Cellular Retinol-binding Protein Research Articles

Regulation of circulating thyroid hormone levels by hypothalamic tanycytes and hypophysial pars tuberalis-specific cells and their morphological and gene- and protein-expression changes under different photoperiods.

Thyroid hormone in the hypothalamus acts as a key determinant of seasonal transitions. Thyroid hormone-levels in the brain are mainly regulated by the hypothalamic tanycytes and pituitary pars tuberalis (PT)-specific cells. TSHβ produced by the PT-specific cells stimulates Dio2 expression and decreases Dio3 expression of the tanycytes. Both tanycytes and PT-specific cells in photosensitive animals exhibit remarkable changes of morphological appearance and expressions of genes and proteins under different photoperiods. Long photoperiods induce increased gene- and protein-expressions and active features. Short photoperiods cause the decreased gene- and protein-expressions and inactive features. In the PT, expressions of TSHβ, common α-subunit of glycoprotein hormones (α-GSU), and MT1 receptor of melatonin receptors and eyes absent 3 change under different photoperiods. Diurnal rhythms of α-GSU mRNA expression are observed in the PT of Djungarian hamsters. Hes1, Nkx2.1, and LIM homeodomain gene 2 (Lhx2) are involved in the differentiation of PT. In the hypothalamic tanycytes, expressions of Dio2, Dio3, vimentin, serine/threonine kinase 33, GPR50, Nestin, Retinoid signaling genes (retinaldehyde dehydrogenase 1, cellular retinol binding protein 1, and Stra6), monocarboxylate transporter 8, and neural cell adhesion molecule change under different photoperiods. Rax, Lhx2, Nfia/b/x, and fibroblast growth factor 10 are involved in the differentiation of tanycytes.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

Comparative molecular dynamics study on the features of binding and non-binding modes of retinoic acid in cellular retinol-binding protein (I)

Retinoids play crucial roles in various biological processes by interacting with their carrier proteins such as cellular retinol-binding protein (CRBP). Understanding the molecular interactions between retinoids and CRBP enables their pharmacological and biomedical applications. Experimentally, CRBP(I) does not bind to retinoic acid, but when arginine is introduced into 108th residue instead of glutamine (Q108R), it binds to retinoic acid. Here, molecular dynamics simulations were performed to understand the differences in the microscopic and dynamic behaviors of the non-binding wild-type CRBP(I)-retinoic acid and binding Q108R variant-retinoic acid complexes. The ligand RMSD and RMSF, the binding poses of binding motif amino acids, and the number of hydrogen bonds and salt-bridges revealed the relative instability of the non-binding complex. In particular, the ligand's terminal group showed very different dynamics and interactions. So far, most studies have focused on the binding characteristics of retinoids, but the features of their non-binding modes have not been studied well. This study provides some structural insights into the non-binding modes of a retinoid in CRBP, which may be applicable in retinoid-based drug discovery and protein engineering through computational modeling.

Reduced Expression of RBP7 is Associated with Resistance to Tamoxifen In Luminal A Breast Cancer.

Tamoxifen is the most commonly used hormonal treatment for ERα-positive breast cancer. Tamoxifen resistance is still a big problem for ERα target therapy. RBP7 is a member of the cellular retinol-binding protein family. This study aims to investigate the prognostic role of RBP7 and the relationship between RBP7 expression and sensitivity or resistance to tamoxifen in ERα-positive breast cancer. A bioinformatics method was used to investigate RBP7 expression and the prognostic value of RBP7 in different subtypes of breast cancer. The relationship between RBP7 expression and sensitivity or resistance to tamoxifen was studied using clinical data (GSE1379) and cell line data (GSE27473, GSE2645923, GSM3715281, and GSM3715282). Transfection of RBP7 experiments was used to testify to the function of RBP7 in MCF7 cell. RBP7 is a member of the family of cellular retinol-binding proteins. RBP7 expression was down-regulated at both mRNA and protein levels in breast cancer and was not associated with different TNM (Tumor, Node, Metastasis) stages. High expression of RBP7 was significantly related to good relative percent survival in the luminal A subtype, but in negative breast cancer, the result was opposite. The ROC plot showed that RBP7 had a significant predictive value for the tamoxifen response in the luminal A subtype. The expression of RBP7 from patients with recurrence treated with tamoxifen was significantly reduced. Gene Expression Omnibus showed that RBP7 expression was reduced considerably in tamoxifen-resistant MCF7 cells and T47D cells. The expression of RBP7 was positively correlated with some microRNAs involved in negatively regulating tamoxifen-resistant breast cancer. We also found that the expression of RBP7 decreased significantly in tamoxifen-resistant MCF7 cells, and transfection of RBP7 increased the sensitivity of resistant cells to tamoxifen. Reduced expression of RBP7 is associated with resistance to tamoxifen in luminal A breast cancer. Our research may help to explore the mechanisms of resistance of breast cancer to tamoxifen.

