Ubiquitin-like processing of TUG proteins as a mechanism to regulate glucose uptake and energy metabolism in fat and muscle

Abstract

In response to insulin stimulation, fat and muscle cells mobilize GLUT4 glucose transporters to the cell surface to enhance glucose uptake. Ubiquitin-like processing of TUG (Aspscr1, UBXD9) proteins is a central mechanism to regulate this process. Here, recent advances in this area are reviewed. The data support a model in which intact TUG traps insulin-responsive "GLUT4 storage vesicles" at the Golgi matrix by binding vesicle cargoes with its N-terminus and matrix proteins with its C-terminus. Insulin stimulation liberates these vesicles by triggering endoproteolytic cleavage of TUG, mediated by the Usp25m protease. Cleavage occurs in fat and muscle cells, but not in fibroblasts or other cell types. Proteolytic processing of intact TUG generates TUGUL, a ubiquitin-like protein modifier, as the N-terminal cleavage product. In adipocytes, TUGUL modifies a single protein, the KIF5B kinesin motor, which carries GLUT4 and other vesicle cargoes to the cell surface. In muscle, this or another motor may be modified. After cleavage of intact TUG, the TUG C-terminal product is extracted from the Golgi matrix by the p97 (VCP) ATPase. In both muscle and fat, this cleavage product enters the nucleus, binds PPARγ and PGC-1α, and regulates gene expression to promote fatty acid oxidation and thermogenesis. The stability of the TUG C-terminal product is regulated by an Ate1 arginyltransferase-dependent N-degron pathway, which may create a feedback mechanism to control oxidative metabolism. Although it is now clear that TUG processing coordinates glucose uptake with other aspects of physiology and metabolism, many questions remain about how this pathway is regulated and how it is altered in metabolic disease in humans.

Keywords: GLUT4; adipocyte; glucose; insulin; membrane trafficking; muscle; p97/VCP ATPases; ubiquitin-like.

Figure 1

Ubiquitin-like proteolytic processing of TUG. Intact TUG protein is 60 kDa and has the domain structure that is shown. UBL1 and UBL2 are ubiquitin-like domains and UBX is a ubiquitin-regulatory X domain. The residues in the protein are numbered, as indicated, according to the mouse sequence. The N-terminal region binds GLUT4 Storage Vesicle (GSV) proteins, and the C-terminal region binds Golgi matrix proteins, as indicated. In fat and muscle cells, insulin stimulates endoproteolytic cleavage of intact TUG at the bond linking residues 164 and 165. Cleavage is mediated by the Usp25m protease and produces TUGUL (TUG Ubiquitin-Like), an 18 kDa ubiquitin-like protein modifier. The C-terminal product is 42 kDa and it is modified to a 54 kDa form to a variable degree. This modification is not understood. In 3T3-L1 adipocytes, TUGUL is covalently attached to a single target protein, the 110 kDa KIF5B kinesin motor, to produce a 130 kDa conjugate. In muscle, the TUGUL-modified (“tugulated”) protein has not been identified, and it is hypothesized that it is KIF5B or another kinesin motor. TUG cleavage liberates the GLUT4 storage vesicles from the Golgi matrix, and tugulation of the kinesin causes the movement of these vesicles toward the cell surface on these microtubule motors. The figure is modified from Habtemichael EN, et al., (11).

Figure 2

Actions by which insulin stimulation causes increased plasma membrane GLUT4. Insulin stimulates the mobilization of GLUT4 Storage Vesicles (GSVs), and also redirects endocytosed GLUT4 so that it returns directly to the plasma membrane from endosomes. In unstimulated cells, most GLUT4 accumulates in GSVs. These vesicles are trapped at the Golgi matrix by the action of intact TUG with other proteins, and they may participate in a cycle involving internal membranes (as described in the text). Insulin stimulates the acute proteolytic processing of TUG to mobilize the GSVs on microtubule motors directed toward the cell surface. The GSVs fuse at the plasma membrane, and the liberated GLUT4 is then able to populate the endosomal system. During ongoing insulin exposure, recycling of GLUT4 in endosomes becomes a significant source of GLUT4 arriving at the plasma membrane. The model that is depicted is based primarily on studies in adipocytes; data suggest that it holds in muscle as well. In skeletal muscle, data from TUG knockout mice imply that other sites at which insulin modulates GLUT4 trafficking are less important for its overall distribution at the cell surface. ERGIC, Endoplasmic Reticulum–Golgi Intermediate Compartment; ERES, Endoplasmic Reticulum Exit Sites. The figure is modified from Bogan JS, (1).

