Journal: Materials Today Bio

Article Title: Application of a novel myristoylproteomics approach identifies GLIPR2 as a key pro-ferroptotic substrate in non-small cell lung cancer

doi: 10.1016/j.mtbio.2026.102945

Figure Lengend Snippet: Augmented myristoylation is a feature and consequence of ferroptosis in sensitive cells. A. Workflow for metabolic labeling with YnMyr and detection of myristoylated proteins via CuAAC. B. Viability of Calu-1 cells pretreated with DMSO or 1 μM IMP-1088, then exposed to erastin or ML162 ± Fer-1. C. The impact of ferroptosis to N-myristoylation was detected by western blot. Calu-1 and H460 cells were first incubated with YnMyr for 18 h. For the final 8 h of YnMyr incubation, the cells were co-treated with 5 μM erastin in the presence or absence of 2 μM Fer-1. In a separate experiment, for the final 2 h of YnMyr incubation, the cells were co-treated with 1 μM RSL3 with or without 2 μM Fer-1. D. Samples were analyzed by TAMRA (top) or enriched by pull-down on streptavidin beads and analyzed by Western blot (bottom). The sample before pull-down (Input), pull-down sample (PD) and the supernatant from the pull-down (Spnt) were analyzed. C-SRC, PRKACA and CHCHD3 were enriched in the pull-down samples. GAPDH: loading control. E-F. The impact of ferroptosis to NMT1 or NMT2 was detected by western blot. Cells (Calu-1, E; H460, F) were treated with 5 μM erastin or 1 μM RSL3 for indicated time.Experiments in B were repeated in triplicate.

Article Snippet: IMP-1088 (#HY-112258), erastin (#HY-15763) were purchased from MedChemExpress (Monmouth, NJ, USA).

Techniques: Labeling, Western Blot, Incubation, Control

Journal: Materials Today Bio

Article Title: Application of a novel myristoylproteomics approach identifies GLIPR2 as a key pro-ferroptotic substrate in non-small cell lung cancer

doi: 10.1016/j.mtbio.2026.102945

Figure Lengend Snippet: Myristoylation-dependent ER localization is required for GLIPR2 to promote ferroptosis. A-B. Dose-response curves of GLIPR2 knockout Calu-1 cells with or without GLIPR2-WT or G2A overexpressed, following treated with erastin (A) or ML162 (B) for 24 h. Viability was assessed and normalized to control. C. Western blot analysis validating the expression of GLIPR2 (WT or G2A) in reconstituted GLIPR2-knockout Calu-1 cells. EV, empty vector. D. Western blot analysis of GLIPR2 myristoylation in GLIPR2-knockout Calu-1 cells reconstituted as indicated, and treated with or without 1 μM IMP-1088. E. Immunofluorescence analysis showing the subcellular localization of re-introduced wild-type GLIPR2 and the G2A mutant in GLIPR2-knockout Calu-1 cells. Scale bar, 20 μm. F-G. Dose-response curves of GLIPR2-knockout Calu-1 cells reconstituted with GLIPR2 (WT) or the indicated subcellular localization mutants, treated with erastin (F) or ML162 (G) for 24 h. H. Immunofluorescence analysis of the subcellular localization of the indicated GLIPR2 mutants in GLIPR2-knockout Calu-1 cells. Scale bar, 20 μm. I. Western blot analysis of the expression levels of GLIPR2 (WT) and the indicated mutants in GLIPR2-knockout Calu-1 cells. Data are mean ± SD; n = 3 (A–B and F-G).

Article Snippet: IMP-1088 (#HY-112258), erastin (#HY-15763) were purchased from MedChemExpress (Monmouth, NJ, USA).

Techniques: Knock-Out, Control, Western Blot, Expressing, Plasmid Preparation, Immunofluorescence, Mutagenesis

Journal: Materials Today Bio

Article Title: Application of a novel myristoylproteomics approach identifies GLIPR2 as a key pro-ferroptotic substrate in non-small cell lung cancer

doi: 10.1016/j.mtbio.2026.102945

Figure Lengend Snippet: Augmented myristoylation is a feature and consequence of ferroptosis in sensitive cells. A. Workflow for metabolic labeling with YnMyr and detection of myristoylated proteins via CuAAC. B. Viability of Calu-1 cells pretreated with DMSO or 1 μM IMP-1088, then exposed to erastin or ML162 ± Fer-1. C. The impact of ferroptosis to N-myristoylation was detected by western blot. Calu-1 and H460 cells were first incubated with YnMyr for 18 h. For the final 8 h of YnMyr incubation, the cells were co-treated with 5 μM erastin in the presence or absence of 2 μM Fer-1. In a separate experiment, for the final 2 h of YnMyr incubation, the cells were co-treated with 1 μM RSL3 with or without 2 μM Fer-1. D. Samples were analyzed by TAMRA (top) or enriched by pull-down on streptavidin beads and analyzed by Western blot (bottom). The sample before pull-down (Input), pull-down sample (PD) and the supernatant from the pull-down (Spnt) were analyzed. C-SRC, PRKACA and CHCHD3 were enriched in the pull-down samples. GAPDH: loading control. E-F. The impact of ferroptosis to NMT1 or NMT2 was detected by western blot. Cells (Calu-1, E; H460, F) were treated with 5 μM erastin or 1 μM RSL3 for indicated time.Experiments in B were repeated in triplicate.

