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Cells Jun 2024Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs)....
Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in , encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of , the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in cells by transduction with gene replacement (ShH10-CMV-) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.
Topics: Choroideremia; Humans; Retinal Pigment Epithelium; Autophagy; Induced Pluripotent Stem Cells; rab GTP-Binding Proteins; Models, Biological; Signal Transduction; Mechanistic Target of Rapamycin Complex 1; Adaptor Proteins, Signal Transducing
PubMed: 38920696
DOI: 10.3390/cells13121068 -
Signal Transduction and Targeted Therapy Jun 2024Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific...
Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.
Topics: Humans; Respiratory Syncytial Virus Infections; Pyridines; Mice; Animals; Respiratory Syncytial Virus, Human; Viral Fusion Proteins; Farnesyltranstransferase; Antiviral Agents; Piperidines; Mice, Inbred BALB C; Protein Conformation; Dibenzocycloheptenes
PubMed: 38853183
DOI: 10.1038/s41392-024-01858-5 -
Angewandte Chemie (International Ed. in... Jun 2024Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic...
Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether-macrocyclic peptide (teMP) library construction/selection technology, so-called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp-C3-prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp-containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo-natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo-natural prenylated peptides, which is readily applicable to other drug targets.
PubMed: 38837490
DOI: 10.1002/anie.202409973 -
Molecular Metabolism Jul 2024Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins...
OBJECTIVE
Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. In this study, we examined the requirement of SREBP target genes for pancreatic ductal adenocarcinoma (PDAC) tumor growth.
METHODS
Here we constructed a custom CRISPR knockout library containing known SREBP target genes and performed in vitro 2D culture and in vivo orthotopic xenograft CRISPR screens using a patient-derived PDAC cell line. In vitro, we grew cells in medium supplemented with 10% fetal bovine serum (FBS) or 10% lipoprotein-deficient serum (LPDS) to examine differences in gene essentiality in different lipid environments. In vivo, we injected cells into the pancreata of nude mice and collected tumors after 4 weeks.
RESULTS
We identified terpenoid backbone biosynthesis genes as essential for PDAC tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that geranylgeranyl diphosphate synthase 1 (GGPS1) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model.
CONCLUSIONS
These findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.
Topics: Humans; Animals; Pancreatic Neoplasms; Mice; Mice, Nude; Cell Line, Tumor; Cell Proliferation; Polyisoprenyl Phosphates; Carcinoma, Pancreatic Ductal; Sterol Regulatory Element Binding Proteins; Clustered Regularly Interspaced Short Palindromic Repeats
PubMed: 38823776
DOI: 10.1016/j.molmet.2024.101964 -
Anticancer Research Jun 2024Thymic carcinoma is a rare cancer type with limited treatment options. Our previous study demonstrated that statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A...
BACKGROUND/AIM
Thymic carcinoma is a rare cancer type with limited treatment options. Our previous study demonstrated that statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, can prevent thymic carcinoma. However, the mechanisms through which statins affect intracellular events in cancer cells are not well understood. The aim of the study was to determine how thymic carcinoma modulates the intracellular signals in response to statin administration.
MATERIALS AND METHODS
We analyzed statin-induced protein phosphorylation in Ty82 human thymic carcinoma cells, which were cultured with fluvastatin, and protein phosphorylation was examined using western blotting.
RESULTS
Treating Ty82 with fluvastatin led to ERK5 phosphorylation via protein prenylation attenuation. The antitumor effects of fluvastatin on thymic carcinoma were enhanced when combined with an ERK5 inhibitor.
CONCLUSION
Statin therapy in combination with ERK5 inhibition may be a promising therapeutic approach for treating thymic carcinoma.
Topics: Fluvastatin; Humans; Thymus Neoplasms; Cell Line, Tumor; Mitogen-Activated Protein Kinase 7; Phosphorylation; Indoles; Fatty Acids, Monounsaturated; Thymoma; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Animals
PubMed: 38821590
DOI: 10.21873/anticanres.17057 -
Disease Models & Mechanisms May 2024Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections....
Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections. Prenylated proteins with a C-terminal CaaX sequence are targeted by CaaX-type prenyltransferases and proteases. To aid investigations of these enzymes and their targets, we developed Saccharomyces cerevisiae strains that express these human enzymes instead of their yeast counterparts. These strains were developed in part to explore human prenyltransferase specificity because of findings that yeast FTase has expanded specificity for sequences deviating from the CaaX consensus (i.e. atypical sequence and length). The humanized yeast strains displayed robust prenyltransferase activity against CaaX sequences derived from human and pathogen proteins containing typical and atypical CaaX sequences. The system also recapitulated prenylation of heterologously expressed human proteins (i.e. HRas and DNAJA2). These results reveal that substrate specificity is conserved for yeast and human farnesyltransferases but is less conserved for type I geranylgeranyltransferases. These yeast systems can be easily adapted for investigating the prenylomes of other organisms and are valuable new tools for helping define the human prenylome, which includes physiologically important proteins for which the CaaX modification status is unknown.
Topics: Humans; Saccharomyces cerevisiae; Protein Prenylation; Substrate Specificity; Amino Acid Sequence; Dimethylallyltranstransferase; Viral Proteins; Alkyl and Aryl Transferases
PubMed: 38818856
DOI: 10.1242/dmm.050516 -
The Journal of Biological Chemistry May 2024Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested...
Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.
PubMed: 38815864
DOI: 10.1016/j.jbc.2024.107423 -
International Journal of Molecular... May 2024Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase...
Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.
Topics: Humans; Computational Biology; Peptide Library; Substrate Specificity; Farnesyltranstransferase; Oligopeptides; Amino Acid Sequence; Crystallography, X-Ray; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Protein Binding
PubMed: 38791363
DOI: 10.3390/ijms25105324 -
BioRxiv : the Preprint Server For... May 2024Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins...
Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. Using parallel in vitro and in vivo CRISPR knockout screens, we identified terpenoid backbone biosynthesis genes as essential for pancreatic ductal adenocarcinoma (PDAC) tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that ( ) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model. These findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.
PubMed: 38746286
DOI: 10.1101/2024.05.03.592368 -
American Journal of Physiology.... Jul 2024Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only...
Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a. The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.
Topics: Polyisoprenyl Phosphates; Humans; Simvastatin; Chemotactic Factors; Phagocytes; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Complement C5a; Protein Prenylation; Animals; Mice; Sesquiterpenes
PubMed: 38717364
DOI: 10.1152/ajpendo.00359.2023