Kelch-like protein 28 (KLHL28) is a protein that is encoded by the KLHL28 gene in humans. It is a member of the Kelch-like gene family, which comprises 42 different genes. Aberrant activation of KLHL28 results in increased likelihood of hypertension, hyperkalemia, and cancer. The KLHL28 gene, also known as BTBD5, has orthologs in vertebrates and some marine invertebrates, and has been well-conserved over evolutionary timescales.
Gene The KLHL28 gene is located on the negative strand of human Chromosome 14 (14q21.2) and spans 7019 base pairs in its complete isoform (isoform 1).
Transcription factors Hundreds of transcription factors are predicted to bind to the promoter region. Many of the highly scored transcription factors from the JASPAR database via the University of California, Santa Cruz Genome Browser are listed in the table below. +Transcription Factors for Human KLHL28Transcription factorBinding RegionStrandFunctionZNF652 (Zinc Finger Protein 652)aagagtt+Transcription repressorFOXH1 (Forkhead Box H1)aatcccaa−Transcription in nodal expressionETV4 (ETS Variant 4)accggagct+Promotes proliferationFEZF2 (FEZ Family Zinc Finger 2)cccagg+Regulates corticospinal motor neuron genesTGIF2LY (TGFB Induced Factor Homeobox 2-like Y-linked)ctccagttgtcc+Regulates RNA Polymerase II in malesTGIF2 (TG-Interacting Factor 2)tgaccacgatct−Regulates brain developmentZBED4 (Zinc Finger BED-Type Containing 4)ttctctccgc−Regulates genes involved in retinal functionZNF708 (Zinc Finger Protein 708)tgtagaa−Regulates RNA Polymerase II, carcinomaELF1 (E74-like ETS TF1)ctaggaaag−Regulates homeostasis and vascular developmentMsgn1 (Mesogenin 1)cacaaatcgg+Regulates mesoderm fateKLF2 (Krüppel-like Factor 2)ccccgg−Regulates differentiationELK1 (ETS-like Kinase 1)aaat−Regulates early gene experession via serum responseKLF14 (Krüppel-like Factor 14)tggga−Regulates lipid metabolismSP4 (Specificity Protein 4)gtag−Regulates cytochrome C in primary neuronsSpi1 (Spleen Focus Forming Virus Proviral Integration Oncogene)aaagaaatgttgc−Regulates development and function of microgliaTBX20 (T-Box TF20)taggtctgttt+Regulates cardiac developmentZNF530 (Zinc Finger Protein 530)ggcggagagggaa−Regulates RNA Polymerase IIMAZ (Myc-associated Zinc Finger Protein)cccctccg+Regulates transcription in neural stem cellsZNF263 (Zinc Finger 263)gggaggc−Upregulates IL-33, promotes lung cancerSPIB (Spi-B TF)tcacttgcggt+Regulates M-cell differentiation
Expression KLHL28 is ubiquitously expressed under normal physiological conditions in humans and has been found not to be monallelically expressed. DNA microarray data also suggests that gene expression of KLHL28 is elevated in the brain and heart.
Under hypoxic conditions, DNA microarray data illustrated increased expression of KLHL28. Further, in a DNA microarray study of small-cell lung cancer, KLHL28 was expressed at significantly higher levels than the control. These data indicate that the gene's transcription is impacted by the tumor microenvironment, which is typically not well-vascularized and often hypoxic.
Mutations Missense single nucleotide polymorphisms (SNPs) have been identified in both the coding and noncoding regions of the gene. The SNPs with the potential for most clinical significance are those affecting the composition of the KLHL28 protein, specifically within Kelch motif 1 (rs117295933, C>A / C>G / C>T) and motif 3 (rs35352691, T>G).
mRNA and transcriptional variants
There are a total of seven transcriptional variants: two isoforms and five spliced transcript variants, all of which are based on the longest transcript, isoform 1. Isoform 1 contains five exons, and all transcriptional variants contain at least exon 2. The six conserved Kelch motifs in the KLHL28 protein are found through the end of exon 2 through exon 5.
