# KLHL28

Kelch-like Homolog 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 KLHL28 :
| Transcription Factor | Binding Region | Strand | Function |
| ZNF652 (Zinc Finger Protein 652) | aagagtt | + | Transcription repressor |
| FOXH1 (Forkhead Box H1) | aatcccaa | - | Transcription in nodal expression |
| ETV4 (ETS Variant 4) | accggagct | + | Promotes proliferation |
| FEZF2 (FEZ Family Zinc Finger 2) | cccagg | + | Regulates corticospinal motor neuron genes |
| TGIF2LY (TGFB Induced Factor Homeobox 2-like Y-linked) | ctccagttgtcc | + | Regulates RNA Polymerase II in males |
| TGIF2 (TG-Interacting Factor 2) | tgaccacgatct | - | Regulates brain development |
| ZBED4 (Zinc Finger BED-Type Containing 4) | ttctctccgc | - | Regulates genes involved in retinal function |
| ZNF708 (Zinc Finger Protein 708) | tgtagaa | - | Regulates RNA Polymerase II, carcinoma |
| ELF1 (E74-like ETS TF1) | ctaggaaag | - | Regulates homeostasis and vascular development |
| Msgn1 (Mesogenin 1) | cacaaatcgg | + | Regulates mesoderm fate |
| KLF2 (Krüppel-like Factor 2) | ccccgg | - | Regulates differentiation |
| ELK1 (ETS-like Kinase 1) | aaat | - | Regulates early gene experession via serum response |
| KLF14 (Krüppel-like Factor 14) | tggga | - | Regulates lipid metabolism |
| SP4 (Specificity Protein 4) | gtag | - | Regulates cytochrome C in primary neurons |
| Spi1 (Spleen Focus Forming Virus Proviral Integration Oncogene) | aaagaaatgttgc | - | Regulates development and function of microglia |
| TBX20 (T-Box TF20) | taggtctgttt | + | Regulates cardiac development |
| ZNF530 (Zinc Finger Protein 530) | ggcggagagggaa | - | Regulates RNA Polymerase II |
| MAZ (Myc-associated Zinc Finger Protein) | cccctccg | + | Regulates transcription in neural stem cells |
| ZNF263 (Zinc Finger 263) | gggaggc | - | Upregulates IL-33, promotes lung cancer |
| SPIB (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 KLHL28 :
| KLHL28 | pI | MW (in kDa) | Residues |
| Whole Protein | 5.90 | 65.8 | 585 |
| BTB Domain | 4.92 | 13.2 | 118 |
| BACK Domain | 4.43 | 13.1 | 113 |
| Kelch 1 | 4.11 | 5.3 | 48 |
| Kelch 2 | 5.71 | 6.1 | 55 |
| Kelch 3 | 8.95 | 4.9 | 44 |
| Kelch 4 | 5.38 | 5.4 | 49 |
| Kelch 5 | 7.02 | 5.4 | 48 |
| Kelch 6 | 5.30 | 4.8 | 44 |

