Membrane-spanning Domains Research Articles

Novel gain-of-function mutants identify a critical region within CFTR membrane-spanning domain 2 controlling cAMP-dependent and ATP-independent channel activation

CFTR is an anion channel that has evolved from the mold of an ABC transporter. It possesses specific structural features, including a lateral portal between the cytoplasmic extensions of its transmembrane helices TM4 and TM6. This TM4-TM6 portal is lined by basic residues attracting anions from the cytosol towards the intracellular vestibule. Even though a symmetric, open portal is not observed at the level of the TM10/TM12 interface, basic amino acids are also present at this level, exposed to solvent in the vicinity of the regulatory R region, whose phosphorylation enables channel activation. Here, using all-atom molecular dynamics simulations in combination with functional and biochemical assays, we investigate the importance of these basic amino acids (R1158 and R1030), and of a neighboring aromatic amino acid (W846) in the regulation of CFTR activity. Results indicate that mutation of these amino acids globally increased channel activity and enabled channel opening by potentiators without the need to elevate cAMP levels. These effects (i) were observed even when the binding site of the potentiator VX-770 was mutated, revealing a probable independent mechanism, and (ii) were additive to one gain-of-function mutant within the selectivity filter. Taken together, our results indicate that the region of the membrane-spanning domain 2 (MSD2), symmetric to the lateral portal located between MSD1 TM4 and TM6, is a novel critical actor of CFTR regulation.

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

A uniquely efficacious type of CFTR corrector with complementary mode of action.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

Structural basis for excitatory neuropeptide signaling

Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily is diverse in terms of its gating stimuli, with some DEG/ENaCs gated by neuropeptides, and others gated by pH, mechanical force or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. Here we dissected the structural basis for neuropeptide-gated activity of a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. Structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes of channel gating, which we verified with complementary mutagenesis experiments. Our results illuminate channel gating in DEG/ENaCs and offer a structural template for experimental dissection of channel pharmacology and ion conduction.

A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli–based in vitro coupled transcription–translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2.

Molecular characterization of gonadotropin-releasing hormone receptor and its expression profiling in response to HCG stimulation in striped murrel, Channa striata (Bloch, 1793)

The gonadotropin releasing hormone receptors (GnRHR) play an important role in the activation of GnRH and elicit the release of gonadotropins that plays an important role in maturation and spawning. In the present work, molecular characterization of the GnRHR gene of snakehead murrel, Channa striata was carried out. The full-length cDNA of GnRH receptors of the 1581 bp sequence, which encodes 440 amino acids was identified by RACE-PCR. Signal peptide analysis confirmed that GnRHR is a non-secretory protein, and hydropathy profiles indicated its hydrophobic nature. The deduced amino acid sequences of the GnRHR showed the expected conserved domains, which include seven membrane-spanning domains, two potential N-linked glycosylation sites (631 Asp, 561 Asp), and 32 sites phosphorylation sites of serine and threonine residues. A homology search of GnRHR cDNA sequence revealed 88 % identity with Channa argus, followed by 85 % and 83 % sequence identities with Lates calcarifer, and Anabas testudineus at the protein level. Phylogenetic analysis depicted a close association with Channa argus and other fish species. Our findings showed differential GnRHR expression during different reproductive stages, with higher expression after HCG administration. Expression profiling revealed that GnRHR is highly expressed in the brain and testis at 16 h post-HCG injection, while it was neutrally regulated in female ovarian tissues. This study provides full-length coding sequence information of the GnRHR gene and will help to understand the reproductive biology of murrel using expression studies and devise strategies for induced breeding.

The LytS-type histidine kinase BtsS is a 7-transmembrane receptor that binds pyruvate.

Here, we studied the LytS-type histidine kinase BtsS of E. coli and identified the pyruvate binding site within the membrane-spanning domains. It is a small cavity, and pyruvate forms interactions with the side chains of Arg72, Arg99, Cys110, and Ser113 located in transmembrane helices III, IV, and V, respectively. Our results can serve as a starting point to convert BtsS into a sensor for structurally similar ligands such as lactate, which can be used as biosensor in medicine.

