Membrane phospholipids activate the inflammatory response in macrophages by various mechanisms.

Exosomes, which are small membrane-encapsulated particles derived from all cell types, are emerging as important mechanisms for intercellular communication. In addition, exosomes are currently envisioned as potential carriers for the delivery of drugs to target tissues. The natural population of exosomes is very variable due to the limited amount of cargo components present in these small vesicles. Consequently, common components of exosomes may play a role in their function. We have proposed that membrane phospholipids could be a common denominator in the effect of exosomes on cellular functions. In this regard, we have previously shown that liposomes made of phosphatidylcholine (PC) or phosphatidylserine (PS) induced a robust alteration of macrophage (Mϕ) gene expression. We herewith report that these two phospholipids modulate gene expression in Mϕs by different mechanisms. PS alters cellular responses by the interaction with surface receptors, particularly CD36. In contrast, PC is captured by a receptor-independent process and likely triggers an activity within endocytic vesicles. Despite this difference in the capture mechanisms, both lipids mounted similar gene expression responses. This investigation suggests that multiple mechanisms mediated by membrane phospholipids could be participating in the alteration of cellular functions by exosomes.

Human HSP70-escort protein 1 (hHep1) interacts with negatively charged lipid bilayers and cell membranes.

Human Hsp70-escort protein 1 (hHep1) is a cochaperone that assists in the function and stability of mitochondrial HSPA9. Similar to HSPA9, hHep1 is located outside the mitochondria and can interact with liposomes. In this study, we further investigated the structural and thermodynamic behavior of interactions between hHep1 and negatively charged liposomes, as well as interactions with cellular membranes. Our results showed that hHep1 interacts peripherally with liposomes formed by phosphatidylserine and cardiolipin and remains partially structured, exhibiting similar affinities for both. In addition, after being added to the cell membrane, recombinant hHep1 was incorporated by cells in a dose-dependent manner. Interestingly, the association of HSPA9 with hHep1 improved the incorporation of these proteins into the lipid bilayer. These results demonstrated that hHep1 can interact with lipids also present in the plasma membrane, indicating roles for this cochaperone outside of mitochondria.

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes.

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

Interaction of HSPA5 (Grp78, BIP) with negatively charged phospholipid membranes via oligomerization involving the N-terminal end domain.

Heat shock proteins (HSPs) are ubiquitous polypeptides expressed in all living organisms that participate in several basic cellular processes, including protein folding, from which their denomination as molecular chaperones originated. There are several HSPs, including HSPA5, also known as 78-kDa glucose-regulated protein (GRP78) or binding immunoglobulin protein (BIP) that is an ER resident involved in the folding of polypeptides during their translocation into this compartment prior to the transition to the Golgi network. HSPA5 is detected on the surface of cells or secreted into the extracellular environment. Surface HSPA5 has been proposed to have various roles, such as receptor-mediated signal transduction, a co-receptor for soluble ligands, as well as a participant in tumor survival, proliferation, and resistance. Recently, surface HSPA5 has been reported to be a potential receptor of some viruses, including the novel SARS-CoV-2. In spite of these observations, the association of HSPA5 within the plasma membrane is still unclear. To gain information about this process, we studied the interaction of HSPA5 with liposomes made of different phospholipids. We found that HSPA5 has a high affinity for negatively charged phospholipids, such as palmitoyl-oleoyl phosphoserine (POPS) and cardiolipin (CL). The N-terminal and C-terminal domains of HSPA5 were independently capable of interacting with negatively charged phospholipids, but to a lesser extent than the full-length protein, suggesting that both domains are required for the maximum insertion into membranes. Interestingly, we found that the interaction of HSPA5 with negatively charged liposomes promotes an oligomerization process via intermolecular disulfide bonds in which the N-terminus end of the protein plays a critical role.

Human HSPA9 (mtHsp70, mortalin) interacts with lipid bilayers containing cardiolipin, a major component of the inner mitochondrial membrane.

