lipids and membranes [5]. The sHSP family is considered the most functionally diverse of the HSPs, consisting of proteins homologous to each other mostly in their alpha-crystallin domain, forming large hetero/homo-oligomeric complexes [6]. The initial characterization of sHSP interaction with lipids and their subsequent role in preserving the integrity of membranes via these interactions has been established by studying the HSP17 protein of the

**Citation:** Csoboz, B.; Gombos, I.; Kóta, Z.; Dukic, B.; Klement, É.; Varga-Zsíros, V.; Lipinszki, Z.; Páli, T.; Vígh, L.; Török, Z. The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes. *Int. J. Mol. Sci.* **2022**, *23*, 7317. https://doi.org/10.3390/ ijms23137317

Academic Editor: Masoud Jelokhani-Niaraki

Received: 30 May 2022 Accepted: 29 June 2022 Published: 30 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

blue-green algae *Synechocystis* PCC 6803. The greater proportion of HSP17 was found to be associated with thylakoid membranes [7,8]. Its interaction with lipids increased the microviscosity of large unilamellar vesicles, consisting of synthetic or cyanobacterial lipids [9] and stabilized the lamellar liquid crystalline phase at the expense of the non-lamellar phase in membranes composed of the non-bilayer lipid, di-elaidoyl-phosphatidylethanolamine [10]. These studies [8–10] were among the first to demonstrate that HSP17 not only had an affinity towards specific membrane lipids, but also a previously unrecognized ability to stabilize membranes by modulating their lipid phase behavior. Several other examples, such as the sHSP proteins, IbpA and IbpB, of *Escherichia coli* [11] and HSP16 of *Mycobacterium tuberculosis* [12] reinforced the idea of the association of sHSPs with membranes. Moreover, the sHSP, Lo18, of the lactic acid bacterium, *Oenococcus oeni*, was upregulated and localized to the membrane fraction upon administration of the membrane fluidizer, benzyl alcohol (BA) [13]. Lo18 was also shown to interact with liposomes formed from lipids of *Oenococcus oeni* membranes and to reduce membrane fluidity of these vesicles at elevated temperatures [13]. Examples of the lipid interaction of sHSPs in other organisms are sparse. So far, only a few mammalian sHSPs were found to be associated with membranes. While HSP11 [14] and the muscle-specific HSPB2 [15] were linked to mitochondrial membranes, another mammalian sHSP, α-crystallin, was shown to associate with the plasma membranes of fiber cells in the lens of the eye [16] and to have a stabilizing effect on model membranes formed from synthetic lipids [10].

While it is clear that members of the sHSP family interact with membrane lipid vesicles in vitro, it is unclear whether this is a general property of sHSPs among different species and of different members of the sHSP family within a species. Moreover, detailed knowledge of how sHSPs affect membrane physical properties and whether they restore membrane functionality during/after heat shock is still lacking with only few reports. The existing studies sugges<sup>t</sup> that the functional consequences of the association of sHSPs with membranes may include a reduced level of fluidity [9], elevated bilayer stability [10], and the overall restoration of membrane functionality during heat stress. Based on the above studies, we hypothesized that sHSPs may have a membrane-protective role across species to keep the membrane structure intact during elevated temperatures or other membrane-perturbing conditions. The association between sHSPs and membranes may constitute a general mechanism that preserves membrane integrity when the lipid order is compromised. One of the most ubiquitously expressed mammalian sHSPs, HSPB1, appears to be a potential candidate that could serve as a general membrane-stabilizing protein in mammalian cells. HSPB1's membrane association was initially described in the context of its interaction with the membrane-connected cytoskeletal network [17–19], but recently, it has been reported to associate with synthetic liposome membranes [20]. Other physiological observations further substantiated the possible association between HSPB1 and the plasma membrane by demonstrating that HSPB1 was among the few HSPs that were upregulated upon the treatment of mouse cells with the membrane fluidizing compound, BA [21,22].

In this study, we aimed to describe the potential lipid/membrane interaction of HSPB1. We were particularly interested in determining if HSPB1 could affect the physical state of membranes and the mobility of lipids to a similar degree as described for other sHSPs. Overall, we aimed to find out if HSPB1 contributed to membrane integrity during and after stress conditions.
