KLHL36

Kelch-Like Protein 36 (abbreviated KLHL36) is a protein that in humans is encoded by the KLHL36 gene.^[5] Although the gene's specific function has yet to be characterized, it is predicted to serve as a substrate adaptor for Cullin-RING E3 ubiquitin ligase (CRL3) complexes, contributing to proteostasis.^[6]

In humans, the KLHL 36 gene consists of 5 exons and 4 introns spanning 19,176 base pairs on the positive strand of chromosome 16 (16q24.1).^[5]

The gene is a member of the Kelch-like gene family, which is characterized by a conserved BTB-BACK-Kelch domain architecture. Kelch-like genes encode proteins that recognize and bind specific protein substrates, bind the scaffold protein cullin 3 in the CRL3 complex, and pass the substrate protein to the complex to facilitate ubiquitylation and degradation at the proteasome.^[6]

The KLHL36 gene is highly conserved in vertebrates. Orthologs of KLHL36 are present in jawed vertebrates tracing back to cartilaginous fishes, although no orthologs have been identified in jawless vertebrates or invertebrates.^[7] This suggests that the KLHL36 gene originated in a common ancestor of jawed vertebrates roughly 462 millions years ago.^[8]

KLHL is remarkably conserved across jawed vertebrates, sharing a greater than 77 percent sequence identity with many species of cartilaginous fishes, whose lineages diverged from that of humans roughly 462 million years ago. KLHL36 orthologs are summarized in Table 1.

Among the 20 selected orthologs, the amino acid identity at a given position was conserved in all orthologs at 35.4% of positions.

Figure 2 shows the divergence of KLHL36 in terms of corrected number of amino acid changes over millions of years. KLHL36 is remarkably highly conserved, similar to cytochrome c, with minimal changes in amino acid composition over time.^[9][10] Corrected amino acid changes are calculated as 100 * ln(1 - (M/100)), where M represents the observed number of amino acid changes per hundred amino acids in selected orthologs of KLHL36.

54 paralogs of KLHL36 have been identified, with its closest relatives being KLHL13 and KLHL26.^[11] The KLHL family of genes contains 42 genes from KLHL1 to KLHL42. Paralogs of KLHL36 are summarized in Table 2.

KLHL36 is expressed ubiquitously at moderate levels across all tissues, with moderate variation across tissues.^[5] The highest expression of the protein is observed in the testes, fat, and thyroid.^[12]

Whole body transcriptomic tissue analyses have revealed that, while KLHL36 is expressed at its highest absolute values in the testes, fat, and thyroid, it is expressed at its highest levels relative to other proteins in cells of the immune system, such as B cells, T cells, and monocytes.^[13]

There are 6 known transcript variants of KLHL36, the most common transcript variant being transcript variant 1. Transcript variant 1 encodes the longest and most common isoform, isoform 1.^[5] Transcript variant 1 is 7,559 nucleotides in length and contains 5 exons.^[5] Its 5' untranslated region is 158 nucleotides in length and its 3' untranslated region is 5,556 nucleotides in length.

Transcript Variants X1 and X2 contain the same coding sequence and encode an identical protein to transcript variant 1, with slightly differing 5' untranslated regions. Transcript Variant 2 encodes a slightly different protein product, missing part of the internal amino acid sequence but with the Kelch motifs at the C terminal intact. Transcript Variant X4 is significantly truncated, missing a large portion of the coding sequence and the 3' UTR and containing no Kelch motifs. Transcript Variants of KLHL36 and their corresponding protein isoforms are summarized in Table 3.^[5]

Many RNA binding proteins are predicted to bind to the 5' UTR KLHL36 Transcript Variant 1 mRNA, including YBX1, Vts1, RBM4, KHSRP, and RBMX.^[14] Several additional RNA binding proteins are predicted to bind to the 3' UTR, including YBX1, PUM2, EIF4B, MBNL2, ACO1, KHSRP, RBMY1A1, SFRS13A, YTHDC1, ELAVL1, FUS, ZRANB2, PABPC1, and SFRS9.^[14] RNA binding proteins that are predicted to bind the promoter of KLHL36 include ZNF667, ZNF530, ZIC4, SNF257, TFAP2A, TFAP2C, ZTBT24, KLF10, KLF12, SREBF1, KLF5, ZVED4, and NFYB.^[14]