Figure 3

Proteins that bind and regulate GLUT4 Storage Vesicles (GSVs). A GSV membrane containing IRAP and GLUT4 is shown, and amino acid residues in the cytosolic N-terminus of IRAP are numbered. Tankyrase (TNKS), TUG, and Tbc1D4 (AS160) bind to adjacent peptides in IRAP, as indicated by brackets. TUG also binds the large intracellular loop of GLUT4, and both TUG and TNKS bind Usp25m. Direct binding of TUG to TNKS and to Tbc1D4 has not been shown; these hypothesized interactions are indicated with question marks. Tbc1D1 binds the cytosolic domain of IRAP (residues 1-109), but this interaction has not been localized further; the question mark indicates that it is hypothesized to bind IRAP residues 1-49 similar to Tbc1D4. TUG binds to Golgin-160 and other Golgi matrix proteins through its C-terminus, which is required to trap the vesicles intracellularly in the absence of insulin stimulation.

Figure 4

Coordinated regulation of distinct physiologic effects. Insulin signals through at least two signaling cascades to mobilize GLUT4 storage vesicles (GSVs). One involves Akt and the RabGAPs, Tbc1D4 and Tbc1D1, which regulate distinct Rab proteins on the GSVs (and on other membranes, such as endosomes). A second pathway involves the Rho family GTPase TC10α, which is coupled through its effector PIST to the TUG protease, Usp25m. Insulin stimulates TUG endoproteolytic cleavage to liberate the GSVs from Golgi matrix proteins, including Golgin-160 and others not shown. The N-terminal cleavage product, TUGUL, is a ubiquitin-like modifier. In adipocytes, TUGUL is attached to KIF5B, a microtubule motor that carries the GSVs to the cell surface; in muscle, this or another kinesin may be involved. Fusion of these vesicles at the cell surface results in the insertion of their specific cargo proteins into the plasma membrane. These include GLUT4, which promotes glucose uptake, IRAP which cleaves and inactivates circulating vasopressin, and LRP1 and sortilin, which bind the lipoproteins ApoE and ApoA5 and may regulate lipid metabolism. These proteins may have other effects as well. The TUG C-terminal product is extracted from the Golgi matrix by the action of p97 ATPases and is targeted to the nucleus. It binds the transcriptional regulator PPARγ and its co-factor PGC-1α, and stimulates the expression of genes to promote lipid oxidation and thermogenesis. The stability of the C-terminal product is regulated by the Ate1 arginyltransferase, and the product confers Ate1-dependent stability on PGC-1α. Degradation of these proteins makes their thermogenic effect transient, consistent with the transient increase in thermogenesis that is observed after meals.

Figure 5

The structure of mouse TUGUL as predicted by AlphaFold is shown (accession Q8VBT9). The two ubiquitin-like domains, UBL1 and UBL2, are linked by a short peptide including the Gly87 residue. In both the mouse and human predicted structures, the main α-helix in UBL2 has a bend at residue 122, corresponding to Pro122 in humans and Ala122 in mice. The β-sheet that wraps around this helix has an extended central strand, extending to Lys173 in the predicted structure as shown. Data indicate that the scissile bond is that joining Gly164 and Ser165, which is near the beginning of this strand. This unusual structure may help to confer the cell type -specificity of TUG cleavage, which occurs primarily in fat and muscle cells. See text for details.