5' Untranslated region The 5' untranslated region (5' UTR) of KLHL28 is well-conserved amongst orthologs, similar to the protein sequence. Demonstrating this conservation, the transcription factor ZNF263 was conserved after a five-member mammalian multiple sequence alignment (MSA) using the orthologs golden snub-nosed monkey, green monkey, southern pig-tailed macaque, and thirteen-lined ground squirrel.
3' Untranslated region A four-member MSA was run for the 3' UTR and found that, like the 5' UTR, it was strongly conserved across primates, with the same orthologs as in the 5' UTR section, minus the thirteen-lined ground squirrel.
RNA binding protein interactions An analysis of the miRNA binding capacity of the KLHL28 isoform 1 transcript returned with only one miRNA target that was found on the 3' UTR and conserved through mammals. The miRNA, hsa-miR-182-5p, has been associated with tumorigenesis, specifically in lung tissue.
RNA binding protein binding capacity for the 3' UTR of human KLHL28 was assessed and returned with 12 repeats of ELAVL2, 2 of SNRPA, 3 of ZFP36, and 1 of pum. Based on these highly probable predicted RNA binding proteins, KLHL28 expression at the RNA level is predicted to be connected to neural development during embryogenesis and differentiation.
Protein
Amino acid composition The KLHL28 gene encodes the KLHL28 protein which is 585 amino acids long and has a molecular weight of 65.8kDa. The theoretical isoelectric point (Ip) was predicted to be 5.90 based on the amino acid composition.
An analysis of the whole protein indicated that it is tyrosine-rich (5.3%); however, amino acids at the domain level were expressed differently. The broad-complex, tramtrack, and bric-á-brac (BTB) and BACK (BTB and C-terminal Kelch) domains were extremely glycine-poor (2.4%) and also histidine-rich (4.7%). On the other hand, the Kelch domains were rich in both valine (9.7%) and tyrosine (5.6%).
Protein domains The protein can be broken down into eight domains: the Broad-complex, Tramtrack, and Bric-á-brac (BTB) domain (amino acids 31-148); the BACK domain (amino acids 151-253); and Kelch domains 1 (amino acids 298-345), 2 (amino acids 346-400), 3 (amino acids 401-444), 4 (amino acids 445-493), 5 (amino acids 494-541), 6 (amino acids 542-585). The high predicted isoelectric point of Kelch domain 3 indicates it may have an important role in forming the Cullin3-RING E3 ubiquitin ligase complex. +Annotated Protein Domains in KLHL28KLHL28pIMW (in kDa)ResiduesWhole Protein5.9065.8585BTB Domain4.9213.2118BACK Domain4.4313.1113Kelch 14.115.348Kelch 25.716.155Kelch 38.954.944Kelch 45.385.449Kelch 57.025.448Kelch 65.304.844
Post-translational modifications Predicted post-translational modifications (PTMs) of the protein include a number of phosphorylation sites, visualized in the linear protein schematic to the right.
Tertiary structure The BTB and BACK domains are predicted with high confidence to mostly fold into alpha helices. Meanwhile, the Kelch domains in the protein form into beta sheets, which are then expected to complex into a beta barrel. No hydrophobic regions were identified on the protein, indicating that it is most likely not cell or organelle membrane-localized.
Protein localization KLHL28 protein is soluble, localized in the cytoplasm, and is predicted to contain a nuclear localization signal.
Function Based on the protein-protein interaction (PPI) network below, the KLHL28 protein is predicted to play a critical role in the formation and function of the E3 ubiquitin ligase complex involved in protein degradation and recycling. Additionally, the component domains of the protein suggest it may also be involved in orienting the substrate protein entering the Cullin3-based E3 ligase complex.
