The Role Of Centrin In The Recognition Process Of Nucleotide Excision Repair

Ca²⁺serves as one of the most ubiquitous and critical second messengers regulating a plethora of downstream cellular signaling events.As an intracellular signalling ion,Ca²⁺is involved in an array of cellular functions from fertilization,contraction,cell differentiation and proliferation,to apoptosis and,in the case of disregulation,cancer.Free Ca²⁺within cells is tightly regulated in the nanomolar range?50¹00 nm?and large of Ca²⁺in any living cell would be catastrophic.It is necessary for cell to properly regulation of Ca²⁺flux to maintain Ca²⁺homeostasis.Therefore,cells have adapted multiple mechanisms to accomplish this,including the evolutionary emergence of numerous Ca²⁺pumps and channels associated with plasma,mitochondrial,and intracellular membranes as well as a plethora of Ca²⁺-binding proteins.Ca²⁺binding proteins?CaBPs?are central players in all aspects of Ca²⁺signaling events,with diverse roles ranging from controlling the opening and closing of Ca²⁺channels to modulating the intensity and duration of Ca²⁺signals to transducing these signals into biochemical and biomechanical responses.The importance of CaBPs is evident from their direct association with diseases ranging from Alzheimer's disease to cardiac arrhythmia syndromes,complications of diabetes,chronic inflammatory disorders,and cancer.CaBPs is distributed throughout all parts of the cell.At present,over1000 species of CaBPs have been identified from their unique sequence signatures.Among CaBPs,Calmodulin and centrin become the focus of attention of Ca²⁺sensor currently.They contain the common structure basis,EF-hand motif.The functional diversity and specificity of this small size proteins make them a perfect model for the study of protein structure and functional relationship and Ca²⁺binding.Centrin is acidic centrosomal or contractile fiber-associated protein,which belongs to the subfamily within the superfamily of Ca²⁺binding proteins.It has been found that centrin is ubiquitous in all eukaryote species,ranging from unicellular organisms to vertebrates cells,since it was first identifed in unicellular green algae,such as Tatraselmis striata and Chlamydomonas reinhardtii.Similarly with calmodulin being a calcium signal protein,centrin is composed of two domains,N-and C-terminal domains,linked by a flexible central?-helix.The crystal structure of centrin reveals a dumbbell-shaped protein with two globular domains.In general,it contains four helix-loop-helix topology structures,the so-called EF-hand sub-domains.The EF-hand structural motif was devised recognised in 1973and is the most common calcium binding motif found in proteins.Ca²⁺ions are coordinated by ligands within the loop region?usually about 12 amino acids?.Upon Ca²⁺binding,EF-hand protein undergoes a large conformational change from“closed”to“open”and exposes more hydrophobic surface.Besides,the presence of Ca²⁺can lead to more spherical shaped structure from elongated shape of Ca²⁺-free centrins.Despite sharing similarity with calmodulins,centrin has mass different properties,such as self-assembly based on a disordered region of first 20 amino acids.The region of centrin confers its functional diversity.Research has shown that centrins can form multimers in the Ca²⁺loaded state above a protein concentration of 10?M.As a major component of contraction fibers,centrin plays a fundamental role in various cellular processes.Extensive researches have proved that centrin is tightly linked to the regulation of the microtubule organizing center?MTOC?duplication and separation during the cell cycle.And only about10%of the centrins are concentrated on the MTOC.Centrin distributed throughout the cell is strongly associated with the light transduction cascade in photoreceptor cells,the nuclear mRNA export in yeast and the ciliary voltage-gated Ca²⁺channel in Paramecium in Tetrahymena.Moreover,centrin may play a functional role in pre-mRNA splicing and regulation of neuron-specific K⁺channel.Recently,related investigations have reported the participation of centrins in a recognition process in nucleotide excision repair?NER?.Xeroderma pigmentosum group C?XPC?protein,the human homologue of Rad23B?HR23B?protein and centrin ternary complex behaves as a damage detector that initiated the global genome NER.However,it is still unclear about the molecular mechanisms implicated in the process,especially the process of centrin complicated.Understanding of centrin functions requires a detailed description of the molecular and structural properties of the isolated proteins and of their interactions with relevant cellular targets.It may provide new insights into the multifunctionality of centrin.Based on the previous research in our laboratory,we conducted the following research with Euplotes octocarinatus centrin?EoCen?as the research object.To explore the possible role of centrin,we initiated a physicochemical study of the N-terminal domain of Euplotes octocarinatus centrin?N-EoCen?with DNA in the absence or presence of Ca²⁺in 10 mM Hepes at pH 7.4.N-EoCen shows unusual affinity for double-stranded DNA.The interaction results in the protein exposing more hydrophobic surface along with a certain perturbation taking place in the double helix structure.The coordination of apoN-EoCen with Ca²⁺can promote the binding to DNA.Then,the affinity of DNA to N-EoCen,C-EoCen and EoCen were compared.It was found that N-EoCen has a relative high affinity to DNA.To explore the possible role of DNA in the process of centrin self-assembly,the aggregation properties of N-EoCen in the presence of DNA with or without metal ions are investigated.It is verified that double strand DNA?dsDNA?is capable of binding N-EoCen,changing conformation of protein and modulating centrin aggregation,as demonstrated by extensive biophysical assays.Interestingly,the open conformation of protein induced by metal ions may be favour of the interaction of protein with dsDNA.Nevertheless,the randomly coiled single strand DNA?ssDNA?is completely inefficient to the aggregation regulation.Furthermore,results reveal that hydrophobic site could play important role in the process.This finding may link to the potent roles of centrin in the NER process.Interestingly,N-EoCen exhibits endonuclease-like activities via a hydrolysis pathway,which induces DNA strand breaks,such as supercoiled DNA into nicked circular and linear DNA.Importantly,mutation of serine?Ser?and threonine?Thr?to alanine?Ala?demonstrates that Ser and Thr,in particular Ser located at 22?Ser22?,may be the key residues responsible for DNA cleavage activity.The coordination of apoN-EoCen with Ca?II?can raise the cleavage activities.In contrast,the binding to Ca?II?of mutant proteins may trigger a conformational change so that the cleavage activity decreases dramatically,as confirmed by protein hydrolysis activity experiments.This is first report of the endonuclease-like activity of centrin,which provides valuable information for understanding a novel property of centrin,as well as knowledge of the functional diversity of centrin.The xeroderma pigmentosum group C protein?XPC?,centrin2 and human homologue of Rad23B?HR23B?heterotrimer are the primary initiators of global genome nucleotide excision repair?NER?.To understand the possible molecular and structural properties of the multiprotein process,the interactions of Euplotes octocarinatus centrin?EoCen?,melittin,and DNA are described.EoCen shares a sequence identity of 66%with centrin2.Melittin possessing inverse direction hydrophobic triads?LLW?which are responsible for centrin binding is applied as a natural peptide to mimic centrin target peptide.As a result,it is proved that the integrated protein shows an endonuclease-like activity to DNA.Melittin is capable of interaction with both EoCen and DNA.More importantly,it is found that melittin displays an inhibitory effect on the endonuclease-like activity of centrin when it co-exists with EoCen and DNA in solution.Meanwhile,the DNA-melittin-EoCen ternary complex forms in the process.Quantitative analyses demonstrated by extensive biophysical assays reveal that binding of the peptide to DNA or centrin modulates the binding properties of it to another component.Furthermore,a possible positioning model of DNA and EoCen on melittin is proposed.This finding may constitute a model for that existing between centrin and its target peptide in NER process.