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Prenylation process of the CAAX proteins. The protein prenylation process includes 3 steps: polyisoprenylation, proteolysis, and carboxyl methylation. Polyisoprenylation is the attachment of an isoprenoid lipid by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) to CAAX box. In the second step, the CAAX residues are proteolysed by prenyl protein peptidase RCE1 family to release-AAX. This is followed by subsequent endoproteolytic trimming and carboxyl methylation significantly increases the hydrophobicity of the C-termini of CAAX-modified proteins.
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CAAX proteins are widely involved in global cellular functions such as proliferation, differentiation, and carcinogenesis. As an important modulator of biological activity, signal transduction via protein prenylation is a crucial step for most CAAX protein functions, particularly for anchoring these CAAX proteins to cellular membrane system. With a...
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... Enzymes related to Rce1 and MmRce1 are not expected to be one-step transpeptida ses, as sortases are known to be, and archaeosortase, exosortase, and rhombosortase all might be. The CAAX (Cys-aliphatic-aliphatic-any) motif, occurring at the very end of a protein, has an absolute requirement for a Cys residue, which must be post-translation ally modified by attachment of a large prenyl-derived moiety to its side chain, either a geranylgeranyl (20-carbon) or farnesyl (15-carbon) group, before the IMP can cleave the polypeptide distal to that Cys residue (27). Rce1-related proteins are known more generally as CPBP intramembrane proteases. ...
... We were highly intrigued to find CPBP family intramembrane proteases regularly in close association with client proteins possessing the JDVT-CTERM domain, a small C-terminal putative protein-sorting signal domain with a conspicuous invariant Cys residue, shared by proteins that often shared no other regions of homology. Based on prior knowledge of the Rce1 (27) and MmRce1 (19) proteins, this association immedi ately suggested that the protease performs a critical step in sorting and maturation, including a cleavage step likely to act on a site within the sorting domain, most likely just C-terminal to the Cys residue. This cleavage step is likely dependent on a modification on the Cys side chain prior to the completion of the proteolysis. ...
The LPXTG protein-sorting signal, found in surface proteins of various Gram-positive pathogens, was the founding member of a growing panel of prokaryotic small C-terminal sorting domains. Sortase A cleaves LPXTG, exosortases (XrtA and XrtB) cleave the PEP-CTERM sorting signal, archaeosortase A cleaves PGF-CTERM, and rhombosortase cleaves GlyGly-CTERM domains. Four sorting signal domains without previously known processing proteases are the MYXO-CTERM, JDVT-CTERM, Synerg-CTERM, and CGP-CTERM domains. These exhibit the standard tripartite architecture of a short signature motif, a hydrophobic transmembrane segment, and an Arg-rich cluster. Each has an invariant cysteine in its signature motif. Computational evidence strongly suggests that each of these four Cys-containing sorting signals is processed, at least in part, by a cognate family of glutamic-type intramembrane endopeptidases related to the eukaryotic type II CAAX-processing protease Rce1. For the MYXO-CTERM sorting signals of different lineages, their sorting enzymes, called myxosortases, include MrtX (MXAN_2755 in Myxococcus xanthus ), MrtC, and MrtP, all with radically different N-terminal domains but with a conserved core. Related predicted sorting enzymes were also identified for JDVT-CTERM (MrtJ), Synerg-CTERM (MrtS), and CGP-CTERM (MrtA). This work establishes a major new family of protein-sorting housekeeping endopeptidases contributing to the surface attachment of proteins in prokaryotes.
IMPORTANCE
Homologs of the eukaryotic type II CAAX-box protease Rce1, a membrane-embedded endopeptidase found in yeast and human ER and involved in sorting proteins to their proper cellular locations, are abundant in prokaryotes but not well understood there. This bioinformatics paper identifies several subgroups of the family as cognate endopeptidases for four protein-sorting signals processed by previously unknown machinery. Sorting signals with newly identified processing enzymes include three novel ones, but also MYXO-CTERM, which had been the focus of previous experimental work in the model fruiting and gliding bacterium Myxococcus xanthus . The new findings will substantially improve our understanding of Cys-containing C-terminal protein-sorting signals and of protein trafficking generally in bacteria and archaea.