SPTLC1 p.Leu38Arg, a novel mutation associated with childhood ALS

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neuromuscular disease. Recently, several gain-of-function mutations in SPTLC1 were associated with juvenile ALS. SPTLC1 encodes for a subunit of the serine-palmitoyltransferase (SPT) - the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL). SPT activity, and thus SL de novo synthesis, is tightly controlled by a homeostatic feedback mechanism mediated by ORMDL proteins. Here we report a novel SPTLC1p.L38R mutation in a young Chinese girl with a signature of juvenile ALS. The patient presented with muscular weakness and atrophy, tongue tremor and fasciculation, breathing problems and positive pyramidal signs. All SPTLC1-ALS mutations including the SPTLC1 p.L38R are located within a single membrane-spanning domain of the protein and impede the interaction with the regulatory ORMDL subunit of SPT. Pertinent to the altered homeostatic control, lipid analysis showed overall increased SL levels in the patient plasma. An increased SPT activity and SL de novo synthesis was confirmed in p.L38R expressing HEK293 cells. Particularily dihydro-sphingolipids (dhSL) were signficantly increased in patient plasma and p.L38R mutant expressing cells. Increased dhSL formation has been previously linked to neurotoxicity and may be involved in the pathomechanism of SPTLC1-ALS mutations.

Impact of transmembrane peptides on individual lipid motions and collective dynamics of lipid bilayers

The fluid nature of lipid bilayers is indispensable for the dynamic regulation of protein function and membrane morphology in biological membranes. Membrane-spanning domains of proteins interact with surrounding lipids and alter the physical properties of lipid bilayers. However, there is no comprehensive view of the effects of transmembrane proteins on the membrane's physical properties. Here, we investigated the effects of transmembrane peptides with different flip-flop-promoting abilities on the dynamics of a lipid bilayer employing complemental fluorescence and neutron scattering techniques. The quasi-elastic neutron scattering and fluorescence experiments revealed that lateral diffusion of the lipid molecules and the acyl chain motions were inhibited by the inclusion of transmembrane peptides. The neutron spin-echo spectroscopy measurements indicated that the lipid bilayer became more rigid but more compressible and the membrane viscosity increased when the transmembrane peptides were incorporated into the membrane. These results suggest that the inclusion of rigid transmembrane structures hinders individual and collective lipid motions by slowing down lipid diffusion and increasing interleaflet coupling. The present study provides a clue for understanding how the local interactions between lipids and proteins change the collective dynamics of the lipid bilayers, and therefore, the function of biological membranes.

PIN structures shed light on their mechanism of auxin efflux.

Polar auxin transport is a quintessential feature of higher plant physiology and it has been known for many years that some of the primary drivers of polar auxin transport are the PIN-formed (PIN) auxin efflux proteins. Formative research established many key biochemical features of the transport system and discovered inhibitors such as 1-naphthylphthalamic acid (NPA), but the mechanism of action of PINs has remained elusive. This changed in 2022 with the publication of high-resolution structures of the membrane-spanning domains of three PIN proteins. The atomic structures and associated activity assays reveal that PINs use an elevator mechanism to transport auxin anions out of the cell. NPA was shown to be a competitive inhibitor that traps PINs in their inward-open conformation. The secrets of the hydrophilic cytoplasmic loop of PIN proteins remain to be discovered.

Novel tricyclic pyrrolo-quinolines as pharmacological correctors of the mutant CFTR chloride channel

F508del, the most frequent mutation in cystic fibrosis (CF), impairs the stability and folding of the CFTR chloride channel, thus resulting in intracellular retention and CFTR degradation. The F508del defect can be targeted with pharmacological correctors, such as VX-809 and VX-445, that stabilize CFTR and improve its trafficking to plasma membrane. Using a functional test to evaluate a panel of chemical compounds, we have identified tricyclic pyrrolo-quinolines as novel F508del correctors with high efficacy on primary airway epithelial cells from CF patients. The most effective compound, PP028, showed synergy when combined with VX-809 and VX-661 but not with VX-445. By testing the ability of correctors to stabilize CFTR fragments of different length, we found that VX-809 is effective on the amino-terminal portion of the protein that includes the first membrane-spanning domain (amino acids 1–387). Instead, PP028 and VX-445 only show a stabilizing effect when the second membrane-spanning domain is included (amino acids 1–1181). Our results indicate that tricyclic pyrrolo-quinolines are a novel class of CFTR correctors that, similarly to VX-445, interact with CFTR at a site different from that of VX-809. Tricyclic pirrolo-quinolines may represent novel CFTR correctors suitable for combinatorial pharmacological treatments to treat the basic defect in CF.