Retinyl Ester Storage Is Normal in Transgenic Mice with Enhanced Expression of Cellular Retinol-Binding Protein Type I

This report describes the production and characterization of transgenic mice with high expression of human cellular retinol-binding protein type I [hCRBP(I)]. In initial experiments, overexpression of hCRBP(I) was driven by the strong promoter SRα, but no transgenic offspring were produced. When we used the less efficient mouse metallothionein I promoter fused to the hCRBP(I) cDNA for microinjection, we obtained 12% transgenic offspring. Two of these transgenic mice (409/1 and 401/2) expressed mRNA and immunoreactive hCRBP(I) in several organs. Both lines had relatively high contents of hCRBP(I) in intestine, testis and epididymis. On the other hand, only 401/2 transgenic mice had high contents of hCRBP(I) in kidney. Effects on storage of vitamin A were studied by measuring the concentration of retinyl esters in different organs. The concentrations of retinyl esters in liver, lung and kidney did not significantly differ between transgenic and control mice, and the concentration of total retinol in plasma was within the normal range in transgenic mice. Furthermore, feeding mice a diet with high or low concentrations of vitamin A for 2 wks resulted in no marked differences in the concentrations of retinyl esters in liver, kidney, lung, intestine and testis in transgenic mice compared with control mice. Therefore, in spite of high expression of hCRBP(I) in several organs, the transgenic mice had normal storage of retinyl esters in all organs studied. The present in vivo study indicates that the CRBP(I) content alone does not control retinyl ester storage.

Light controlled reversible Michael addition of cysteine: a new tool for dynamic site-specific labeling of proteins.

Cysteine-based Michael addition is a widely employed strategy for covalent conjugation of proteins, peptides, and drugs. The covalent reaction is irreversible in most cases, leading to a lack of control over the process. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrate Michael addition of an engineered cysteine residue in human Cellular Retinol Binding Protein II (hCRBPII) with a coumarin analog that creates a non-fluorescent complex. UV-illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. This series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON-OFF control of fluorescence. The details of the mechanism of photoswitching was illuminated by recapitulation of the process in light irradiated single crystals, confirming the mechanism at atomic resolution.

Cellular retinol-binding protein 1: a therapeutic and diagnostic tumor marker.

Cellular Retinol Binding Protein 1 (CRBP1) gene is a protein coding gene located on human chromosome 3q21, which codifies a protein named CRBP1. CRBP1 is widely expressed in many tissues as a chaperone protein to regulate the uptake, subsequent esterification and bioavailability of retinol. CRBP1 combines retinol and retinaldehyde with high affinity to protect retinoids from non-specific oxidation, and transports retinoids to specific enzymes to promote the biosynthesis of retinoic acid. The vital role of CRBP1 in retinoids metabolism has been gradually discovered, which has been implicated in tumorigenesis. However, the precise functions of CRBP1 in different diseases are still poorly understood. The purpose of this review is to provide an overview of the role of CRBP1 in various diseases, especially in both the promotion and inhibition of cancers, which may also offer a novel biomarker and potential therapeutic target for human diseases.