... In addition, the C-terminus of Ras proteins harbor a CAAX domain that acts as a signal for the post-translational addition of a farnesyl isoprenoid (Fernandez-Medarde et al., 2021). Prenylation involves isoprenoid lipid attachment to the cysteine of the CAAX sequence, followed by proteolysis of the -AAX sequence, and then carboxyl methylation (Gao et al., 2009). This promotes anchoring of Ras to membranes (Michaelson et al., 2005). ...
Ras GTPases regulate many developmental and physiological processes and mutations in Ras are associated with numerous human cancers. Here, we report the function, levels, and localization of an N-terminal knock-in of mNeonGreen (mNG) into C. elegans LET-60 /Ras. mNG:: LET-60 interferes with some but not all LET-60 /Ras functions. mNG:: LET-60 is broadly present in tissues, found at different levels in cells, and concentrates in distinct subcellular compartments, including the nucleolus, nucleus, intracellular region, and plasma membrane. These results suggest that mNG:: LET-60 can be a useful tool for determining LET-60 levels and localization once its functionality in a developmental or physiological process is established.
... The C-terminal region containing the CAAX motif varies considerably among different members of the RAS family. However, this motif is essential for post-translational modification (35). RAS is activated by guanine nucleotide exchange factors (GEFs) and transduces signals to downstream pathways. ...
Three rat sarcoma (RAS) gene isoforms, KRAS, NRAS, and HRAS, constitute the most mutated family of small GTPases in cancer. While the development of targeted immunotherapies has led to a substantial improvement in the overall survival of patients with non-KRAS-mutant cancer, patients with RAS-mutant cancers have an overall poorer prognosis owing to the high aggressiveness of RAS-mutant tumors. KRAS mutations are strongly implicated in lung, pancreatic, and colorectal cancers. However, RAS mutations exhibit diverse patterns of isoforms, substitutions, and positions in different types of cancers. Despite being considered “undruggable”, recent advances in the use of allele-specific covalent inhibitors against the most common mutant form of RAS in non-small-cell lung cancer have led to the development of effective pharmacological interventions against RAS-mutant cancer. Sotorasib (AMG510) has been approved by the FDA as a second-line treatment for patients with KRAS-G12C mutant NSCLC who have received at least one prior systemic therapy. Other KRAS inhibitors are on the way to block KRAS-mutant cancers. In this review, we summarize the progress and promise of small-molecule inhibitors in clinical trials, including direct inhibitors of KRAS, pan-RAS inhibitors, inhibitors of RAS effector signaling, and immune checkpoint inhibitors or combinations with RAS inhibitors, to improve the prognosis of tumors with RAS mutations.
... The luciferase activities from the HiBiT constructs of both variants 1 and 2 were significantly reduced by CXCL12 stimulation, but the luciferase activity of variant 5 was not altered (Fig 5C). The CAAX motif in membrane-attached proteins enables plasma membrane localization through isoprenylation at the cysteine residue and proteolysis [41]. Thus, LgBiT-CAAX can localize to the intracellular surface of the plasma membrane and bind to C-terminal SmBiT-tagged GPCRs localized on the membrane. ...
C-X-C motif chemokine ligand 12(CXCL12) is an essential chemokine for organ development and homeostasis in multiple tissues. Its receptor, C-X-C chemokine receptor type 4(CXCR4), is expressed on the surface of target cells. The chemokine and receptor are expressed almost ubiquitously in human tissues and cells throughout life, and abnormal expression of CXCL12 and CXCR4 is observed in pathological conditions, such as inflammation and cancer. CXCR4 is reportedly translated into five splicing variants of different lengths, which each have different amino acids in the N-terminus. As the N-terminus is the first recognition site for chemokines, CXCR4 variants may respond differently to CXCL12. Despite these differences, the molecular and functional properties of CXCR4 variants have not been thoroughly described or compared. Here, we explored the expression of CXCR4 variants in cell lines and analyzed their roles in cellular responses using biochemical approaches. RT-PCR revealed that most cell lines express more than one CXCR4 variant. When expressed in HEK293 cells, the CXCR4 variants differed in protein expression efficiency and cell surface localization. Although variant 2 demonstrated the strongest expression and cell surface localization, variants 1, 3, and 5 also mediated chemokine signaling and induced cellular responses. Our results demonstrate that the N-terminal sequences of each CXCR4 variant determine the expression of the receptor and affect ligand recognition. Functional analyses revealed that CXCR4 variants may also affect each other or interact during CXCL12-stimulated cellular responses. Altogether, our results suggest that CXCR4 variants may have distinct functional roles that warrant additional investigation and could contribute to future development of novel drug interventions.
