BTBD9 gene and TS
While researching autophagy, I stumbled upon BTBD9 gene again. I think I’ve posted earlier about association of mutations in this gene and TS.
Surprisingly, several studies describe association of mutations in this gene, iron status and tics severity.
This definitely invites for further research of the gene’s function, and how it’s related to iron and tics/TS/RLS.
Some clues
To begin, Freeman et al. (R9) identified the fly homologue of BTBD9 and created two null mutations in the gene. When they examined sleep in these mutants, they found that both mutant alleles caused severely fragmented nighttime sleep and an increase in the amount of waking after sleep onset, as is seen in human patients with restless legs syndrome. (R8)
Interestingly, they found that flies mutant for dBTBD9 show dramatically reduced brain dopamine levels and that the abnormal sleep phenotype was completely rescued by administering mutant flies the dopamine D2 receptor agonist Pramipexole, which is used clinically in humans to treat restless legs syndrome.
Importantly, Freeman et al. (R9) used RNA interference (RNAi) to show that they could recapitulate restless legs syndrome symptoms by knocking-down dBTBD9 expression in a subset of dopaminergic neurons. Finally, the authors over-expressed BTBD9 in HEK cells to confirm that the gene does indeed play a role in iron homeostasis, acting via regulation of iron regulatory protein-2 (IRP2). (R8)
Adaptor for Cul-3
These data support BTBD9’s function as a Cul-3 adaptor and suggest that stringent stoichiometric ratios between BTBD9 and Cul-3 might be maintained under physiological conditions. (R9)
Cul-3 and CRL3
Cullin-RING Ligases (CRLs), one major type of E3 ubiquitin ligases, regulate about 20% cellular proteins degradation by ubiquitin-proteasome system. … In particular, Cullin3-RING ligases (CRL3s), without receptor proteins, utilize substrates specific Bric-a-Brac/Tramtrack/Broad (BTB) domain proteins to recognize their corresponding substrates and to regulate various biological processes.
Dysregulation of CRL3s leads to tumorigenesis and tumor development.
Furthermore, CRL3s play pivotal roles in the innate immune response to infection. For example, knockdown of the component of Cullin3-Keap1 complex activates NF-κB and drives the expression of pro-inflammatory cytokines. (R12)
TNFAIP1
More importantly, Cul3-ROC1 (CRL3), a subfamily of CRLs, was identified to specifically interact with TNFAIP1 and promote its polyubiquitination and degradation.
Mechanistically, BTBD9, a specific adaptor component of CRL3 complex, was further defined to bind and promote the ubiquitination and degradation of TNFAIP1 in cells.
As such, downregulation of BTBD9 promoted lung cancer cell migration by upregulating the expression of TNFAIP1, whereas TNFAIP1 deletion abrogated this effect. Finally, bioinformatics and clinical sample analyses revealed that BTBD9 was downregulated while TNFAIP1 was overexpressed in human lung cancer, which was associated with poor overall survival of patients.
Taken together, these findings reveal a previously unrecognized mechanism by which the CRL3BTBD9 ubiquitin ligase controls TNFAIP1 degradation to regulate cancer cell migration. (R11)
BTBD9 Modulates IRP2
Mechanistically, the iron-responsive element-binding protein, IREB2 or IRP2 inhibits ferritin translation in response to changes in cellular iron, such that iron abundance leads to IRP2 degradation and a resultant increase in ferritin translation.
We therefore tested whether BTBD9 controls levels of IRP2. Western blots in Figure 4C show that both under basal and iron-chelation conditions, BTBD9 reduces IRP2 levels. This observation is consistent with the effect of BTBD9 on ferritin and profers a pathway by which BTBD9 modulates ferritin levels through regulation of IRP2 (R9)
BTBD9 acts together with Cullin-3 to regulate IRP2 levels in the cell. This in turn controls ferritin expression and iron metabolism. Under normal conditions, BTBD9 inhibits IRP2 and promotes ferritin expression.
BTBD9 also maintains dopamine biosynthesis either directly, or indirectly through iron, by currently unknown mechanisms. Together, this ensures normal motor drive and sleep consolidation. However, in RLS, loss of BTBD9 leads to increased IRP2, and reduced ferritin, thereby altering iron metabolism. It also negatively impacts dopamine synthesis leading to inappropriate motor activity and sleep fragmentation. (R9)
Dopamine neurons
In the mammalian brain, BTBD9 is strongly expressed in dopaminergic neurons of the substantia nigra and A11 neurons (R9)
BTBD9 and its variants
rs9357271
Accumulating evidence revealed that some variants related to genes not directly implicated in iron homeostasis might also show an association with iron status parameters. For example, rs9357271 SNP in the BTB domain-containing protein-9 gene (BTBD9) was associated with decreased serum ferritin in patients and their relatives; despite the emergence of conflicting results 5,6.