Article Snippet: IMP-1088 (#HY-112258), erastin (#HY-15763) were purchased from MedChemExpress (Monmouth, NJ, USA).

Techniques: Labeling, Western Blot, Incubation, Control

Journal: Materials Today Bio

Article Title: Application of a novel myristoylproteomics approach identifies GLIPR2 as a key pro-ferroptotic substrate in non-small cell lung cancer

doi: 10.1016/j.mtbio.2026.102945

Figure Lengend Snippet: Comparative myristoylome profiling identifies candidates associated with ferroptosis sensitivity. A. Experimental strategy to identify N-myristoylated proteins with higher levels in ferroptosis-sensitive Calu-1 vs. H460 cells. B. Volcano plot for Calu-1 cells shows YnMyr incorporation versus significance in the presence of IMP-1088. Confidence categories: high (blue), medium (green; circle, rhombus, triangle), low (yellow), potential (purple), non-substrates (gray). C. Corresponding volcano plot for H460 cells. D. Comparison of YnMyr intensities between Calu-1 and H460 cells. Subtypes: Calu-1 > H460 (red), unique to Calu-1 (yellow), unique to H460 (green), H460 > Calu-1 (blue), common (purple), non-substrates (gray). E. Schematic for selecting myristoylated substrates linked to ferroptosis sensitivity. F. Viability of Calu-1 cells after siRNA knockdown of candidate genes and treatment with 0.04 μM ML162. Data are mean ± SD; n = 3 (F). Statistics: unpaired t-test (F). ∗p < 0.05, ∗∗p < 0.01, NS, not significant.

Article Snippet: IMP-1088 (#HY-112258), erastin (#HY-15763) were purchased from MedChemExpress (Monmouth, NJ, USA).

Techniques: Comparison, Knockdown

Journal: Materials Today Bio

Article Title: Application of a novel myristoylproteomics approach identifies GLIPR2 as a key pro-ferroptotic substrate in non-small cell lung cancer

doi: 10.1016/j.mtbio.2026.102945

Figure Lengend Snippet: Myristoylation-dependent ER localization is required for GLIPR2 to promote ferroptosis. A-B. Dose-response curves of GLIPR2 knockout Calu-1 cells with or without GLIPR2-WT or G2A overexpressed, following treated with erastin (A) or ML162 (B) for 24 h. Viability was assessed and normalized to control. C. Western blot analysis validating the expression of GLIPR2 (WT or G2A) in reconstituted GLIPR2-knockout Calu-1 cells. EV, empty vector. D. Western blot analysis of GLIPR2 myristoylation in GLIPR2-knockout Calu-1 cells reconstituted as indicated, and treated with or without 1 μM IMP-1088. E. Immunofluorescence analysis showing the subcellular localization of re-introduced wild-type GLIPR2 and the G2A mutant in GLIPR2-knockout Calu-1 cells. Scale bar, 20 μm. F-G. Dose-response curves of GLIPR2-knockout Calu-1 cells reconstituted with GLIPR2 (WT) or the indicated subcellular localization mutants, treated with erastin (F) or ML162 (G) for 24 h. H. Immunofluorescence analysis of the subcellular localization of the indicated GLIPR2 mutants in GLIPR2-knockout Calu-1 cells. Scale bar, 20 μm. I. Western blot analysis of the expression levels of GLIPR2 (WT) and the indicated mutants in GLIPR2-knockout Calu-1 cells. Data are mean ± SD; n = 3 (A–B and F-G).

Article Snippet: IMP-1088 (#HY-112258), erastin (#HY-15763) were purchased from MedChemExpress (Monmouth, NJ, USA).

Techniques: Knock-Out, Control, Western Blot, Expressing, Plasmid Preparation, Immunofluorescence, Mutagenesis

