Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Structure of Rheb-GDP shown in cartoon with helices colored blue and β-strands light purple. The G-domain is from PDB 1XTQ while the farnsylated C-terminal hypervariable region (HVR) was model-built (see Methods) and attached to a hypothetical membrane for visualization of prenyl insertion into the hydrophobic core of the membrane. Helices α3 (residues 90–107), α4 (residues 131–140), and α5 (residues 153–170) are labeled and the effector binding Switch I (residues 33–41) and Switch II (residues 63–79) regions are highlighted in orange and yellow, respectively. ( B ) Initial model Rheb-GDP in a bilayer of mixed lipids (mol % as indicated) with Rheb shown in gray cartoon except for the farnesyl acyl chain that is in a stick representation (cyan). POPC = palmitoyl-oleoyl-glycero-phosphocholine, POPE = palmitoyl-oleoyl-glycero-phosphoethanolamine, SAPI = stearoyl-arachidonoyl-phosphatidylinositol (also called phosphatidylinositol or PI), and Chol = cholesterol. Water and ions are omitted for clarity.

Article Snippet: For siRNA transfections, 2 μL of RNAiMAX (ThermoFisher), 100 μL of OptiMEM (ThermoFisher) and 2 μL of 20 μM Rheb siRNA (#14267; CST, Danvers, MA) stock per well was added to 100,000 cells seeded in a 12-well dish and incubated for 48 hours before plasmid transfection.

Techniques: Membrane, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Illustration of the tilt angle θ, measured between the membrane normal (N) and a vector along helix α5 (V H5 ; Pro168 Cα → Arg161 Cα). ( B ) Visualization of rotation angle φ on a circular plot (see main text) with an outer wheel highlighting Rheb G-domain structural elements facing the membrane at a given φ. ( C ) Joint normalized probability distribution of φ and θ (P(φ, θ)) projected onto a circular grid with φ along the concentric and θ along the radial coordinate. High-density regions are defined as discrete orientation states (OS), and a representative snapshot of each OS (OS1, OS2, and OS3) is shown at the top with the bilayer in tan surface and Rheb in gray cartoon with a semi-transparent silhouette. Structural regions facing the membrane are colored according to the mapping in panel B. The N-terminus (residues 1-5) and inter-switch loop (residues 50-54) are colored in magenta and sky blue in the relevant orientation.

Article Snippet: For siRNA transfections, 2 μL of RNAiMAX (ThermoFisher), 100 μL of OptiMEM (ThermoFisher) and 2 μL of 20 μM Rheb siRNA (#14267; CST, Danvers, MA) stock per well was added to 100,000 cells seeded in a 12-well dish and incubated for 48 hours before plasmid transfection.

Techniques: Membrane, Plasmid Preparation

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Illustration of MD-guided selection of fluorophore labeling sites. Representative structures of Rheb in two orientations attached to a hypothetical membrane (light blue line), with lobe 1 (residues 1-85) in orange and lobe 2 (residues 86-170) in light blue. A136 and S180 highlighted in purple and green spheres, respectively, represent sites of Cys mutation for fluorophore labeling with the FRET efficiency (E A ) predicted to increase and decrease as the catalytic domain becomes proximal and distal from the membrane. ( B ) Normalized histograms of E A obtained from 34 individual ND-HA-Rheb-A136C/S180C particles (left) and R eff obtained from a 40 μs aggregate MD simulation of GDP- and GTP-bound Rheb (right). See main text for state assignments of the smFRET data and GMM fits of the MD data. ( C ) Comparison of ensemble-averaged MD-derived R eff (x-axis) and smFRET distances (y-axis) for the four states of panel B, with the dashed line indicating an ideal agreement. Standard deviations are shown for R eff (horizontal bars) and smFRET distance (vertical bars). ( D ) Comparison of ensemble sizes with solid bars denoting smFRET and striped bars representing MD.

Article Snippet: For siRNA transfections, 2 μL of RNAiMAX (ThermoFisher), 100 μL of OptiMEM (ThermoFisher) and 2 μL of 20 μM Rheb siRNA (#14267; CST, Danvers, MA) stock per well was added to 100,000 cells seeded in a 12-well dish and incubated for 48 hours before plasmid transfection.