... To test whether mutations in rgp1 disrupt Rab6a activation and vesicle movement in our zebrafish model, we generated and injected a Tol2 DNA construct to overexpress mCherry-fused WT zebrafish Rab6a (WT zRab6a) under a Col2a1a promoter (57,58,68). We co-injected the construct along with transposase into the single-cell progeny of heterozygote mutant fish carrying a transgene that labels chondrocyte membrane with caax-eGFP (Figure 2A) (69). Because transposase integration is mosaic, not all cells express the construct, and the mCherry fusion protein highlights specific vesicular compartments. ...
... Craniofacial chondrocytes predominantly secrete collagen II during larval craniofacial development (68). To assess secretion of collagen II, we performed immunofluorescence with a collagen II antibody in WT and rgp1 -/larvae crossed to a transgenic zebrafish line expressing membrane-bound caax-eGFP under the Col2a1a promoter ( Figure 3A) (57,69,70). The caax-eGFP transgene labeled the cell membrane boundary to distinguish intracellular from extracellular space. ...
Rgp1 was previously identified as a component of a guanine nucleotide exchange factor (GEF) complex to activate Rab6a-mediated trafficking events in and around the Golgi. While the role of Rgp1 in protein trafficking has been examined in vitro and in yeast, the role of Rgp1 during vertebrate embryogenesis and protein trafficking in vivo is unknown. Using genetic, CRISPR-induced zebrafish mutants for Rgp1 loss-of-function, we found that Rgp1 is required for craniofacial cartilage development. Within live rgp1-/- craniofacial chondrocytes, we observed altered movements of Rab6a+ vesicular compartments, consistent with a conserved mechanism described in vitro. Using transmission electron microscopy (TEM) and immunofluorescence analyses, we show that Rgp1 plays a role in the secretion of collagen II, the most abundant protein in cartilage. Our overexpression experiments revealed that Rab8a is a part of the post-Golgi collagen II trafficking pathway. Following loss of Rgp1, chondrocytes activate an Arf4b-mediated stress response and subsequently respond with nuclear DNA fragmentation and cell death. We propose that an Rgp1-regulated Rab6a-Rab8a pathway directs secretion of ECM cargoes such as collagen II, a pathway that may also be utilized in other tissues where coordinated trafficking and secretion of collagens and other large cargoes is required for normal development and tissue function.
... The CAAX box is composed of a cysteine residue (C), two aliphatic residues (AA), and any C-terminal amino acid (X), and this sequence can undergo prenylation, a process in which post-translational lipid additions occur, thereby creating a hydrophobic C-terminus and thus greatly increasing the ability of the protein to interact with the plasma membrane. 43,44 We then stably incorporated our membrane-tethered GCaMP6s-CAAX sensor into our Ca V 3/K ir 2.3 cell lines and demonstrated GCaMP6s-CAAX localization at the plasma membrane ( Figure 2B). Using the high-throughput fluorometric imaging plate reader (FLIPR), the membrane-tethered GCaMP6s-CAAX resulted in a dramatically reduced baseline signal before stimulation compared to the untethered GCaMP6s and the calciumsensitive dye Fluo-4AM ( Figure 2C). ...
T-type voltage-gated Ca2+ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca2+ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca2+ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca2+ channels are largely inactivated and unable to open to allow Ca2+ influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express Kir2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca2+ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca2+ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.
