Comprehensive Proteomic Analysis of Nitrogen-Starved

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Comprehensive Proteomic Analysis of Nitrogen Starved Mycobacterium smegmatis #pup Reveals the Impact of Pupylation on Nitrogen Stress Response Giuseppina Fascellaro, Agnese Petrera, Zon Weng Lai, Paolo Nanni, Jonas Grossmann, Sibylle Burger, Martin L. Biniossek, Alejandro Gomez-Auli, Oliver Schilling, and Frank Imkamp J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00378 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 10, 2016

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Journal of Proteome Research

Comprehensive

Proteomic

Analysis

of

NitrogenStarved

Mycobacterium

smegmatisΔpupReveals theImpact of Pupylation onNitrogen Stress Response Giuseppina Fascellaro1, Agnese Petrera2, Zon Weng Lai2, Paolo Nanni3, Jonas Grossmann3, Sibylle Burger1, Martin L. Biniossek4, Alejandro Gomez-Auli2, Oliver Schilling2,4, 5, Frank Imkamp1*

1

Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

2

Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg,

Germany 3

Functional Genomic Center, University of Zurich/ ETH, Zurich,Switzerland

4

BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany

5

German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ),

Heidelberg, Germany

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ABSTRACT Pupylation isa bacterial ubiquitin-like protein modification pathway, whichresults in the attachment of the small protein Pup to specific lysine residues of cellular targets. Pup was shown to serve as a degradation signal, directing proteins towards the bacterial proteasome for turn-over. Recently, it was hypothesized that pupylation and proteasomal protein degradation support the survival of Mycobacterium smegmatis (Msm) duringnitrogen starvation by supplying recycledamino acids. In the present study we generated a Pupdeletion strain to investigate the influence of pupylation onMsm proteomein the absence of nitrogensources. Quantitative proteomic analyses revealed a relativelylow impact of Pup on MsmΔpupproteome immediately after exposure to growth medium lacking nitrogen. Less than 5.4% of the proteins displayed alteredcellular levels when compared to Msmwild type. In contrast, post24 h of nitrogen starvation 501 proteins (41% of the total quantified proteome) ofMsmpupdeletion strain showed significant changes in abundance. Noteworthy, important players involved in nitrogen assimilation were significantly affectedin MsmΔpup. Furthermore, we quantified pupylated proteins of nitrogen starved Msmto gain more detailed insights in the role of pupylation in surviving and overcoming the lack of nitrogen.

Keywords:

pupylation,

Mycobacterium

smegmatis,

nitrogen

proteomics


starvation,

quantitative

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INTRODUCTION Bacteria, when confronted with continuously changing environmental conditions, require fast and efficient adaptation in order to survive. Adaptive processes include adjustments at the transcriptional level as well as at the level of the proteome1.Proteomic changes are accomplished by a multitude of proteolytic complexes such asClp, Lon or FtsH2,3 that ensure the coordinated removal of cellular proteins2,3.Whilstall prokaryotes harbour a subset of these proteases, members of the phyla Actinobacteria and Nitrospira additionallycarrya proteasome. In order to direct substrates towards the proteasome for degradation, these organisms make use of a posttranslational protein modification pathway termed pupylation, which was initially discovered and described in the human pathogen Mycobacterium tuberculosis(Mtb) and its non-pathogenic relative M. smegmatis (Msm)4,5.Although pupylation is reminiscent of eukaryotic ubiquitination,it features distinct enzymes and mechanisms. In a two-step process,the prokaryotic ubiquitin-like protein Pup is covalently attached to a specific lysine residue of a target protein via an isopeptide bond4-6.Initially, Pup is rendered competent for conjugation by the action of Dop (Deamidase of Pup), which catalyses the deamidation of Pup C-terminal glutamine to glutamate. The formation of the isopeptide bond between this glutamate residue and the ε-amino group of a substrate lysine residue is then mediated by the Pup-ligase PafA6-10.Pup is eventually recognized by the proteasomal chaperone (termed Mpa in mycobacteria, or ARC in other actinobacterial species), which mediates the ATP-dependent unfolding of the bound protein substrate and the subsequent translocation of the unfolded polypeptide chain to the proteolytic core of the proteasome11-13. In eukaryotes, ubiquitination is a reversible process since deubiquitinases counterregulateubiquitination by acting on the isopeptide bond between the substrate protein and the covalently attached ubiquitin14. In an analogous manner, pupylation is not a unidirectional process. Interestingly, Dop not only catalyses the deamidation of Pup, thereby initiating the



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pupylation process, but acts also as a de-pupylase,subsequently mediating the removal of Pup from pupylated proteins8,15. Mtb, the causative agent of tuberculosis,employs a series of resistance mechanisms to overcome the hostdefencesand to prevent its own eradication. The pathogen is transmitted by cough-induced aerosols.Once inhaled,the bacteriumreaches the pulmonary alveoli where it is phagocytized by alveolar macrophages. Inside the macrophage,Mtb faces a multitude of challenges, like nutrient limitation, drastic shifts in oxygen availability as well as reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI), which are generated by the hostimmune response16,17.Strikingly, pupylation and the proteasomal degradation machinery were shown to be crucial for Mtb virulence as they ensureintracellular growthand contribute tolong-term persistence18-22. However, theunderlying mechanisms still remain elusive. Various studies have investigatedthe identity of pupylated proteins, the so-called “pupylome”, in Mtb and Msmto gain insight into the precise function of the Pup-proteasome system in pathogenic and non-pathogenic mycobacteria23-26. Amongst others, the identified targets comprisedproteins involved in intermediary metabolism, virulence and detoxification. Interestingly, not all proteins annotated as part of the “pupylome”seem to be degraded by the proteasome