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Phylogenetic tree of 83 Eurotiale order fungal IMPDHs, including 62 IMPDH-As and 21 IMPDH-Bs constructed using BALi-Phy . Branches with ≥80% support are shown. Five major subclades are found: Byssochlamys and Talaromyces (olive green), Aspergillus (purple), Penicillium IMPDH-As (blue), IMPDH-Bs (red), and outgroups composed closely related Trichophyton , Microsporum , and Coccoidiodes. Bi-partition branch supports are listed to the left of their node of origin. PbreA, PnalA, PbreB and PnalB are highlighted to illustrate the incongruence of the IMPH-A and IMPDH-B phylogeny. Resurrected ancestors are denoted by the number along the branches. MPA producers are denoted with stars. Branch length scale is shown at the bottom. Abbreviations: Acan , Aspergillus candidus ; Apse , Aspergillus pseudoglaucus ; BsAF , Byssochlamys sp AF001B; Pani , Paecilomyces nivea ; Pant , Penicillium antarcticum ; Pbre , P. brevicompactum ; Pbra, P. brasilianum ; Pcam , P. camemberti ; Pcar, P. carneum ; Pcop, P. coprophilum ; Pdig, P. digitatum ; Pexp , P. expansum ; Pfre , P. freii ; Pgris , P. griseofulvum ; Pita , P. italicum ; Pnal , P. nalgiovense ; Pnor, P. nordicum ; Ppar, P. parvum ; Ppol, P. polonicum ; Proq , P. roqueforti ; PruW, P. rubens Wisconsin ; Psam , P. samsonianum ; Psol , P. solitum ; Pste , P. steckii ; Pvul , P. vulpinum . B-G. The values of K iapp for MPA ( B ), k cat ( C ), K IMP ( D ), K NAD ( E ), k cat /K IMP ( F ) and k cat /K NAD ( G ) are shown for IMPDH-Bs from producers (Prod, magenta) and nonproducers (Non, teal) and ancestors (Anc, colored as noted), as well as MPA-sensitive IMPDHs from the literature (MPA S , black, defined as K iapp ≤ 30 nM), See , and and and for data and parameters for each enzyme. **, p ≤ 0.01. **, p ≤ 0.01. B. IMPDH-Bs and Anc3-Anc6 are MPA-resistant while Anc1 and Anc2 are MPA S . C. The values of k cat are similar for IMPDH-Bs and MPA S enzymes. D. The values of K IMP are larger for IMPDH-Bs than MPA S enzymes. E. The values of K NAD are comparable among IMPDH-Bs and MPA S enzymes. F. The values of k cat /K IMP of MPA S IMPDHs are greater than IMPDH-Bs. Producer IMPDH-Bs tend to be more active than nonproducer enzymes (p = 0.18; p= 0.02 if the thermolabile Pani B value is omitted). G. The values of k cat /K NAD of MPA S IMPDHs are greater than nonproducer IMPDH-Bs.

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Construct

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: Enzyme concentrations are listed in the Methods section. Reactions were with fixed substrate concentrations as listed and at 25°C unless otherwise specified. The average and range of duplicate reactions are plotted. Data was analyzed in GraphPad Prism. The values of K IMP were determined by fits to the Michaelis-Menten equation, values of K NAD were determined by fits to Michealis-Menten or substrate inhibition equation as appropriate. The values of K iapp for MPA were determined as described in the Methods. CI, 95% confidence intervals. Values are listed in .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Inhibition

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A . Clustal Omega sequence alignment of ancestral IMPDHs . Ancestors listed in order of appearance in the tree in . First change at a given position denoted in yellow, second change in green, third change in magenta. B. AlphaFold model of Anc1 colored by pLDDT . Inset shows PAE. C. Anc1 is shown in purple ribbon, substitutions in Anc2-7 are shown colored as in the key. K + , pink ball. IMP, gray sticks. MPA, olive sticks. These ligands were positioned by aligning Anc1 with the C. griseus E-XMP*•MPA complex (PDB: 1JR1, ).

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Sequencing

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Timelines for the changes in resistance (values of K iapp for MPA and RVP) and catalytic efficiency ( k cat /K IMP and k cat /K NAD ) for the resurrected ancestors. B. Ancestor substitutions near the MPA binding site. The structure of Anc1 (purple) was predicted using AlphaFold2 ( ; ). Substitutions in Anc2-7 are shown colored as in the key in panel A . K + , pink ball; IMP, gray sticks; MPA, olive sticks. These ligands were positioned by aligning Anc1 with the C. griseus E-XMP*•MPA complex (PDB: 1JR1, ). C-E. Timelines for the changes in resistance (values of K iapp for MPA and RVP) and catalytic efficiency ( k cat /K IMP and k cat /K NAD ) for the ancestors and their extant descendants. MPA producers are magenta, nonproducers are teal, ancestors are colored as in the key in panel A .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Linear free energy relationship between the values of k cat /K IMP and MPA resistance ( K iapp ). MPA S , K iapp ≤ 30 nM; MPA-moderately resistant (MPA M ), 1 μM ≥ K iapp > 30 nM; MPA R , K iapp > 1 μM. See Tables S1 and S2 for all values. B. Linear free energy relationship between k cat /K NAD and MPA resistance ( K iapp ). Key as in panel A . C. E•RVP mimics E-XMP*. The structures of E-XMP*•MPA (cornflower blue, PDB: 1JR1) and E•RVP•MAD (olive, PDB: 1NF7; MAD = mycophenolate adenine dinucleotide) are overlaid. E-XMP* is shown in cyan, MPA in spring green, RVP in magenta, and MAD in dark magenta. Figure rendered in UCSF Chimera .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques:

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: Enzyme concentrations are listed in Methods section. Reactions performed in duplicate at 0.5* K IMP and 1.8* K NAD and 1 mM RVP at 25°C unless otherwise noted. Anc6, Pani B and Pfre B were assayed at 18°C. Pcam B was assayed at 11.5°C. Gray denotes representative eukaryotic IMPDHs, magenta denotes enzymes from MPA producers, teal denotes enzymes from nonproducers and ancestors are colored as in - .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: