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• Due to its cancer-specific expression, DDX43 (DEAD-box helicase 43) is considered a potential target molecule for cancer therapy. • Biochemical assays to determine the enzymatic activities and properties of DDX43 protein, including its KH domain, link domain, and ATPase/helicase core domain. • Identify the specific nucleic acids sequence bound by its KH domain using SELEX, ChIP-seq, CLIP-seq, and NMR. • Structural characterization of the KH domain and full-length DDX43 protein aided by Phyre 2, NMR, and AphaFold. • Emerging roles of DDX43 in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. • Structural and functional studies of DDX43 will facilitate anti-cancer drug development by targeting DDX43. DDX43 (DEAD-box helicase 43), also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a K homology (KH) domain in its N terminus, a helicase core domain in its C terminus, and a flexible linker domain in between. DDX43 expression is low or undetectable in normal tissue, but is overexpressed in many tumors; therefore, it is considered a potential target molecule for cancer therapy. We, along with other groups, have shown that DDX43 is an ATP-dependent RNA and DNA helicase, and the KH domain is required for its ATPase and unwinding activity. Electrophoretic mobility shift assay (EMSA), SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, crosslinking immunoprecipitation (CLIP)-seq, and nuclear magnetic resonance (NMR) showed that the KH domain prefers to bind pyrimidine-rich ssDNA and ssRNA, such as TTGT in the promoter regions of genes. Moreover, the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase. No animal model has been generated for DDX43; cellular studies have revealed that DDX43 has roles in piRNA amplification, tumorigenesis, RAS signaling, and innate immunity. Structural and functional studies of DDX43 will not only advance our understanding of DEAD-box helicases and KH domains, but also shed light on the application of DDX43 as therapeutics, where its key binding sites can be targeted by small molecules and natural products as an alternative approach in treating DDX43 overexpressed cancers. [ABSTRACT FROM AUTHOR]

• The cDNA sequence of DDX43 was identified from tilapia (Oreochromis niloticus). • Expressions of DDX43 mRNAs were altered after Streptococcus agalactiae , LPS, and poly I:C stimulation. • The overexpression of OnDDX43 could significantly upregulate IFN-β activity in 293 T cells. • OnDDX43 interacted with both IPS-1 and TRIF. DDX43 is one of the members of the DExD/H-box protein family, and emerging data suggest that it may play an important role in antiviral immunity across mammals. However, little is known about DDX43 in the fish immune response. In this study, we isolated the cDNA sequence of ddx43 in Nile tilapia (Oreochromis niloticus). The ddx43 gene was 2338 bp in length, contained an open reading frame (ORF) of 2064 bp and encoded a polypeptide of 687 amino acids. The predicted protein of On DDX43 has three conserved domains, including the RNA binding domain KH, DEAD-like helicase superfamily DEXDc and C-terminal HELICc domain. In healthy Nile tilapia, the Onddx43 transcript was broadly expressed in all examined tissues, with the highest expression levels in the muscle and brain and the lowest in the liver. After challenge with Streptococcus agalactiae , lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (Poly I:C), the expression level of Onddx43 mRNA was upregulated or downregulated in all of the tissues tested. Overexpression of On DDX43 in 293 T cells showed that it has a positive regulatory effect on IFN-β. The subcellular localization showed that On DDX43 was expressed in the cytoplasm. We performed further pull-down assays and found that On DDX43 interacted with both interferon-β promoter stimulator1 (IPS-1) and TIR domain-containing adaptor inducing interferon-β (TRIF). [ABSTRACT FROM AUTHOR]

DDX43 is a cancer/testis antigen and is thought to stimulate oncogenic pathways in cell proliferation, while its specific function in cancer development is largely unexplored. DDX43 is the member of RNA helicase in DEAD-box family, consists of conserved helicase core and a single K-homology (KH) domain in its N-terminus. In this paper, we expressed and purified human DDX43 protein in E. coli and demonstrated that this protein is a homogeneous monomer. To understand the role and explore the substrates preference of DDX43 in vitro , we systematically studied its binding properties. We found that DDX43 prefers single-strand DNA or RNA with length longer than 12 nt and much prefers guanosine than the other three nucleotides. Achievement of the full binding affinity of protein to substrate needs the existence of all domains, and they must be connected. The absence of either of them or the disjunction can result in a decreased binding affinity to substrates, approximately reduced 10-fold. We also found that the unwinding ability of DDX43 in vitro was neither efficient nor sustainable. • DDX43 prefers single strand G-rich substrate. • Both KH domain and helicase core are required in substrates binding. • KH domain and helicase core must be linked for DDX43 high affinity to substrates. [ABSTRACT FROM AUTHOR]

Abstract: DEAD box polypeptide 43 (DDX43), a cancer/testis antigen (CTA), has been found to be overexpressed in various solid tumors and some hematologic malignancies. In the present work hypomethylation of the DDX43 gene was detected in 15% (32/214) of primary acute myeloid leukemia (AML) using real-time quantitative methylation-specific PCR (RQ-MSP). The level of DDX43 expression was correlated with DDX43 hypomethylation (R =0.277, P =0.014). Moreover, bisulfite sequencing confirmed the significant correlation between the methylation density and the level of DDX43 hypomethylation. Additionally, restoration of DDX43 expression in the K562 cell line by 5-aza-2′-deoxycytidine treatment confirmed a direct contribution of methylation in regulating the DDX43 gene. DDX43 hypomethylation was observed more frequently in favorable group (21.4%) and intermediate group (15.8%) than in poor group (0%) (P =0.009). AML patients with DDX43 hypomethylation had a better overall survival (median not obtained) than those with DDX43 methylation (median 8 months, 95% confidence interval 5.6–10.4 months) (P =0.014). In summary, the DDX43 gene is activated by promoter hypomethylation and DDX43 hypomethylation may be a favorable prognostic factor in AML. [Copyright &y& Elsevier]

The article presents information on effect of abnormal hypomethylation of DDX43, a cancer/testing gene, enhances myelodysplastic syndrome (MDS). It mentions that patients with DDX43 methylation and patients without DDX43 methylation differed only in their clinical characteristic of hemoglobin level. A table depicting DDX43 hypomethylation in MDS patient and two graphs depicting survival of MDS patient are presented.