Methylation-Mediated Silencing of RBP7 Promotes Breast Cancer Progression through PPAR and PI3K/AKT Pathway.

Retinoid-binding protein7 (RBP7) is a member of the cellular retinol-binding protein (CRBP) family, which is involved in the pathogenesis of breast cancer. The study aims to illustrate the prognostic value and the potential regulatory mechanisms of RBP7 expression in breast cancer. Bioinformatics analysis with the TCGA and CPTAC databases revealed that the mRNA and protein expression levels of RBP7 in normal were higher compared to breast cancer tissues. Survival analysis displayed that the lower expression of RBP7, the worse the prognosis in ER-positive (ER+) breast cancer patients. Genomic analysis showed that low expression of RBP7 correlates with its promoter hypermethylation in breast cancer. Functional enrichment analysis demonstrated that downregulation of RBP7 expression may exert its biological influence on breast cancer through the PPAR pathway and the PI3K/AKT pathway. In summary, we identified RBP7 as a novel biomarker that is helpful for the prognosis of ER+ breast cancer patients. Promoter methylation of RBP7 is involved in its gene silencing in breast cancer, thus regulating the occurrence and development of ER+ breast cancer through the PPAR and PI3K/AKT pathways.

Identification of SEC14 like lipid binding 2(SEC14L2) sequence and expression profiles in the Chinese tree shrew (Tupaia belangeri chinensis).

The product of the SEC14L2 (SEC14 Like Lipid Binding 2) gene belongs to a family of lipid-binding proteins including Sec14p, alpha-tocopherol transfer protein, and cellular retinol-binding protein. SEC14L2 expression enables replication of clinical hepatitis C virus (HCV) isolates in several hepatoma cell lines, and mutations in SEC14L2 may enhance HCV replication in vitro. The Chinese tree shrew (Tupaia belangeri chinensis) is a potential animal model for studying HCV replication, however, the cDNA sequence, protein structure, and expression of the Chinese tree shrew SEC14L2 gene have yet to be characterized. In the present study, we cloned the full-length cDNA sequence of the SEC14L2 in the Chinese tree shrew by using rapid amplification of cDNA ends technology. This led us to determine that, this is 2539 base pairs (bp) in length, the open reading frame sequence is 1212bp, and encodes 403 amino acids. Following this, we constructed a phylogenetic tree based on SEC14L2 molecules from various species and compared SEC14L2 amino acid sequence with other species. This analysis indicated that the Chinese tree shrew SEC14L2 protein (tsSEC14L2) has 96.28% amino acid similarity to the human protein, and is more closely related to the human protein than either mouse or rat protein. The Chinese tree shrew SEC14L2 mRNA was detected in all tissues, and showed highest expression levels in the pancreas, small intestine and trachea, however the tsSEC14L2protein abundance was highest in the liver and small intestine. The Chinese tree shrew SEC14L2 gene was closer in evolutionary relation to humans and non-human primates and expression of the tsSEC14L2protein was highest in the liver and small intestine. These results may provide useful information for tsSEC14L2 function in HCV infection.

Retinol-binding protein 2 (RBP2): More than just dietary retinoid uptake

Retinol-binding protein 2 (RBP2, also known as cellular retinol-binding protein 2 (CRBP2)) is a member of the fatty acid-binding protein family and has been extensively studied for its role in facilitating dietary vitamin A (retinol) uptake and metabolism within enterocytes of the small intestine. RBP2 is present in highest concentrations in the proximal small intestine where it constitutes approximately 0.1–0.5% of soluble protein. Recent reports have established that RBP2 binds monoacylglycerols (MAGs) with high affinity, including the canonical endocannabinoid 2-arachidonoylglycerol (2-AG). Crystallographic studies reveal that retinol, 2-AG, or other long-chain MAGs alternatively can bind in the retinol-binding pocket of RBP2. It also has been demonstrated recently that Rbp2-deficient mice are more susceptible to developing obesity and associated metabolic phenotypes when exposed to a high fat diet, or as they age when fed a conventional chow diet. When subjected to an oral fat challenge, the Rbp2-deficient mice release into the circulation significantly more, compared to littermate controls, of the intestinal hormone glucose-dependent insulinotropic polypeptide (GIP). These new findings regarding RBP2 structure and actions within the intestine are the focus of this review.

