Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a moonlighting protein

The function and regulation of protein phosphatase Z in Candida albicans

A protein foszfatáz Z egy új típusú szerint/treonin specifikus protein foszfatáz, amely csak a gombákban mutatható ki. Az opportunista patogén Candida albicans-ban egy gén, a CaPPZ1 kódolja a CaPpz1 fehérjét, amely a jól ismert Saccharomyces cerevisiae ScPpz1 ortológjához hasonlóan szerepet játszik a sejtfal integritás és ozmotikus stabilitás kialakításában, a kation homeosztázisban és az oxidatív stresszválaszban. Munkánk során tanulmányoztuk a CaPpz1 további funkcióit és regulációs mechanizmusát. A CaPpz1 fehérje szerepét a fonalas gomba hifaképzésben klasszikus mikroszkópia és a folyamatos követést biztosító videomikroszkópia módszerével vizsgáltuk. Mind a két kísérlet azt mutatta, hogy cappz1 foszfatáz deléciós mutánsban csökken a hifanövekedés sebessége, valamint a gombasejtek felületi adhéziója. Tehát sikerült kimutatnunk a CaPpz1 eddig nem ismert morfológiai szerepét, ami összefüggésbe hozható a C. albicans fertőzőképességével. S. cerevisiae-ben korábban már leírták a ScHal3 fehérjét, mint a ScPpz1 foszfatáz inhibitorát. Nemrégiben azt is kimutatták, hogy a ScHal3 fehérje két paralógjával (ScVhs3 és ScCab3) kiegészülve a heterotrimer foszfopantotenoil-cisztein dekarboxiláz (PPCDC) enzim alegysége, amely esszenciális szerepet játszik a CoA bioszintézisben. Bioinformatikai kereséssel C. albicans-ban két ScHal3 ortológot találtunk, amelyeket S. cerevisiae homológjukkal mutatott szekvencia hasonlóság alapján CaHal3 és CaCab3 fehérjéknek neveztünk. Ezen két fehérje fiziológiás szerepét vizsgálva igazoltuk, hogy mindkét ScHal3 ortológ megőrizte esszenciális funkcióját, és részt vesz a PPCDC holoenzim felépítésében. Ezen kívül rekombináns fehérjékkel végzett in vitro kísérletekkel bizonyítottuk, hogy mindkét fehérje kötődik a CaPpz1 enzimhez és képes annak protein foszfatáz aktivitását gátolni. A megfelelő S. cerevisiae deléciós mutánsok komplementálása alapján megállapítottuk, hogy a CaCab3 a ScHal3 fehérjéhez hasonló feladatokat lát el a foszfatáz gátlásához köthető vizsgálati körülmények között, ezzel szemben a CaHal3 fehérje hatástalan. Tehát in vivo körülmények között csak a CaCab3 bizonyult hatékony CaPpz1 regulátor fehérjének. Eredményeink szerint a CaHal3 és CaCab3 fehérjék PPCDC aktivitásban betöltött esszenciális szerepe konzerválódott az evolúció során, de a CaCab3 másodlagos foszfatázgátló funkciója ettől független úton alakult ki.Protein phosphatase Z is a so called “novel” type Ser/Thr phosphatase that is restricted to the fungal kingdom. In the opportunistic pathogen Candida albicans there is only one gene (CaPPZ1) that codes for the CaPpz1 protein. The latter, like its better known Saccharomyces cerevisiae counterpart ScPpz1, is involved in the determination of cell wall integrity, preservation of osmotic stability, and cation homeostasis as well as in the mechanism of oxidative stress response. In the present study we investigated its additional roles and the ways of its regulation. The function of CaPpz1 in the hypha formation of the filamentous fungus was analyzed by classical microscopy and by long term videomicroscopy. Both of the methods revealed a significant delay in the rate of hyphal outgrowth in the cappz1 deletion mutant relative to the control strain. In addition the knockout mutant cell exhibited slower adhesion to solid surfaces. Thus we identified some novel morphological functions of CaPpz1 that may be related to the virulence of C. albicans. The ScHal3 protein was originally described in S. cerevisiae as a specific ScPpz1 inhibitor. Recently, it has been reported that ScHal3 together with its two paralogs (ScVhs3 and ScCab3) acts as an essential subunit of the heterotrimeric phosphopanthotenoyl cysteine decarboxylase (PPCDC) enzyme that is required for CoA biosynthesis. In a database search we found two Hal3 orthologs in C. albicans that we termed CaHal3 and CaCab3 based on their amino acid sequence similarities to the S. cerevisiae counterparts. In the course of molecular genetic functional analysis we found that both of the Candida proteins preserved their essential function as building blocks of the PPCDC enzyme. In addition we demonstrated by in vitro experiments with recombinant proteins that both CaHal3 and CaCab3 bind to the CaPpz1 enzyme and are able to inhibit its protein phosphatase activity. The complementation of S. cerevisiae deletion mutants revealed that overexpression of CaCab3 was able to replace ScHal3 in functional tests that are related to phosphatase inhibition. In contrast, the CaHal3 protein proved to be ineffective under the same conditions. Our in vivo experiments suggest that only CaCab3 can act as a physiological regulator of CaPpz1. According to our results the essential functions of the CaHal3 and CaCab3 proteins as PPCDC subunits have been well conserved but the moonlighting function of CaCab3 as a phosphatase inhibitor evolved separately during evolution.N

Egy új típusú gomba specifikus protein foszfatáz, a CaPPZ1 vizsgálata

Egy új típusú gomba specifikus protein foszfatáz, a CaPPZ1 vizsgálataBscbiológiag

Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a moonlighting protein

Analysis of Two Putative C<i>andida albicans</i> Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles - Fig 3

<p><b>Inhibition of CaPpz1 (A) and its catalytic domain CaPpz1-Cter (B) by CaHal3 and CaCab3 proteins.</b> Protein phosphatase activity was determined with [³²P]-labeled myosin light chain substrate in the absence (100% relative activity) or in the presence of increasing concentrations of CaHal3 (●) or CaCab3 (○). The mean ± SD of three determinations are shown in this representative figure. Similar results were obtained with at least two independent sets of recombinant protein preparations.</p