The protein coded by this gene is ubiquitously expressed both in the central nervous system and in the periphery, and both during development and adulthood. 16,17
Although the BTBD9 variants may directly influence iron metabolism and may be associated with proteins that participate in iron regulation signaling pathways (Figure 1A), yet the mechanism is not known (R1)
Results: The study provided 3 single-nucleotide polymorphisms within BTBD9 associated with TS (chi(2) = 8.02 [P = .005] for rs9357271), with the risk alleles for restless legs syndrome and periodic limb movements during sleep overrepresented in the TS cohort.
We stratified our group of patients with TS according to presence or absence of obsessive-compulsive disorder and/or attention-deficit disorder and found that variants in BTBD9 were strongly associated with TS without obsessive-compulsive disorder (chi(2) = 12.95 [P < .001] for rs9357271).
Furthermore, allele frequency of rs9357271 inversely correlated with severity of obsessive-compulsive disorder as measured by the Yale-Brown Obsessive Compulsive Scale score. (R7)
rs9296249 ❗️
This intronic variant is quite common - about 30% of population has it / GnomAD link / 39 papers mentioning this variant on LitVar.
Results: Homozygosity for the T-allele of BTBD9 rs9296249 was associated with lower serum ferritin. The odds ratio for low serum ferritin was 1.35 (95% confidence interval, 1.02-1.77; p = 0.03) when comparing donors with the TT genotype with donors with the CT genotype.
Conclusion: A frequent polymorphism in BTBD9 was significantly associated with serum ferritin. This polymorphism has previously been associated with RLS, but not low iron stores in blood donors. (R6)
There was a statistically significant association between the variant rs9296249 of the BTBD9 gene and the TS phenotype. However, no statistically significant associations were found between the other four variants (rs4714156, rs9357271, rs518147, and rs3813929) and the TS phenotype (P>0.05). (R2)
Links
MEIS1
Furthermore, this gene shows a lower expression of mRNA and protein in blood and thalamus of individuals with the MEIS1 RLS risk haplotype.
Simulating this reduced MEIS1 expression in mouse models resulted in circadian hyperactivity, a phenotype compatible with RLS. While MEIS1 shows a strong association with RLS, the protein’s function that is directly linked to an RLS biological pathway remains to be discovered.
The links to iron and the enhancer activity of the HCNRs of MEIS1 suggest promising links to RLS pathways (R6)
IRP2
The expression levels of TfR1, DMT1, ferritin and FPN1 are post-transcriptionally regulated by binding of IRP1 or IRP2 to IREs in the transcripts that encode these iron metabolism proteins. … IRP2 has an additional cysteine-rich 73 amino acid domain with unknown function (Bourdon et al., 2003; Wang et al, 2004). Both IRP1 and IRP2 are expressed ubiquitously.
The expression of Irp1 is dominant in kidney, liver and brown fat, whereas the expression of Irp2 is dominant in the central nervous system (Meyron-Holtz et al., 2004). The activities of these iron regulatory proteins are also regulated by iron, but through different mechanisms (Rouault, 2006). … In contrast, at high iron concentrations, IRP2 undergoes proteasomal degradation by an E3 ubiquitin ligase complex that contains an F-box protein, FBXL5, which is activated when iron and oxygen bind to a hemerythrin domain in FBXL5 (Salahudeen et al., 2009; Vashisht et al., 2009; reviewed in Rouault, 2009).
Therefore, both the activity and protein level of IRP2 decrease when the cells are iron-replete (reviewed in Rouault, 2006). … In contrast, if the IRE is located in the 3′UTR of the target mRNAs, binding of IRPs can increase protein expression by stabilizing the mRNA.
TfR1 and DMT1 have the IREs in the 3′UTRs of their transcripts. Thus, when the cells are iron depleted, expression levels of the mRNAs of the iron import proteins TfR1 and DMT1 increase, whereas translation of the iron storage protein ferritin, the iron export protein FPN1, the first enzyme for heme synthesis eALAS, the protein involved in energy production ACO2, and the hypoxia and erythropoiesis sensor and effector protein HIF2α decreases. … Loss of Irp2 caused decreased TfR1 and increased ferritin expression in motor neurons. The resulting functional iron deficiency led to decreased activities of iron-sulfur containing Complex I and Complex II (but not Complex IV which does not contain an Fe-S cluster). As a result, Irp2−/− mice had decreased mitochondrial function and swollen mitochondria (Jeong et al., 2011). (R10)