Techniques: Selection, Labeling, Membrane, Mutagenesis, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Schematic of the spherical polar coordinate system (R eff , φ, θ) used to describe Rheb G-domain membrane orientation, defined by the polar angle θ, azimuthal angle φ, and radial distance R eff . ( B ) View from bottom of the normalized 3D probability density distribution (P(R eff , φ, θ)) derived from a 10-component GMM analysis of the combined GDP- and GTP-Rheb simulations. Contours indicate iso-surfaces at density levels of 0.3, 0.5, and 0.7. ( C ) Equatorial view of P(R eff , φ, θ) with representative structures corresponding to the four major orientation states (OS1-OS4) shown around the sphere. Contour levels are the same as in panel B.

Article Snippet: For siRNA transfections, 2 μL of RNAiMAX (ThermoFisher), 100 μL of OptiMEM (ThermoFisher) and 2 μL of 20 μM Rheb siRNA (#14267; CST, Danvers, MA) stock per well was added to 100,000 cells seeded in a 12-well dish and incubated for 48 hours before plasmid transfection.

Techniques: Membrane, Derivative Assay

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Residue contact frequency (RCF) profiles in each orientation state ensemble (OS1–OS4), highlighting specific lipid-protein interaction patterns. Contact is measured as any protein side chain heavy atom within 4 Å of lipid heavy atom, and frequency is calculated as the proportion of frames where a residue has ≥ 1 contact. Colored vertical bands denote key structural regions: NT (N-terminus), Switch I (SwI), inter-switch (IS), Switch II (SwII), α3, α4, α5, and HVR using the same color scheme as in . ( B ) Representative snapshots illustrating key lipid interactions in OS2 and OS3 with a portion of Rheb shown as gray cartoon and the bilayer as a semi-transparent surface. Residues mutated to Ala for the experiments in C and D are labeled, along with their contacts with lipid head groups (POPE, teal; SAPI, orange; Chol, sand/yellow). Hydrogen bonds are depicted as yellow dashed lines. ( C ) Representative immunoblots of phosphorylated S6 kinase (pS6K; mTORC1 activity readout), total S6K (tS6K), and Rheb expression in Rheb−/− (dKO) cells reconstituted with WT Rheb or the indicated mutants (OS2: S4A/K5A; OS3: N50A/Q52A). ( D ) Quantification of pS6K normalized to total S6K and Rheb expression, expressed as percentage of the WT condition. Points represent independent biological replicates and bars are mean ± SEM. Statistical significance was assessed using pairwise two-tailed t-tests on the normalized ratios (prior to WT scaling). P < 0.05; ***P < 0.0001.

Article Snippet: For siRNA transfections, 2 μL of RNAiMAX (ThermoFisher), 100 μL of OptiMEM (ThermoFisher) and 2 μL of 20 μM Rheb siRNA (#14267; CST, Danvers, MA) stock per well was added to 100,000 cells seeded in a 12-well dish and incubated for 48 hours before plasmid transfection.

Techniques: Residue, Labeling, Western Blot, Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Silhouette of the experimental Rheb–mTORC1 complex (PDB 9ED4) with major subunits labeled. ( B ) Representative Rheb structures from each orientation state (OS1–OS4) were aligned to the Rheb subunit in the experimental complex, and the same rigid-body transformation was applied to the full mTORC1 assembly to place the complex into the membrane reference frame defined by each simulated orientation. In OS1, OS2, and OS4, mTORC1 sterically clashes the membrane, whereas OS3 positions the complex above the bilayer with minimal clashes. ( C ) Assembly of an mTORC1 complex bound to two Rheb structures in OS3 generated by independently aligning them to the corresponding Rheb structures in the experimental complex, demonstrating that OS3 uniquely permits simultaneous, membrane-compatible engagement of both Rheb binding sites. ( D ) Schematics describing kinetic gateway mechanisms wherein the continuous exchange between the occluded OS1 and the signaling-competent OS3 observed in WT Rheb is disrupted by N-terminal S4A/K5A OS2 mutations, resulting in the system being “locked” in OS1 and unable to access OS2 or the rest of the states and thus reducing signaling. Likewise, the inter-switch N50A/Q52 OS3 mutation may destabilize the intermediate states, which also display significant inter-switch loop-membrane contacts, thereby shifting the population to OS3. ( E ) An alternative mechanism of OS3 mutant gain-of-function, wherein the N50A/Q52A mutation destabilizes OS3 membrane contacts and decrease the population of membrane proximal conformations and increasing the mTORC1-capturable Rheb extended fraction (see ref ).