... Upon dissociation of ERDJ4 and BiP from the IRE1α/BiP/ERDJ4 complex, the IRE1α signaling pathway is initiated causing splicing of Xbp1 mRNA and its translation into XBP1 protein, which travels to the nucleus to activate transcription of chaperones, such as ERdj4 which inhibits protein glycosylation, revealed that ERDJ4 is not glycosylated [9,15]. The C-terminus of ERDJ4 has a CSGQ sequence which-as a CAAX motif-might be a target for prenylation, a mode to anchor proteins to cellular membranes [16]. Whether the cytosolic pool of ERDJ4 protein is possibly attached to, or anchored at a membrane via this CAAX motif has not yet been investigated. ...
Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces a well-orchestrated cellular response to reduce the protein burden within the ER. This unfolded protein response (UPR) is controlled primarily by three transmembrane proteins, IRE1α, ATF6, and PERK, the activity of which is controlled by BiP, the ER-resident Hsp70 protein. Binding of BiP to co-chaperones via their highly conserved J-domains stimulates the intrinsic ATPase activity of BiP, thereby providing the energy necessary for (re-)folding of proteins, or for targeting of misfolded proteins to the degradation pathway, processes specified and controlled by the respective co-chaperone. In this review, our aim is to elucidate the function of the co-chaperone ERDJ4, also known as MDG1, MDJ7, or DNAJB9. Knockout and knockin experiments clearly point to the central role of ERDJ4 in controlling lipogenesis and protein synthesis by promoting degradation of SREBP1c and the assembly of the protein complex mTORC2. Accumulating data reveal that ERDJ4 controls epithelial-to-mesenchymal transition, a central process during embryogenesis, in wound healing, and tumor development. Overexpression of ERdj4 has been shown to improve engraftment of transplanted human stem cells, possibly due to its ability to promote cellular survival in stressed cells. High ERDJ4-plasma levels are specific for fibrillary glomerulonephritis and serve as a diagnostic marker. As outlined in this review, the functions of ERDJ4 are manifold, depending on the cellular (patho-) physiological state, the cellular protein repertoire, and the subcellular localization of ERDJ4.
... Rac1, one of mammalian Rho GTPases, is implicated in regulation of membrane ruffling, cytoskeletal rearrangement, migration, and apoptosis (Nobes and Hall, 1995;Mattila and Lappalainen, 2008). Before performing the proper function, Rac1 needs prenylation modification for anchoring to plasm membranes and interaction with downstream signaling effectors (Gao et al., 2009). However, ZA proves to inhibit FPP synthase, which is essential for the synthesis of geranylgeranyl pyrophosphate (GGPP; the substrate used for Rac1 posttranslational prenylation) (Dunford et al., 2006). ...
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a severe side effect of long-term administration of bisphosphonates such as zoledronic acid (ZA), but its pathogenesis remains unclear. Impairment of the clearance of apoptotic cells (termed “efferocytosis”) by ZA may be associated with the pathogenesis of BRONJ. The aim of this study was to investigate whether ZA might inhibit macrophage efferocytosis and promote osteocytic apoptosis, and the underlying mechanisms responsible for the disturbing balance between clean and generation of osteocytic apoptosis. We found that ZA significantly promoted the apoptosis of osteocyte and pre-osteoblast via BRONJ mouse models and in vitro MC3T3-E1 but also inhibited the efferocytosis of macrophage on apoptotic cells. Moreover, supplement with geranylgeraniol (GGOH), a substrate analog for geranylgeranylation of Rac1, could restore Rac1 homeostasis and rescue macrophage efferocytosis. GGOH partially inhibits MC3T3-E1 apoptosis induced by ZA via downregulation of Rac1/JNK pathway. We also examined the Rac1 distribution and activation conditions in bone marrow-derived macrophages (BMDMs) and MC3T3-E1 under ZA treatment, and we found that ZA impaired Rac1 migration to BMDM membrane, leading to round appearance with less pseudopodia and efferocytosis inhibition. Moreover, ZA simultaneously activated Rac1, causing overexpression of P-JNK and cleaved caspase 3 in MC3T3-E1. Finally, the systemic administration of GGOH decreased the osteocytic apoptosis and improved the bone healing of the extraction sockets in BRONJ mouse models. Taken together, our findings provided a new insight and experimental basis for the application of GGOH in the treatment of BRONJ.