Effect of cellular stress on retinoid homeostasis in the small intestine

Vitamin A is an essential diet‐derived nutrient. Retinoids are compounds with Vitamin A activity and are essential for many biological processes, including proper cell function and tissue organization. The main active metabolite of Vitamin A is all‐trans retinoic acid (RA), which is critical for nuclear receptor mediated signaling for cell proliferation, differentiation, and apoptosis. Retinoid signaling and RA are essential for intestinal immune homeostasis. Intestinal epithelial cells (IEC) produce high levels of RA, which is crucial for RA‐dependent signaling, lineage commitment, and barrier function. Vitamin A deficiency can alter intestinal immune and IEC homeostasis.There are many mechanisms in place to maintain retinoic acid homeostasis, including the expression of the main intracellular chaperone protein cellular retinol binding protein, type 1 (CRBP1). CRBP1 binds to retinol and retinal, protects them from non‐specific oxidation, and facilitates their delivery to the appropriate enzymes for RA biosynthesis. CRBP1 has been shown to be decreased in diseases that display dysfunctional proliferation and differentiation, including cancer, inflammatory disorders, fibrosis, and infection. Reduction of CRBP1 directly correlates with reduction in the active metabolite, RA, and restoration of CRBP1 expression has been shown to increase RA levels and positively impact RA‐dependent outcomes. Therefore, monitoring CRBP1 and retinoid metabolite levels in response to cellular stress will provide useful information about the effect of CRBP1 loss on retinoid signaling.While previous studies have explored the effect of cellular stress on RA levels, the effect of stress on the entirety of the retinoid signaling pathway is not well understood. The objective of these studies is to systematically determine the effect of cellular stress on retinoid homeostasis.We have established three models of cellular stress, including hypoxia, oxidative stress, and inflammation, in two human intestinal epithelial cells lines, HIEC‐6 and Caco‐2. To mimic hypoxia, cells are treated with 0‐100 μM CoCl2 for 24 hours. To mimic oxidative stress, cells are treated with 0‐100 μM H2O2 for 24 hours. To mimic inflammation, cells are treated with 0‐1 μg/mL lipopolysaccharide for 48 hours. These conditions were chosen because there was no effect on cell viability. CRBP1 and RA levels will be measured through targeted mass spectrometry methods developed by our lab. Additionally, expression levels of genes associated with retinoid metabolism will be measured. Our current data shows that RA levels decreased in response to hypoxia, oxidative stress, and inflammation in both HIEC‐6 and Caco‐2 cells.

Role of cellular retinol-binding protein, type 1 and retinoid homeostasis in the adult mouse heart: A multi-omic approach.

The main active metabolite of Vitamin A, all-trans retinoic acid (RA), is required for proper cellular function and tissue organization. Heart development has a well-defined requirement for RA, but there is limited research on the role of RA in the adult heart. Homeostasis of RA includes regulation of membrane receptors, chaperones, enzymes, and nuclear receptors. Cellular retinol-binding protein, type 1 (CRBP1), encoded by retinol-binding protein, type 1 (Rbp1), regulates RA homeostasis by delivering vitamin A to enzymes for RA synthesis and protecting it from non-specific oxidation. In this work, a multi-omics approach was used to characterize the effect of CRBP1loss using the Rbp1-/- mouse. Retinoid homeostasis was disrupted in Rbp1-/- mouse heart tissue, as seen by a 33% and 24% decrease in RA levels in the left and right ventricles, respectively, compared to wild-type mice (WT). To further inform on the effect of disrupted RA homeostasis, we conducted high-throughput targeted metabolomics. A total of 222metabolite and metabolite combinations were analyzed, with 33having differential abundance between Rbp1-/- and WT hearts. Additionally, we performed global proteome profiling to further characterize the impact of CRBP1loss in adult mouse hearts. More than 2606 unique proteins were identified, with 340 proteins having differential expression between Rbp1-/- and WT hearts. Pathway analysis performed on metabolomic and proteomic data revealed pathways related to cellular metabolism and cardiac metabolism were the most disrupted in Rbp1-/- mice. Together, these studies characterize the effect of CRBP1loss and reduced RA in the adult heart.