Article Snippet: For siRNA transfections, 2 μL of RNAiMAX (ThermoFisher), 100 μL of OptiMEM (ThermoFisher) and 2 μL of 20 μM Rheb siRNA (#14267; CST, Danvers, MA) stock per well was added to 100,000 cells seeded in a 12-well dish and incubated for 48 hours before plasmid transfection.

Techniques: Labeling, Transformation Assay, Membrane, Generated, Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Residue contact frequency (RCF) profiles in each orientation state ensemble (OS1–OS4), highlighting specific lipid-protein interaction patterns. Contact is measured as any protein side chain heavy atom within 4 Å of lipid heavy atom, and frequency is calculated as the proportion of frames where a residue has ≥ 1 contact. Colored vertical bands denote key structural regions: NT (N-terminus), Switch I (SwI), inter-switch (IS), Switch II (SwII), α3, α4, α5, and HVR using the same color scheme as in . ( B ) Representative snapshots illustrating key lipid interactions in OS2 and OS3 with a portion of Rheb shown as gray cartoon and the bilayer as a semi-transparent surface. Residues mutated to Ala for the experiments in C and D are labeled, along with their contacts with lipid head groups (POPE, teal; SAPI, orange; Chol, sand/yellow). Hydrogen bonds are depicted as yellow dashed lines. ( C ) Representative immunoblots of phosphorylated S6 kinase (pS6K; mTORC1 activity readout), total S6K (tS6K), and Rheb expression in Rheb−/− (dKO) cells reconstituted with WT Rheb or the indicated mutants (OS2: S4A/K5A; OS3: N50A/Q52A). ( D ) Quantification of pS6K normalized to total S6K and Rheb expression, expressed as percentage of the WT condition. Points represent independent biological replicates and bars are mean ± SEM. Statistical significance was assessed using pairwise two-tailed t-tests on the normalized ratios (prior to WT scaling). P < 0.05; ***P < 0.0001.

Article Snippet: Wild type and S4A/K5A and N50A/Q52A mutants of Rheb were synthesized (Twist Bioscience) and PCR-amplified to obtain constructs with overlapping regions matching pcDNA3.1(-)-Hygromycin (obtained from Travis Moore, UTHealth).

Techniques: Residue, Labeling, Western Blot, Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Rheb membrane orientation dynamics and functional consequences elucidated by molecular simulations, single-molecule-FRET and signaling assays

doi: 10.64898/2026.02.27.708535

Figure Lengend Snippet: ( A ) Silhouette of the experimental Rheb–mTORC1 complex (PDB 9ED4) with major subunits labeled. ( B ) Representative Rheb structures from each orientation state (OS1–OS4) were aligned to the Rheb subunit in the experimental complex, and the same rigid-body transformation was applied to the full mTORC1 assembly to place the complex into the membrane reference frame defined by each simulated orientation. In OS1, OS2, and OS4, mTORC1 sterically clashes the membrane, whereas OS3 positions the complex above the bilayer with minimal clashes. ( C ) Assembly of an mTORC1 complex bound to two Rheb structures in OS3 generated by independently aligning them to the corresponding Rheb structures in the experimental complex, demonstrating that OS3 uniquely permits simultaneous, membrane-compatible engagement of both Rheb binding sites. ( D ) Schematics describing kinetic gateway mechanisms wherein the continuous exchange between the occluded OS1 and the signaling-competent OS3 observed in WT Rheb is disrupted by N-terminal S4A/K5A OS2 mutations, resulting in the system being “locked” in OS1 and unable to access OS2 or the rest of the states and thus reducing signaling. Likewise, the inter-switch N50A/Q52 OS3 mutation may destabilize the intermediate states, which also display significant inter-switch loop-membrane contacts, thereby shifting the population to OS3. ( E ) An alternative mechanism of OS3 mutant gain-of-function, wherein the N50A/Q52A mutation destabilizes OS3 membrane contacts and decrease the population of membrane proximal conformations and increasing the mTORC1-capturable Rheb extended fraction (see ref ).