Identification of Anti-gp41 Monoclonal Antibodies That Effectively Target Cytotoxic Immunoconjugates to Cells Infected with Human Immunodeficiency Virus, Type 1.

We are developing cytotoxic immunoconjugates (CICs) targeting the envelope protein (Env) of the Human Immunodeficiency Virus, type 1 (HIV) to purge the persistent reservoirs of viral infection. We have previously studied the ability of multiple monoclonal antibodies (mAbs) to deliver CICs to an HIV-infected cell. We have found that CICs targeted to the membrane-spanning gp41 domain of Env are most efficacious, in part because their killing is enhanced in the presence of soluble CD4. The ability of a mAb to deliver a CIC does not correlate with its ability to neutralize nor mediate Ab-dependent cellular cytotoxicity. In the current study, we seek to define the most effective anti-gp41 mAbs for delivering CICs to HIV-infected cells. To do this, we have evaluated a panel of human anti-gp41 mAbs for their ability to bind and kill two different Env-expressing cell lines: persistently infected H9/NL4-3 and constitutively transfected HEK293/92UG. We measured the binding and cytotoxicity of each mAb in the presence and absence of soluble CD4. We found that mAbs to the immunodominant helix-loop-helix region (ID-loop) of gp41 are most effective, whereas neutralizing mAbs to the fusion peptide, gp120/gp41 interface, and the membrane proximal external region (MPER) are relatively ineffective at delivering CICs. There was only a weak correlation between antigen exposure and killing activity. The results show that the ability to deliver an effective IC and neutralization are distinct functions of mAbs.

Standard binding free-energy calculation of glycophorin a dimer in non-isotropic media sheds light on the transmembrane alpha-helices association mechanism.

The transmembrane (TM) protein domain recognition and association processes happening in lipid bilayers are pivotal for understanding membrane protein biogenesis. The most straightforward system to study the transmembrane protein binding mechanism is the glycophorin A (GpA) dimeric TM domain. The GpA is a glycoprotein ubiquitous to the human erythrocyte membrane, which forms a non-covalent dimer through the reversible association of its membrane-spanning domain sequences, creating a crossing angle between two alpha-helices. In our study, the membrane protein complex is primarily located in the phosphatidylcholine (POPC) lipid bilayer, surrounded by water. Applying a rigorous theoretical framework of the standard binding free-energy calculation that combines molecular dynamics and potential-of-mean-force calculations, the so-called “geometrical route”, we investigated this complex in non-isotropic media. Our analysis shows that at large separation distances, the helices are pushed together by the lipids, which triggers the interhelical interactions to occur. This observation is in agreement with the “two-stage” model of integral membrane protein folding. Additionally, we observed that the monomers, while reversibly associated, go through a meta-stable state, where the GpA alpha-helices form a different shape compared to the initial one. From the theoretical point of view, we suggest that for the membrane proteins in the non-isotropic environment, the standard binding free-energy estimation via the geometrical route should be analyzed from another perspective compared to those of protein-protein complexes in water media. We revised the separation PMF evaluation, assuming that the lipid bilayer forms a pseudo-cylindrical zone for the reversible separation of the dimer. The methodology employed herein successfully addresses the challenging transmembrane protein-protein binding problem while offering promising perspectives for the binding affinity characterization of different protein complexes in lipid bilayers.

Signaling mechanism of growth hormone-releasing hormone receptor.

The hypothalamic peptide growth hormone-releasing hormone (GHRH) stimulates the secretion of growth hormone (GH) from the pituitary through binding and activation of the pituitary type of GHRH receptor (GHRH-R), which belongs to the family of G protein-coupled receptors with seven potential membrane-spanning domains. Splice variants of GHRH-Rs (SV) in human tumors and other extra pituitary tissues were identified and their cDNA was sequenced. Among the SVs, splice variant 1 (SV1) possesses the greatest similarity to the full-length GHRH-R and remains functional by eliciting cAMP signaling and mitogenic activity upon GHRH stimulation. A large body of work have evaluated potential clinical applications of agonists and antagonists of GHRH in diverse fields, including endocrinology, oncology, cardiology, diabetes, obesity, metabolic dysfunctions, Alzheimer's disease, ophthalmology, wound healing and other applications. In this chapter, we briefly review the expression and potential function of GHRH-Rs and their SVs in various tissues and also elucidate and summarize the activation, molecular mechanism and signalization pathways of these receptors. Therapeutic applications of GHRH analogs are also discussed.