In silico Study on Binding Specificities of Cellular Retinol Binding Protein and Its Q108R Mutant

In silico Study on Binding Specificities of Cellular Retinol Binding Protein and Its Q108R Mutant

Design of Large Stokes Shift Fluorescent Proteins Based on Excited State Proton Transfer of an Engineered Photobase.

The incredible potential for fluorescent proteins to revolutionize biology has inspired the development of a variety of design strategies to address an equally broad range of photophysical characteristics, depending on potential applications. Of these, fluorescent proteins that simultaneously exhibit high quantum yield, red-shifted emission, and wide separation between excitation and emission wavelengths (Large Stokes Shift, LSS) are rare. The pursuit of LSS systems has led to the formation of a complex, obtained from the marriage of a rationally engineered protein (human cellular retinol binding protein II, hCRBPII) and different fluorogenic molecules, capable of supporting photobase activity. The large increase in basicity upon photoexcitation leads to protonation of the fluorophore in the excited state, dramatically red-shifting its emission, leading to an LSS protein/fluorophore complex. Essential for selective photobase activity is the intimate involvement of the target protein structure and sequence that enables Excited State Proton Transfer (ESPT). The potential power and usefulness of the strategy was demonstrated in live cell imaging of human cell lines.

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid–binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the kcat/Km values either decreased 5-fold or were equal to the respective free retinoid values. The kcat/Km value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in kcat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.

Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands

Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format. By examining chemical libraries of bioactive lipids, we provided evidence for the selective interaction of CRBP2 with a subset of nonretinoid ligands with the highest affinity for sn-1 and sn-2 MAGs that contain polyunsaturated C18-C20 acyl chains. We also elucidated the structure-affinity relationship for nonretinoid ligands of this protein. We further dissect the molecular basis for this ligand's specificity by analyzing high-resolution crystal structures of CRBP2 in complex with selected derivatives of MAGs. Finally, we identify T51 and V62 as key amino acids that enable the broadening of ligand selectivity to MAGs in CRBP2 as compared with retinoid-specific CRBP1. Thus, our study provides the molecular framework for understanding the lipid selectivity and diverse functions of CRBPs in controlling lipid homeostasis.

Cellular retinol-binding protein II binds monoacylglycerols and modulates gut endocrine signaling

Cellular retinol-binding protein II binds monoacylglycerols and modulates gut endocrine signaling

Front Cover: Human Cellular Retinol Binding Protein II Forms a Domain‐Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering (ChemBioChem 22/2020)

Front Cover: Human Cellular Retinol Binding Protein II Forms a Domain‐Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering (ChemBioChem 22/2020)

Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Human Cellular Retinol Binding Protein II Forms a Domain‐Swapped Trimer Representing a Novel Fold and a New Template for Protein Engineering

Domain-swapping is a mechanism for evolving new protein structure from extant scaffolds, and has been an efficient protein-engineering strategy for tailoring functional diversity. However, domain swapping can only be exploited if it can be controlled, especially in cases where various folds can coexist. Herein, we describe the structure of a domain-swapped trimer of the iLBP family member hCRBPII, and suggest a mechanism for domain-swapped trimerization. It is further shown that domain-swapped trimerization can be favored by strategic installation of a disulfide bond, thus demonstrating a strategy for fold control. We further show the domain-swapped trimer to be a useful protein design template by installing a high-affinity metal binding site through the introduction of a single mutation, taking advantage of its threefold symmetry. Together, these studies show how nature can promote oligomerization, stabilize a specific oligomer, and generate new function with minimal changes to the protein sequence.