Review Post-Translational Modification & Subcellular Compartmentalization: Emerging Concepts on the Regulation … of RhoGTPases, 2021, Navarro-Lérida+

[SNT Gatchaman]

Post-Translational Modification and Subcellular Compartmentalization: Emerging Concepts on the Regulation and Physiopathological Relevance of RhoGTPases
Navarro-Lérida, Inmaculada; Sánchez-Álvarez, Miguel; del Pozo, Miguel Ángel

Cells and tissues are continuously exposed to both chemical and physical stimuli and dynamically adapt and respond to this variety of external cues to ensure cellular homeostasis, regulated development and tissue-specific differentiation. Alterations of these pathways promote disease progression—a prominent example being cancer.

Rho GTPases are key regulators of the remodeling of cytoskeleton and cell membranes and their coordination and integration with different biological processes, including cell polarization and motility, as well as other signaling networks such as growth signaling and proliferation. Apart from the control of GTP–GDP cycling, Rho GTPase activity is spatially and temporally regulated by post-translation modifications (PTMs) and their assembly onto specific protein complexes, which determine their controlled activity at distinct cellular compartments.

Although Rho GTPases were traditionally conceived as targeted from the cytosol to the plasma membrane to exert their activity, recent research demonstrates that active pools of different Rho GTPases also localize to endomembranes and the nucleus.

In this review, we discuss how PTM-driven modulation of Rho GTPases provides a versatile mechanism for their compartmentalization and functional regulation. Understanding how the subcellular sorting of active small GTPase pools occurs and what its functional significance is could reveal novel therapeutic opportunities.

Web | DOI | PDF | Cells | Open Access

 

[SNT Gatchaman]

I thought this was worth indexing in our library for reference. Their lens is cancer and cell migration / metastasis but there are overlap features that are of potential relevance to our research, eg Endoplasmic Reticulum, cell signalling, cell deformation, cell-ECM interaction.

Cell motility is essential for tissue development and homeostasis in multicellular organisms […] The actin cytoskeleton is the engine that drives polarized cell behavior through the spatial and temporal control of cell protrusiveness, adhesion, contractility and rear retraction. Changes in the actin cytoskeleton modulate not only focal adhesion dynamics and cell contraction but also the physical properties (i.e., rigidity, compliance) of the cell, including its nucleus.

The basic molecular machinery underlying the assembly and disassembly of actin filaments consists of a variety of actin-binding proteins that regulate the dynamic behavior of the cytoskeleton in response to different signals. Rho small GTPases act as master molecular switches regulating cytoskeletal remodeling. ‘Classic’ models depict active Rho GTPases proteins as residing and functioning predominantly at the plasma membrane (PM), but recent research supports different pools of active Rho GTPases can also operate in intracellular compartments, including the cell nucleus. […] the dynamic modulation of RhoGTPases is critically dependent on their post-translational modifications. Lipid modifications control protein localization and activity. The functional dependence on prenylation and palmitoylation of different Rho GTPases suggests that PTMs could critically control their subcellular trafficking across spatially distant compartments

The Rho family of proteins make up a major branch of the Ras superfamily of small GTPases. […] the best characterized ‘classical’ members being RhoA, Rac1 and Cdc42.

As molecular switches, Rho small GTPases cycle between an active GTP-bound form and an inactive GDP-bound state […] The control of the balance between the GTP-bound active state and the GDP-bound inactive state constitutes a first essential regulatory layer onto which several mechanisms converge. Guanine nucleotide exchange factors (GEFs) promote GDP-to-GTP exchange, leading to GTPase activation.

Conversely, GTPase-Activating Proteins (GAPs) stimulate GTPase activity, favoring an inactive state. Thus, GAPs and GEFs can be considered negative and positive regulators of small GTPases, respectively.

A second mechanism regulating Rho GTPase activation state is driven by Guanine nucleotide Dissociation Inhibitors (GDIs). […] Many GDIs bind the inactive, GDP-bound form of Rho-family signaling, impairing GTP exchange by GEFs and maintaining GTPases in an inactive state. […] The second and most general activity of GDIs is their ability to solubilize GTPases from cellular membranes.

The third and perhaps most complex mechanism of modulation of GTPases is through subcellular compartmentalization. A large body of literature considers most signaling initiated by Rho GTPases occurs at or around the PM [Plasma Membrane]; however, it is becoming clear that functionally active Rho GTPases, their GEFs and GAPs, and their effectors can be localized to different intracellular compartments beyond the PM. Despite the potential relevance of these novel intracellular pools, little is known about the mechanisms underlying its modulation.

Post-translational modifications of small GTPases are an additional regulatory layer that could integrate functional modulation and subcellular localization for the tight spatiotemporal control of GTPase activity.

A wide range of PTMs was reported for small GPTases, including phosphorylation, ubiquitylation, SUMOylation and lipid modifications, which must be tightly controlled to ensure appropriate GTPase signaling. Moreover, it is increasingly unclear to which extent PM microdomain formation explains the regulatory impact of lipids on small GTPases and signaling lipid species and fatty acid modification seem to interplay with specific hydrophobic motifs in these proteins to achieve both reversible and irreversible modulation.