Figure 4 consists of a conceptual translation of KLHL36 Transcript Variant 1 coding sequence, which demonstrates the layout of the mRNA and the corresponding amino acids, protein domains, and exon boundaries.^[5]

There are 3 protein isoforms of KLHL36, the most common and longest being isoform 1.^[5] Isoform 1 is a kelch-like protein that is 616 amino acids in length with a molecular mass of roughly 70 kilodaltons and an isoelectric point of 5.5.^[16] The charge of the protein in a neutral environment is -12, with no charge clusters identified.^[17]

The protein contains a BTB/POZ domain of 135 amino acids at its N-terminus, which is predicted to mediate homodimerization and be involved in cullin 3 binding.^[6][18] At its C-terminus, the protein contains 6 Kelch-motif domains which form a β-propeller that is predicted to be involved in substrate binding.^[19][20]

There are no unusually scarce or common amino acids present in KLHL36.^[17] The secondary structure of the protein is dominated by alpha helices towards the N-terminus in the region of the BTB/POZ Domain and the BACK domain, and by beta sheets towards the C-terminus in the β-propeller domain.^[21]

Immunofluorescence staining of KLHL36 in the A-549 cell line suggests that the protein localizes to the cytoplasm.^[22] DeepLoc-2.0 analysis of KLHL36 supports this finding, calculating its highest likelihood of subcellular localization to be the cytoplasm at 0.70, followed by the nucleus at 0.48 and the lysosome at 0.25.^[23] Analysis of KLHL36's primary structure reveals no signal peptide, no transmembrane domains, no mitochondrial targeting, no nuclear localization sequence, and no tracking motifs.^[17]

PhosPhoSite Plus and DTU Health Tech Bioinformatic Services predict many post-translational modifications of KLHL36, including phosphorylation, ubiquitylation, and acetylation.^[24][25] These modifications likely contribute to regulating the function of KLHL36 by inducing conformational changes and regulating KLHL36 abundance through ubiquitylation and subsequent proteasomal degradation.

Cullin 3 has been shown to directly bind multiple BTB domains through a conserved amino terminal domain.^[26] It is likely that cullin 3 binds the BTB domain present at the N-terminal of KLHL36 to mediate ubiquitylation and degradation of substrate proteins bound by KLHL36. It is not currently known what proteins KLHL36 might bind to facilitate their degradation.

STRING Functional Protein Association Database identifies 10 proteins with which KLHL36 is predicted to interact. These proteins are summarized in Table 4.^[27]

UniProt identifies 770 variants of the KLHL36 gene, with 94 marked as "pathogenic" or "likely pathogenic".^[11] A single nucleotide polymorphism downstram of KLHL36 (rs12716755) has been reported as a risk variant for early onset Alzheimer's disease.^[28] A genome-wide association study (GWAS) reported an aggregation of 134 single nucleotide polymorphisms implicated in psychological resilience and stress-related psychiatric outcomes in the region of KLHL36 on chromosome 16.^[29]

Additionally, transcriptomic analyses have revealed disease-associated changes in KLHL36 expression. KLHL36 mRNA levels have been shown to be lower in gastrointestinal tumors, and increased expression is correlated with positive prognoses in pancreatic cancer and cholangiocarcinoma.^[30] Additionally, elevated KLHL36 levels have been associated with reduced tumor proliferation in pancreatic adenocarcinoma models, suggesting a possible tumor suppressor role.^[31] Analyses of triple-negative breast cancer tumors have found that KLHL36 expression can be induced by TNFα stimulation, and that induction of expression can be reversed by the flavonoid apigenin.^[32]

KLHL36 has also been implicated in psychiatric contexts. In rodent models, antidepressant treatment and exercise have been negatively correlated with expression of KLHL36 in the brain.^[33] One sequencing analysis found that levels of KLHL36 were significantly increased in both individuals affected by seasonal affective disorder and suicidal ideation.^[34]