... Prenylated proteins include members of the Ras, Rab, and Rho families; lamins; and the γ subunits of some G proteins (Lane and Beese, 2006). Hence, disruption of prenylation leads to the development of not only various pathologies of genetic origin, e.g., cancer, but also to osteoporosis and precocious aging (Gao et al., 2009). The use of farnesyltransferase inhibitors (FTIs) to disrupt the functionality of the oncogene Ras, e.g., appears to be a promising therapeutic approach. ...
... In this study, we investigated the effect of the FTIs lonafarnib and tipifarnib on different prokaryotes for the first time. In eukaryotes, farnesylation plays a major role in many diseases and its inhibition by FTIs is under investigation for the treatment of diseases such as cancer, progeria, and hepatitis delta (Gao et al., 2009;Koh et al., 2015;Gordon et al., 2018;Waller et al., 2019). ...
Farnesyltransferase inhibitors (FTIs) are focus for the treatment of several diseases, particularly in the field of cancer therapy. Their potential, however, goes even further, as a number of studies have evaluated FTIs for the treatment of infectious diseases such as malaria, African sleeping sickness, leishmaniosis, and hepatitis D virus infection. Little is known about protein prenylation mechanisms in human pathogens. However, disruption of IspA, a gene encoding the geranyltranstransferase of Staphylococcus aureus (S. aureus) leads to reprogramming of cellular behavior as well as impaired growth and decreased resistance to cell wall-targeting antibiotics. We used an agar well diffusion assay and a time kill assay and determined the minimum inhibitory concentrations of the FTIs lonafarnib and tipifarnib. Additionally, we conducted cell viability assays. We aimed to characterize the effect of these FTIs on S. aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis (S. epidermidis), Escherichia coli (E. coli), Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Streptococcus pneumoniae (S. pneumoniae). Both the FTIs lonafarnib and tipifarnib were capable of inhibiting the growth of the Gram-positive bacteria S. aureus, MRSA, S. epidermidis, and S. pneumoniae, whereas no effect was observed on Gram-negative bacteria. The analysis of the impact of lonafarnib and tipifarnib on common human pathogens might lead to novel insights into their defense mechanisms and therefore provide new therapeutic targets for antibiotic-resistant bacterial infections.
... CAAX proteins are often prenylated before entering the plasma or nucleus membrane, which facilitates the adhesion of these proteins to the cell membrane. [28] CXX1 has a CAAX motif as a prenylation substrate. [29] The reduced expression level of CXX1 is associated with the hypermethylated pattern in colon cancer, while the frequency of gene methylation in normal colon tissues is also relatively high. ...
Aims:
The hypermethylation of CpG islands in the promoter of tumor-suppressor genes (TSGs) leads to silencing the transcription of tumor suppressors, which lead to the development of cancer. The hypermethylation of CXX1 and CDH1 genes, as TSGs, plays an essential role in the development of various types of cancer, i.e., colorectal and gastric cancer. This study aims at evaluating the expression level of CXX1 and CDH1 genes and the methylation status of CXX1 CpG island's promoter in breast cancer (BC).
Materials and methods:
In this study, the expression level of the CXX1 and CDH1 genes and the promoter methylation status of the CXX1 gene were evaluated in 30 paraffin-embedded tissue blocks of malignant BC and 18 benign breast lesions, using quantitative reverse transcription-PCR and methylation-specific (MS)-PCR assays, respectively.
Results:
The CXX1 gene was downregulated in the malignant tissues due to the hypermethylation of the CpG islands in the promoter, compared to the control group (P = 0.031). The downregulation of CDH1 gene expression was observed in the patient group compared to control, but this reduction was not statistically significant. The results show that the risk of BC is increased with aging (P < 0.001). Furthermore, the benign breast lesions (controls) had more mobility in comparison with the malignant breast tumors (P < 0.001). In the malignant samples, the size of the mass was larger than control's mass samples (P = 0.006).
Conclusions:
In the pathophysiological state of BC, the aberrant DNA hypermethylation in CpG island of CXX1 promoter is responsible for the reduction of its expression level in BC patients.