 

[SNT Gatchaman]

Ubiquitylation, the covalent attachment of ubiquitin to Lys residues in a target protein, can lead to degradation of the substrate or the modulation of its subcellular compartmentalization and/or activity. Several Rho GTPases undergo ubiquitylation, including RhoA, Rac1, Rac1b, Cdc42, RhoB and RhoBTB2. Ubiquitylation was proposed as a mechanism to control the local activity of Rho GTPases

While ubiquitylation is classically conceived as a major route for the turnover of proteins through the proteasome, the importance of autophagy in the regulated turnover of specific proteins, as opposed to the ubiquitin-proteasome system (UPS), has recently emerged. Intriguingly, different studies support an interplay between them modulating RhoGTPases.

Small Ubiquitin-like Modifiers (SUMO) are small proteins remarkably similar in their structure to ubiquitin, although divergent in their amino acid sequence that is covalently attached to lysine residues in the target protein through an analogous but distinct process and machinery.

SUMOylation is a means for regulating nucleocytoplasmic shuttling, and a major share of SUMOylation substrates are nuclear proteins; to date, we do not know whether SUMOylated Rac1 pools partition specifically to this or other compartments.

With the exception of RhoBTB1 and RhoBTB2, post-translational modification by lipids was described for all Rho GTPases, modulating their location and interaction with specific GEFs, and consequently their feeding into downstream signaling pathways. Among them, prenylation and palmitoylation are particularly relevant.

Prenylation confers hydrophobicity to the C-terminus of GTPases, dramatically increasing their capacity to interact with cellular membranes.

Statins have emerged as a means to pharmacologically modulate GTPase signaling.

S-palmitoylation is the covalent attachment of a palmitate molecule (16-carbon, saturated fatty acid) to the side chain of Cys residues. This PTM regulates the subcellular localization and trafficking of substrates. It may also directly modulate protein–protein interactions by physically masking binding sites or by forcing a binding site into close membrane proximity

Regarding the two other main members of the Rho family, RhoA is not susceptible to palmitoylation, while the ubiquitously expressed major Cdc42 protein isoform is not palmitoylated, a novel brain-specific alternative splicing variant enriched in dendritic spines was reported as a palmitoylation substrate.

 

[SNT Gatchaman]

The nuclear envelope (NE) is essential for maintaining the unique biochemical identity of the nucleus. The nucleus is a special membrane-bound compartment: two concentric lipid bilayers, continuous with the ER system—the ONM (outer-nuclear-membrane) and the INM (inner-nuclear-membrane), separated by the perinuclear space (PNS)—and with each other at nuclear pores, act as a strong physical barrier. Despite their similarities in lipid composition, the INM and ONM are biochemically distinct: while the ONM has a certain continuity with the rough endoplasmic reticulum (RER), the INM exhibits a distinct set of integral membrane proteins, which provide docking sites for lamins, chromatin and via interaction with ONM proteins, the cytoskeleton.

PTMs regulating small GTPases can regulate the nuclear import and function of many nuclear proteins.

Palmitoylation can modulate the nuclear import of transcription factors, including all sex steroid receptors (SR), which have highly conserved palmitoylation motifs of nine amino acids: for example, estrogen receptor (ER) α translocates from the PM to the nucleus upon de-S-palmitoylation.

The additional, prominent potential functions for nuclear actin, closely related to the dynamical control of the cytoskeleton by Rho GTPases, are physically scaffolding and conferring plasticity to the cell nucleus. In fact, classic experiments have shown that tugging on integrin adhesion receptors results in distortion of the nucleus, reflecting a physical linkage between the cell surface and the nucleus through the cytoskeleton.

Recent studies reveal that mechanical stress can induce conformational changes in (and modulate the PTM of) nuclear envelope proteins. For example, force application results in apical-to basal differences in the conformation of Lamin A/C […]. This suggests that mechanical changes could also directly feed onto PTM and functional modulation of Rho GTPases to control their nuclear compartmentalization and signaling. The specific relevance of these mechanisms, and the principles by which they are coordinated, for pathological processes such as cell transformation and tumor cell aggressiveness, remain an important standing question.

Recent findings reveal how mechanical stimulation by stretching induces an increased expression of RhoA and Rac1, with depletion of Rac1 significantly inhibiting cell surface stiffness and 3D migration into extracellular matrices. These data strongly support a bidirectional regulation between mechanical modulation and RhoGTPase function.

An intriguing additional potential layer of coupled regulation arises when considering that nuclear membranes are continuous with the ER. Mechanical stretching of the NE is expected to increase membrane tension at the adjacent RER.

The coincidence that both activities involved in the prenylation of RhoGTPases are associated with the ER raises the possibility that mechanical forces can modulate RhoGTPases through ER organization and the regulation of these PTM activities. These associations might also underpin a coupling between lipid metabolism (to which the ER is exquisitely sensitive) and PTM regulation of Rho GTPases.

The last aspect worth mentioning regarding nuclear membrane dynamics and the regulation of nuclear pools of Rho GTPases pertains to the control of its lipid composition. Despite the presence of a plethora of biologically active lipids at nuclear membranes and nucleoplasmic structures, such as glycosphingolipids, the main phospholipid component is phosphatidylcholine, together with other predominantly negatively charged lipids. Cholesterol is also consistently found at the nuclear membrane—albeit at reduced levels, in accordance with its continuity with the ER—where it favors the formation of microdomains

An emerging concept is the potential role of GTPases in the control of cellular homeostasis through its implication in autophagic processes at the cytosolic level. The possibility exists that the nuclear pool of small GTPases can also contribute to modulate nuclear homeostasis through the control of a novel and unexplored mechanism such as the nucleophagy, by selectively removing damaged or non-essential nuclear material from the cell.