Mitochondrial Hsp70 (HSPA9, mtHsp70, mortalin) in conjunction with a complex set of other proteins is involved in the transport of polypeptides across the mitochondrial matrix. This observation allows us to hypothesize that HSPA9 might interact with membranes directly, similarly to other Hsp70s. Thus, we investigated whether human HSPA9 could also get inserted into lipid membranes. Human HSPA9 was incubated with liposomes made of lipids found within the mitochondrial membrane, such as 1', 3'-bis [1, 2-dimyristoyl-sn-glycero-3-phospho]-glycerol (CL), palmitoyl-oleoyl phosphocholine (POPC), palmitoyl-oleoyl phosphoserine (POPS), and palmitoyl-oleoyl phosphoethanolamine (POPE). HSPA9 displayed a predilection for CL and POPS, and low affinity for POPC and POPE, suggesting that the proteins have high specificity for negatively charged phospholipids. Then, liposomes were made with a composition resembling either the outer or inner mitochondrial membrane (OMM or IMM, respectively). We observed that HSPA9 has a higher affinity for IMM than OMM, which is consistent with the higher content of CL in the IMM. A comparison for the incorporation into POPS or CL liposomes by HSPA9 or HSPA1 indicated that both proteins behaved very similarly when exposed to CL liposomes, but differently with POPS liposomes, which was further corroborated by their susceptibility to proteinase K digestion after incorporation into liposomes. The measurement of thermodynamic parameters also showed that the interaction of both proteins with CL and POPS liposomes was different. Overall, our data showed that HSPA9 is prone to interact with membranes resembling the IMM that may be important for its role in the translocation of proteins into the mitochondria.

The small heat shock proteins, HSPB1 and HSPB5, interact differently with lipid membranes.

Increasing evidence shows that heat shock proteins (hsp) escape the cytosol gaining access to the extracellular environment, acting as signaling agents. Since the majority of these proteins lack the information necessary for their export via the classical secretory pathway, attention has been focused on alternative releasing mechanisms. Crossing the plasma membrane is a major obstacle to the secretion of a cytosolic protein into the extracellular milieu. Several mechanisms have been proposed, including direct interaction with the plasma membrane or their release within extracellular vesicles (ECV). HSPB1 (Hsp27), which belongs to the small hsp family, was detected within the membrane of ECV released from stressed HepG2 cells. To further investigate this finding, we studied the interaction of HSPB1 with lipid membranes using liposomes. We found that HSPB1 interacted with liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), palmitoyl oleoyl phosphatidylcholine (POPC), and palmitoyl oleoyl phosphatidylglycerol (POPG), with different characteristics. Another member of the small hsp family, HSPB5 (αB-crystallin), has also been detected within ECV released from HeLa cells transfected with this gene. This protein was found to interact with liposomes as well, but differently than HSPB1. To address the regions interacting with the membrane, proteoliposomes were digested with proteinase K and the protected domains within the liposomes were identified by mass spectroscopy. We observed that large parts of HSPB1 and HSPB5 were embedded within the liposomes, particularly the alpha-crystallin domain. These observations suggest that the interaction with lipid membranes may be part of the mechanisms of export of these proteins.

Bacterial Hsp70 (DnaK) and mammalian Hsp70 interact differently with lipid membranes.

The cellular response to stress is orchestrated by the expression of a family of proteins termed heat shock proteins (hsp) that are involved in the stabilization of basic cellular processes to preserve cell viability and homeostasis. The bulk of hsp function occurs within the cytosol and subcellular compartments. However, some hsp have also been found outside cells released by an active mechanism independent of cell death. Extracellular hsp act as signaling molecules directed at activating a systemic response to stress. The export of hsp requires the translocation from the cytosol into the extracellular milieu across the plasma membrane. We have proposed that membrane insertion is the initial step in this export process. We investigated the interaction of the major inducible hsp from mammalian (Hsp70) and bacterial (DnaK) species with liposomes. We found that mammalian Hsp70 displayed a high specificity for negatively charged phospholipids, such as phosphatidyl serine, whereas DnaK interacted with all lipids tested regardless of the charge. Both proteins were inserted into the lipid bilayer as demonstrated by resistance to acid or basic washes that was confirmed by partial protection from proteolytic cleavage. Several regions of mammalian Hsp70 were inserted into the membrane with a small portion of the N-terminus end exposed to the outer phase of the liposome. In contrast, the N-terminus end of DnaK was inserted into the membrane, exposing the C-terminus end outside the liposome. Mammalian Hsp70 was found to make high oligomeric complexes upon insertion into the membranes whereas DnaK only formed dimers within the lipid bilayer. These observations suggest that both Hsp70s interact with lipids, but mammalian Hsp70 displays a high degree of specificity and structure as compared with the bacterial form.

Interaction of heat shock protein 70 with membranes depends on the lipid environment.

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.