### 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 Network :
| Gene | Localization | Detection Method | Function |
| CUL3 (Cullin-3) | Cytosol/nucleus | Affinity capture-MS | Core component of E3 ubiquitin ligase complex |
| AKAP11 (A-Kinase Anchoring Protein-11) | Cytosol/nucleus | Affinity capture-MS | Enables protein kinase A binding in somatic and germ cells |
| CUL7 (Cullin-7) | Cytosol/nucleus/Golgi | Affinity capture-MS | Aids in E3 ubiquitin ligase assembly |
| FBXL17 (F-Box and Leucine-rich Repeat Protein 17) | Cytosol/nucleus | Affinity capture-MS | Forms SCF complexes, acts as protein ubiquitin ligases |
| IPP (Inracisternal A Particle-promoted Polypeptide) | Cytosol/cytoskeleton | Affinity capture-MS | Actin organization |
| KLHL13 (Kelch-like Homolog 13) | Cytosol/mitochondrion | Affinity capture-MS | Aids in E3 ubiquitin ligase assembly, necessary for chromosome segregation |
| KLHL14 (Kelch-like Homolog 14) | ER/cytoskeleton/cytosol | Affinity capture-MS | Tumor suppressor, promotes ubiquitination of B cell receptors |
| KLHL21 (Kelch-like Homolog 21) | Cytoskeleton/cytosol | Affinity capture-MS | Implicated with ubiquitin protein transferase activity |
| NSP1 (Novel SH2-containing Protein 1) | Cytosol/nucleus | Two-hybrid | May 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 KLHL28 :
| Common Name | Taxonomic Group | Median Date of Divergence (MYA) | Protein Accession # | Sequence Length (aa) | Identity to Human (%) | Similarity to Human (%) |
| Human | Primates | 0 | NP_001295041.1 | 585 | 100.0 | 100.0 |
| Crab-eating Macaque | Primates | 28.8 | XP_015308707.1 | 585 | 99.2 | 99.3 |
| Canada Lynx | Carnivora | 94 | XP_030174874.1 | 571 | 98.8 | 97.3 |
| Sack-winged Bat | Chiroptera | 94 | XP_066133942.1 | 571 | 96.3 | 97.1 |
| House Mouse | Rodentia | 87 | NP_079983.1 | 571 | 97.0 | 96.8 |
| Orca | Artiodactyls | 94 | XP_004270054.1 | 571 | 99.1 | 96.2 |
| Koala | Diprotodontia | 160 | XP_020842083.1 | 645 | 93.2 | 84.8 |
| Common Garter Snake | Squamata | 319 | XP_013915426.1 | 571 | 87.7 | 95.4 |
| Brown Anole | Squamata | 319 | XP_060644111.2 | 571 | 87.0 | 95.0 |
| Papuan Ground Boa | Squamata | 319 | XP_063146665.1 | 571 | 87.6 | 92.3 |
| American Alligator | Crocodilia | 319 | KYO47230.1 | 585 | 93.2 | 92.0 |
| Kori Bustard | Otidiformes | 319 | NXE21565.1 | 577 | 92.5 | 95.6 |
| Humboldt Penguin | Sphenisciformes | 319 | KAF1420631.1 | 577 | 92.2 | 95.2 |
| Barn Owl | Strigiformes | 319 | XP_042655085.1 | 571 | 92.5 | 94.9 |
| Chicken | Galliformes | 319 | XP_015132273.1 | 571 | 92.6 | 91.8 |
| Gaboon Caecilian | Gymnophiona | 352 | XP_033809266.1 | 574 | 92.5 | 95.0 |
| American Toad | Anura | 352 | XP_040267576.1 | 571 | 88.1 | 92.1 |
| Coelacanth | Latimeriodei | 415 | XP_064420773.1 | 571 | 88.4 | 92.3 |
| Zebrafish | Cypriniformes | 429 | XP_017207216.1 | 571 | 83.2 | 90.4 |
| Small-toothed Sawfish | Rhinoprisitiformes | 462 | XP_051874951.1 | 571 | 85.1 | 89.7 |
| Great White Shark | Lamniformes | 462 | XP_041069815.1 | 571 | 85.5 | 89.9 |
| Sea Lamprey | Petromyzontiformes | 563 | XP_032831289.1 | 611 | 61.4 | 71.0 |
| European Lancelet | Amphioxiformes | 581 | XP_066281657.1 | 574 | 56.6 | 70.3 |
| Crown-of-thorns Starfish | Valvatida | 619 | XP_022083107.1 | 576 | 53.3 | 67.9 |
| Branching Stony Coral | Scleractinia | 685 | XP_029214089.1 | 570 | 49.7 | 65.8 |
| Chiton | Chitonida | 686 | XP_064600849.1 | 521 | 47.8 | 55.5 |

#### Paralogs

Selected Paralogs of KLHL28 :
| Gene Name | Protein Accession # | Sequence Length (aa) | Identity to KLHL28 (%) | Similarity to KLHL28 (%) |
| KLHL28 | NP_001295041.1 | 585 | 100.0 | 100.0 |
| KLHL5 | NP_001007076.1 | 709 | 39.9 | 57.4 |
| KLHL3 | NP_059111.2 | 587 | 38.7 | 54.9 |
| KLHL20 | NP_055273.2 | 609 | 42.8 | 54.3 |
| KLHL17 | NP_938073.1 | 642 | 41.7 | 54.5 |
| KLHL1 | NP_066917.1 | 748 | 40.6 | 53.5 |

## Clinical significance

Based on the function of its encoded protein in protein breakdown and recycling, the KLHL28 gene has a strong clinical significance. Furthermore, the expression data in hypoxic and cancerous conditions suggests the Cullin3-RING E3 ubiquitin ligase complex is involved in protein homeostasis, which can be sabotaged in cancerous cells. Consequently, the KLHL28 gene and its interaction network represent novel targets for gene therapy cancer treatments.