Article Snippet: Wild type and S4A/K5A and N50A/Q52A mutants of Rheb were synthesized (Twist Bioscience) and PCR-amplified to obtain constructs with overlapping regions matching pcDNA3.1(-)-Hygromycin (obtained from Travis Moore, UTHealth).

Techniques: Labeling, Transformation Assay, Membrane, Generated, Binding Assay, Mutagenesis

Journal: Scientific Reports

Article Title: Cancer-Associated fibroblasts regulate the development of cholangiocarcinoma through IL-6/STAT3/AKR1C3 signaling axis

doi: 10.1038/s41598-026-37583-y

Figure Lengend Snippet: CAFs regulated the occurrence and development of CCA by activating the AKR1C3/STAT3 signaling axis. (A) The mRNA expression levels of AKR1C3 in QBC939 and TFK1 cells after co-incubation with CAFs were detected by Q-PCR. ** P < 0.01. (B) The protein expression levels of AKR1C3, P-STAT3, and T-STAT3 in QBC939 cells after co-incubation with CAFs at different times were measured by Western blot. (C) After CAF treatment with or without Ab-IL-6 and (or) AKR1C3 knockdown for 8 h, the expression of AKR1C3, P-STAT3, and T-STAT3 were detected by Western blot in QBC939 cells. (D) After treatment of CAFs with or without Ab-IL-6 and (or) AKR1C3 knockdown for 8 h, the expression of PCNA, P-GP, GLUT-1, and PFK-1 were detected by Western blot in QBC939 cells. The proliferation (E) and glycolysis levels (F) in QBC939 and TFK1 cells after treatment of CAFs with or without Ab-IL-6 and (or) AKR1C3 knockdown were assessed by CCK-8, glucose uptake, and lactate release, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001. The cell colony (G) of QBC939 cells exposed to 40 µM 5-FU after treatment of CAFs with or without Ab-IL-6 and (or) AKR1C3 knockdown were detected by crystal violet staining. Ab-IL-6, 2ug/ml.

Article Snippet: Antibodies against P-STAT3 (9145 S), T-STAT3 (30835 S), PCNA (2586 S), P-GP (13879 S), GLUT-1 (73015 S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Western Blot, Knockdown, CCK-8 Assay, Staining

Journal: Scientific Reports

Article Title: Cancer-Associated fibroblasts regulate the development of cholangiocarcinoma through IL-6/STAT3/AKR1C3 signaling axis

doi: 10.1038/s41598-026-37583-y

Figure Lengend Snippet: CAFs regulated the occurrence and development of CCA via the IL-6/STAT3/AKR1C3 signaling axis in vivo. (A) shAKR1C3-QBC939 cells and control cells were subcutaneously injected in nude mice to establish xenograft tumors. The representative tumors and their volume are depicted graphically. (B) The growth and sensitivity to 5-FU were detected in the mixed xenografts of CAFs and shAKR1C3-QBC939 cells. (C) HE and IHC staining were performed to examine CAF-QBC939 and QBC939 xenografts. (D) The protein expressions of AKR1C3, P-STAT3, PCNA, P-GP, GLUT-1, and PFK-1 in the CAFs-shAKR1C3-QBC939 xenografts with or without the treatment of 5-FU were detected by Western blot. (E) Schematic summary illustrating how CAF-derived IL-6 activates the STAT3/AKR1C3 axis in cholangiocarcinoma cells to drive tumor proliferation, chemoresistance, glycolysis, and metastatic potential.

Article Snippet: Antibodies against P-STAT3 (9145 S), T-STAT3 (30835 S), PCNA (2586 S), P-GP (13879 S), GLUT-1 (73015 S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vivo, Control, Injection, Immunohistochemistry, Western Blot, Derivative Assay