Acting on the CFTR Membrane-Spanning Domains Interface Rescues Some Misfolded Mutants.

ABC transporters are large membrane proteins sharing a complex architecture, which comprises two nucleotide-binding domains (NBDs) and two membrane-spanning domains (MSDs). These domains are susceptible to mutations affecting their folding and assembly. In the CFTR (ABCC7) protein, a groove has been highlighted in the MSD1 at the level of the membrane inner leaflet, containing both multiple mutations affecting folding and a binding site for pharmaco-chaperones that stabilize this region. This groove is also present in ABCB proteins, however it is covered by a short elbow helix, while in ABCC proteins it remains unprotected, due to a lower position of the elbow helix in the presence of the ABCC-specific lasso motif. Here, we identified a MSD1 second-site mutation located in the vicinity of the CFTR MSD1 groove that partially rescued the folding defect of cystic fibrosis causing mutations located within MSD1, while having no effect on the most frequent mutation, F508del, located within NBD1. A model of the mutated protein 3D structure suggests additional interaction between MSD1 and MSD2, strengthening the assembly at the level of the MSD intracellular loops. Altogether, these results provide insightful information in understanding key features of the folding and function of the CFTR protein in particular, and more generally, of type IV ABC transporters.

Structure of the GOLD-domain seven-transmembrane helix protein family member TMEM87A.

TMEM87s are eukaryotic transmembrane proteins with two members (TMEM87A and TMEM87B) in humans. TMEM87s have proposed roles in protein transport to and from the Golgi, as mechanosensitive ion channels, and in developmental signaling. TMEM87 disruption has been implicated in cancers and developmental disorders. To better understand TMEM87 structure and function, we determined a cryo-EM structure of human TMEM87A in lipid nanodiscs. TMEM87A consists of a Golgi-dynamics (GOLD) domain atop a membrane-spanning seven-transmembrane helix domain with a large cavity open to solution and the membrane outer leaflet. Structural and functional analyses suggest TMEM87A may not function as an ion channel or G-protein coupled receptor. We find TMEM87A shares its characteristic domain arrangement with seven other proteins in humans; three that had been identified as evolutionary related (TMEM87B, GPR107, and GPR108) and four previously unrecognized homologs (GPR180, TMEM145, TMEM181, and WLS). Among these structurally related GOLD domain seven-transmembrane helix (GOST) proteins, WLS is best characterized as a membrane trafficking and secretion chaperone for lipidated Wnt signaling proteins. We find key structural determinants for WLS function are conserved in TMEM87A. We propose TMEM87A and structurally homologous GOST proteins could serve a common role in trafficking membrane-associated cargo.

TMEM196 inhibits lung cancer metastasis by regulating the Wnt/β-catenin signaling pathway.

The TMEM196 protein, which comprises four membrane-spanning domains, belongs to the TMEM protein family. TMEM196 was identified as a candidate tumor suppressor gene in lung cancer. However, its role and mechanism in lungcancer metastasis remain unclear. Here, we study the role of TMEM196 in tumor metastasis to further verify the function in lung cancer. In this study, we used qRT-PCR, western blot analysis and immunohistochemistry to examine the expression levels of TMEM196 and related proteins in lung cancer tissues and tumor cells. We utilized Transwell assays, xenograft nude mouse models, and TMEM196-/- mouse models to evaluate the effects of TMEM196 on tumor invasion and metastasis. Finally, we used bioinformatics analysis and dual-luciferase reporter gene assays to explore the molecular mechanism of TMEM196 as a tumor suppressor. We found that TMEM196 mRNA and protein expression levels were significantly decreased in lung cancer tissues and cells. Low expression of TMEM196 in clinical patients was associated with poor prognosis. TMEM196 strongly inhibited tumor metastasis and progression in vitro and in vivo. The primary lung tumors induced by tail vein-inoculated B16 cells in TMEM196-/- mice were significantly larger than those in TMEM196+/+ mice. Mechanistically, TMEM196 inhibited the Wnt signaling pathway and repressed β-catenin promoter transcription. TMEM196 silencing in lung cancer cells and mice resulted in significant upregulation of the expression of β-catenin and Wnt signaling pathway downstream target genes (MMP2 and MMP7). Decreasing β-catenin expression in TMEM196-silenced cancer cells attenuated the antimetastatic effect of TMEM196. Our results revealed that TMEM196 acts as a novel lung cancer metastasis suppressor via the Wnt/β-catenin signaling pathway.

