Identification Of Structure And Inhibitors Of Mammalian Cytosolic Carboxypeptidases

Cytosolic carboxypeptidases（CCP1-6）define a subfamily of M14metallocarboxypeptidases and are all involved in a kind of protein posttranslational modification termed polyglutamylation.Polyglutamylation is a dynamic and reversible reaction.In the process of this modification,its initiation and prolongation are catalyzed by the 9 members of the Tubulin Tyrosine Ligase Like（TTLL1,2,4,5,6,7,9,11,13）family.In contrast,the six cytosolic carboxypeptidases catalyze the shortening and digestion of polyglutamylation chains.Polyglutamylation has been initially discovered onα-andβ-tubulin,the building blocks of microtubules,and later also found on other proteins.The disrupted polyglutamylation causes a broad range of disorders,including neurodegeneration,retinal inflammation,retina degeneration,and ciliopathy.Therefore,controlling the length of polyglutamate chains on protein mediated by polyglutamylases and deglutamylating enzymes plays an important role in neuronal survival and cilia related function.Polyglutamylation-modifying enzyme inhibitors may be used for the development of treatment for the corresponding disease caused by imbalanced polyglutamylation.To our knowledge,no specific inhibitors for the cytosolic carboxypeptidase family have been reported so far.Here,this thesis concerns to identify efficient inhibitors of cytosolic carboxypeptidases.However,the catalytic mechanisms of CCP family and their unique substrates recognition mechanisms are incompletely understood.To this end,we also tried to get the crystal structure of CCP6 that will ultimately provide structural basis for possible drug design.In the first part,this study aimed to solving the structure of mammalian CCP family.The recombinant mouse-origined CCP6 gene was expressed in E.coli.However,expression of CCP6 encountered unexpected problems,such insolubility.The expression condition such as induced temperature and different host stains were carefully optimized.Finally,I found that CCP6 containing an N-terminal HMT-tag（His-tag and MBP（maltose binding protein）-tag）could be expressed in a soluble form in the Arctic Express（DE3）expression system.Arctic Express（DE3）strains contain molecular chaperones Gro EL and Gro ES that help the target protein fold properly.However,I found that Gro EL and CCP6 tightly bound to each other and failed to completely separate the molecular chaperone from the target protein even multiple purification techniques were tried.Subsequently,we attempted to analyze the structure of this purified CCP6-chaperone complex by crystallization.Through the screening of high-throughput crystal growth conditions,we obtained the CCP6crystals by the hanging drop method,but did not obtain the diffraction data by the X-ray diffraction method.At the same time,we detected the purified CCP6-chaperone complex activity.I found that the tubulin deglutamylation activity of CCP6 in complex with the molecular chaperone was very weak or even undetectable.Next,according to the working mechanism of molecular chaperone,we tried to separate the molecular chaperone from the target protein by adding ATP and Mg Cl₂ before purification of the target protein by affinity chromatography and gel filtration technology.The full length of CCP6 obtained by this purification method exhibited tubulin deglutamylation activity.The second part of this study was to identify efficient inhibitors of cytosolic carboxypeptidases.Compared with the traditional M14 metal carboxypeptidase inhibitors and other structural analogs of CCP substrates,we found that 2-PMPA,a potent inhibitor of the M28 family member glutamate carboxypeptidase II（GCPII）,can effectively inhibit the activity of CCP family enzymes.Furthermore,it was found that 2-PMPA could not only inhibit the catalytic activity of the CCP family on protein substrates,such as tubulin,but also inhibit its hydrolysis for synthetic peptides.The enzyme kinetics experiments determined that the half-inhibition concentration(IC₅₀)of 2-PMPA to inhibit Nna1 catalyzed synthesis of peptides was 0.99μM,and the mixed inhibition of K_i and K_i’were 0.11μM and 0.24μM,respectively.Homology modeling revealed that the R-form of 2-PMPA is more favorable to bind Nna1 than its S-form,which is stabilized by intensive interactions of its glutarate moiety with the putative S’-pocket in addition to the coordination of its phosphonate group with the zinc ion in the enzyme.Interestingly,we further found that the substrate of GCPII,neuropeptide N-acetylaspartylglutamate（NAAG）,could be metabolized by CCPs.The purified recombinant mouse Nna1 can metabolizing NAAG with a K_m value of0.43 m M and a k_cat value of 1.9 s^-1.However,whether NAAG is a natural substrate of CCPs still needs to be determined.