Protein-protein interaction network The majority of identified proteins found to interact with the KLHL28 protein in humans are involved in the E3 ubiquitin ligase structure. The table below illustrates proteins interacting with human KLHL28 protein and their detection methods based on a consensus of high-throughput screening data reported from STRING, BioGRID, MINT, and IntAct databases. +KLHL28 Protein-Protein Interaction NetworkGeneLocalizationDetection MethodFunctionCUL3 (Cullin-3)Cytosol/nucleusAffinity capture-MSCore component of E3 ubiquitin ligase complexAKAP11 (A-Kinase Anchoring Protein-11)Cytosol/nucleusAffinity capture-MSEnables protein kinase A binding in somatic and germ cellsCUL7 (Cullin-7)Cytosol/nucleus/GolgiAffinity capture-MSAids in E3 ubiquitin ligase assembly FBXL17 (F-Box and Leucine-rich Repeat Protein 17)Cytosol/nucleusAffinity capture-MSForms SCF complexes, acts as protein ubiquitin ligasesIPP (Inracisternal A Particle-promoted Polypeptide)Cytosol/cytoskeletonAffinity capture-MSActin organizationKLHL13 (Kelch-like Homolog 13)Cytosol/mitochondrionAffinity capture-MSAids in E3 ubiquitin ligase assembly, necessary for chromosome segregationKLHL14 (Kelch-like Homolog 14)ER/cytoskeleton/cytosolAffinity capture-MSTumor suppressor, promotes ubiquitination of B cell receptorsKLHL21 (Kelch-like Homolog 21)Cytoskeleton/cytosolAffinity capture-MSImplicated with ubiquitin protein transferase activityNSP1 (Novel SH2-containing Protein 1)Cytosol/nucleusTwo-hybridMay play a role in JNK (Jun N-terminal kinase) activation under stress conditions
Evolution Based on the ortholog data in the table below, the KLHL28 gene first appeared in some marine invertebrates nearly 700 million years ago. The gene is found in some mollusks, cnidarians, and echinoderms, but not in arthropods nor cephalopods. Other members of the gene family (paralogs of KLHL28, such as KLHL20) have been identified in plants, bacteria, and archaea, indicating that the Kelch-like homologs are highly conserved across evolutionary time and likely serve an important role.
Orthologs +Selected Orthologs of KLHL28Common nameTaxonomic groupMedian Date of Divergence (MYA)Protein Accession #Sequence length (aa)Identity to Human (%)Similarity to Human (%)HumanPrimates0NP_001295041.1585100.0100.0Crab-eating MacaquePrimates28.8XP_015308707.158599.299.3Canada LynxCarnivora94XP_030174874.157198.897.3Sack-winged BatChiroptera94XP_066133942.157196.397.1House MouseRodentia87NP_079983.157197.096.8OrcaArtiodactyls94XP_004270054.157199.196.2KoalaDiprotodontia160XP_020842083.164593.284.8Common Garter SnakeSquamata319XP_013915426.157187.795.4Brown AnoleSquamata319XP_060644111.257187.095.0Papuan Ground BoaSquamata319XP_063146665.157187.692.3American AlligatorCrocodilia319KYO47230.158593.292.0Kori BustardOtidiformes319NXE21565.157792.595.6Humboldt PenguinSphenisciformes319KAF1420631.157792.295.2Barn OwlStrigiformes319XP_042655085.157192.594.9ChickenGalliformes319XP_015132273.157192.691.8Gaboon CaecilianGymnophiona352XP_033809266.157492.595.0American ToadAnura352XP_040267576.157188.192.1CoelacanthLatimeriodei415XP_064420773.157188.492.3ZebrafishCypriniformes429XP_017207216.157183.290.4Small-toothed SawfishRhinoprisitiformes462XP_051874951.157185.189.7Great White SharkLamniformes462XP_041069815.157185.589.9Sea LampreyPetromyzontiformes563XP_032831289.161161.471.0European LanceletAmphioxiformes581XP_066281657.157456.670.3Crown-of-thorns StarfishValvatida619XP_022083107.157653.367.9Branching Stony CoralScleractinia685XP_029214089.157049.765.8ChitonChitonida686XP_064600849.152147.855.5
Paralogs +Selected Paralogs of KLHL28Gene NameProtein Accession #Sequence length (aa)Identity to KLHL28 (%)Similarity to KLHL28 (%)KLHL28NP_001295041.1585100.0100.0KLHL5NP_001007076.170939.957.4KLHL3NP_059111.258738.754.9KLHL20NP_055273.260942.854.3KLHL17NP_938073.164241.754.5KLHL1NP_066917.174840.653.5
Clinical significance