An allosteric modulator activates BK channels by perturbing coupling between Ca2+ binding and pore opening

BK type Ca2+-activated K+ channels activate in response to both voltage and Ca2+. The membrane-spanning voltage sensor domain (VSD) activation and Ca2+ binding to the cytosolic tail domain (CTD) open the pore across the membrane, but the mechanisms that couple VSD activation and Ca2+ binding to pore opening are not clear. Here we show that a compound, BC5, identified from in silico screening, interacts with the CTD-VSD interface and specifically modulates the Ca2+ dependent activation mechanism. BC5 activates the channel in the absence of Ca2+ binding but Ca2+ binding inhibits BC5 effects. Thus, BC5 perturbs a pathway that couples Ca2+ binding to pore opening to allosterically affect both, which is further supported by atomistic simulations and mutagenesis. The results suggest that the CTD-VSD interaction makes a major contribution to the mechanism of Ca2+ dependent activation and is an important site for allosteric agonists to modulate BK channel activation.

Structural Insight into Evolution of the Quinone Binding Site in Complex II.

The Complex II family encompasses membrane bound succinate:quinones reductases and quinol:fumarate reductases that catalyze interconversion of succinate and fumarate coupled with reduction and oxidation of quinone. These enzymes are found in all biological genres and share a modular structure where a highly conserved soluble domain is bound to a membrane-spanning domain that is represented by distinct variations. The current classification of the complex II family members is based on the number of subunits and co-factors in the membrane anchor (types A-F). This classification also provides insights into possible evolutionary paths and suggests that some of the complex II enzymes (types A-C) co-evolved as the whole assembly. Origin of complex II types D and F may have arisen from independent events of de novo association of the conserved soluble domain with a new anchor. Here we analyze a recent structure of Mycobacterium smegmatis Sdh2, a complex II enzyme with two transmembrane subunits and two heme b molecules. This analysis supports an earlier hypothesis suggesting that mitochondrial complex II (type C) with a single heme b may have evolved as an assembled unit from an ancestor similar to M. smegmatis Sdh2.

Functional Characterization of a Flavone Synthase That Participates in a Kumquat Flavone Metabolon.

Flavones predominantly accumulate as O- and C-glycosides in kumquat plants. Two catalytic mechanisms of flavone synthase II (FNSII) support the biosynthesis of glycosyl flavones, one involving flavanone 2-hydroxylase (which generates 2-hydroxyflavanones for C-glycosylation) and another involving the direct catalysis of flavanones to flavones for O-glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat FNSII genes (FcFNSII-1 and FcFNSII-2), based on transcriptome and bioinformatics analysis. Data from in vivo and in vitro assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas FcFNSII-1 showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of FcFNSII-2 in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several O-glycosyl flavones. Moreover, studying the subcellular localizations of FcFNSII-1 and FcFNSII-2 demonstrated that N-terminal membrane-spanning domains were necessary to ensure endoplasmic reticulum localization and anchoring. Protein–protein interaction analyses, using the split-ubiquitin yeast two-hybrid system and bimolecular fluorescence-complementation assays, revealed that FcFNSII-2 interacted with chalcone synthase 1, chalcone synthase 2, and chalcone isomerase-like proteins. The results provide strong evidence that FcFNSII-2 serves as a nucleation site for an O-glycosyl flavone metabolon that channels flavanones for O-glycosyl flavone biosynthesis in kumquat fruits. They have implications for guiding genetic engineering efforts aimed at enhancing the composition of bioactive flavonoids in kumquat fruits.