The N-terminal Of NBPF15 Causes Multiple Types Of Aggregates And Mediates Phase Tran-Sition&The Studies Of DDX43 Enzymatic Properties

Neuroblastoma breakpoint family(NBPF)consists of 24 members that play an im-portant role in neuroblastoma and other cancers.NBPF is an evolutionarily recent gene fam-ily that encodes several repeats of Olduvai domain and an N-terminal region.The Olduvai domain has experienced rapid and large-scale amplification in human lineage,with the highest number of 302 copies in humans,while only one copy in mice,and does not exist in non-mammals.It was reported that the copy number variants(CNVs)of Olduvai domain are related to brain size and have implicated in normal and pathological variation in human brain gray matter volume and neuron number.The amplification of Olduvai domain is related to autism and macrocephaly,while the deficiency of the Olduvai domain is associated with schizophrenia and microcephaly.Olduvai domains are expressed in many tissues of the hu-man,especially in brain regions associated with higher cognitive function and show neu-ron-specific expression.The function and biochemical properties of both Olduvai domain and the N-terminal region remain enigmatic.Human NBPF15 encodes 670 amino acids protein consisting of six clades of Olduvai domains and an N-terminal longer than 100 amino acids in length.In this study,we synthe-sized and expressed full-length NBPF 15 and series of NBPF 15 truncations,and found that the expression of both full-length NBPF 15 and truncations without Olduvai were unsuc-cessful,while the expression of proteins that contained single Olduvai domain were fairly better.We finally have purified ten truncations and analyzed these truncations using dynamic light scattering(DLS),superdex200(S200),small-angle X-ray scattering(SAXS),far-UV circular dichroism(CD)spectroscopy,transmission electron microscope(TEM)and crystal-lography.We found that proteins containing both the N-terminal region and Olduvai domain are heterogeneous with multiple types of aggregates.The longer N-terminal a protein pos-sessed,the more types of aggregates it present.The characteristic of N-terminal proteins be-ing with multiple types of aggregates was gradually unapparent with longer N-terminal had truncated and was finally homogeneous until the entire N-terminal was removed.Proteins that contain only the Olduvai domain are homogeneous extended monomers proved by SAXS.The morphological properties of these N-terminal truncations were examined by TEM and distinctly vary,long N-terminus causes the fibrous and severely aggregated protein.In addition to being with multiple types of aggregates,the N-terminal-containing NBPF15 truncations were found to undergo a phase transition.When protein concentration reached a threshold,generally at 40 mg/ml,they gradually and spontaneously transited from a soluble state to a hydrogel state.We proved that the presence of multiple types of aggregates is be-cause of the N-terminal sequence-intrinsic preference for conformational heterogeneity.NBPF15 is present in numerous intrinsically disordered regions(IDRs),and proteomics and genetic studies suggest that the presence of IDRs in proteins drives phase transitions.The N-terminal of NBPF15 belongs to polyampholytes based on sequence-structural ensemble relationships,which have approximately equal numbers of positive and negative charges and are randomly distributed.The results of CD experiment show that the NBPF15 N-terminal was like a mosaic with ordered helical-rich core and disordered wings.The speed of the phase transitions in the truncations consisted with the content of α-helix.All NBPF proteins have abundant and conserved N-terminal sequence,the biochemical characteristics of the NBPF 15 N-terminus are likely to be similar to all NBPF proteins,which could be associated with Olduvai-related cognitive dysfunction diseases.The second research is about the enzymatic properties of RNA helicase DDX43.DDX43 is a cancer/testis antigen(CTA)gene and is considered as a new cancer biomarker.DDX43 can promote the expression of proto-oncogenes.In melanoma cell,DDX43 contrib-ute to the expression and proliferation of NRAS by opening the secondary structure of NRAS mRNA,while its specific function in cancer development is largely unexplored.DDX43 is the member of RNA helicase in the DEAD-box family,it has a conserved helicase core and a single K-homology(KH)domain in its N-terminus,and a linker of up to 100 amino acids between the helicase core and KH domain.We have expressed and obtained human DDX43 protein in E.coli system and demon-strated that DDX43 is a homogeneous monomer.To understand the role and explore the sub-strates’ 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,Kd<10 nM.For nucleotide preferences,DDX43 much prefers guanosine than the other three nucle-otides,in the order of G>T>C>A,the affinity for G is about ten times that of T.Among the two main domains of DDX43,KH domain exerts the major substrate-binding ability,and its affinity for the substrate is twice that of the helicase core.The linker is essential in main-taining the high affinity of DDX43 to the substrate.The deletion(approximately 30 amino acids)or increases(3C protease insertion)of the linker would not affect the activities of DDX43,while the interruption can result in a sharply decreased binding affinity to substrates.We suspected that DDX43 preferred ssDNA/RNA and required two linked function domains to bind to the substrates probably because ssDNA/RNA was much more flexible and man-ageable and,the linked domains could contribute to the stability of the proper conformation of DDX43 after binding to single-strand DNA/RNA.We also found that the unwinding abil-ity of DDX43 in vitro was neither efficient nor sustainable.In this experiment,we have systematically and quantitatively reported the affinity of full-length DDX43 with the substrate,specifically demonstrated the role of each domain of DDX43 in substrate affinity,and proved that the key to maintaining high affinity of DDX43 with the substrate is the physical connection between the two domains,which adds the foundation of in vitro biochemical research for the clinical research of this new tumor mark-er.