25

.This might indicate a degradation-independent regulatory function of the

pupylation pathway. A recent study in Msmhas highlighted the importance of Pup-dependent protein degradation for survival under nutrient-limiting conditions, especially when nitrogen becomes scarce26. Immunoblot analyses of wild type Msmrevealedthat nitrogen starvation is accompanied by a fast decrease in cellular levels of pupylated proteins.An Msm mutant lacking the genes encoding Pup and the proteasomal subunits PrcA and PrcBdisplayed astrong growth phenotypeand was affected in its ability to withstand nitrogen starvation.Reintroduction of pup in the mutant re-established pupylationand pupylated proteins accumulated in the cell during nitrogen limiting growth conditions.Based on these observations, it was postulated that proteasomal degradation of pupylated proteins is -4ACS Paragon Plus Environment

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employed to recycle amino acids, if usual assimilation mechanisms fail to provide sufficient nitrogen to maintain basal cellular functions. In the present study, we highlighted for the first time two different aspects of pupylation in Msm.Initially, we set out to elucidate changes in the proteome taking place during nitrogen starvation conditions in a pupylation-dependent manner. Therefore, a pup deletion mutant of Msmwas generated and characterized using quantitative proteomic analysis by incorporating a stable isotope dimethyl labelling strategyupon long-term exposure to nitrogenstarvation.In a second approach, we applied labelfree relative quantification to investigate how pupylation itself is changing in the absence of nitrogen.Subsequently, the Msmpup deletion strain was complemented with a StrepII-tagged variant of Pup to isolate pupylated proteins from nitrogen-starved cells and to quantify pupylation at the level of single proteins.

EXPERIMENTAL PROCEDURES Bacterial strains and growth conditions A detailed overview on bacterial strains and plasmids used in this study can be found in SupplementaryTable 1. Generation of MsmΔpup strain is described in details in Supplementary

Figure

S-1.Mycobacterium

smegmatis

was

grown

in

LB medium

supplemented with 0.05% Tween-80 (LB-T). When necessary, media were supplemented with antibiotics at the following concentrations: streptomycin100µg/ml; hygromycin 100µg/ml. To determine in vitro growth characteristics, pre-cultures grown to mid-log phasewere used to inoculate 50ml cultures to an OD600 = 0.1. The cultures were incubated at 37°C in a shaking incubator (180 rpm). At given time points samples were withdrawn to measure the optical density (OD)at 600 nmusing a Bio-Photometer plus (Eppendorf). All nitrogen starvation experiments were conducted in a defined M9 minimal medium devoid of any ammonium source (M9/-N), containing 52 mM Na2HPO4-7H2O, 22 mM -5ACS Paragon Plus Environment


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KH2PO4, 9 mM NaCl, 2 mM MgSO4, 0.1 mM CaCl2, 0.4% (w/v) glucose and 0.05% (v/v) Tween-80. Pre-cultures were initially grown in LB-T to mid-log phase. Cells were then harvested by centrifugation and were washed three times with M9/-N medium. Main cultures were finally inoculated with washed cells to an OD600 = 1.0 and were grown at 37°C in a shaking incubator. For subsequent proteomic analyses cultures were incubated for an additional 24h. To assess the bacteria survival under nitrogen limiting conditions, samples were withdrawn from the cultures at given timepoints and serial dilutions of the samples were spotted onto non-selective LB-agar plates. Plates were incubated at 37°C for three days. Preparation of cell-free lysates For proteomic analyses, bacterial cultures were harvested and washed three times with PBS. Cells were resuspended in resuspension buffer (100mM TRIS-HCl pH 8.0, 300mM NaCl) supplemented with 0.1mg/ml PMSF and 1mM protease inhibitor cocktail (Sigma-Aldrich) and lysed by French press (18,000 psi). Cell debris was removed by two centrifugation steps ((18,000 rpm (SS-34 Sorvall Instruments), 4°C)). Protein concentration was determined by using the Pierce BCA Protein Assay Kit (Thermo Scientific). For immunoblots, the lysates were supplemented additionally with1mM DNAse,1mM MgCl2,1mM Dithiothreitol (DTT), 1mM RNAse. Sample preparation and Liquid chromatography– tandem mass spectrometry forstable isotope dimethyl labelling Lysates of M. smegmatis SMR5 and M. smegmatis∆pup were generated (see previous section). Briefly, proteins were precipitated with trichloroacetic acid (TCA), solubilized and trypsinized(seq. grade trypsin; Worthington, Lakewood, NJ, USA). Cysteine residues werereducedand alkylated. Samples were then labelled with 20 mM either formaldehyde light [d012 C] (12COH2) or heavy [d213 C] (13COD2) in the presence of 20 mM sodium cyanoborohydride. After quenching the reaction with glycine, samples were combined in pairs in 1:1 ratio. Following desalting by C18 solid phase extraction (Sep-Pak C18 Plus Light -6ACS Paragon Plus Environment

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Cartridge, Waters, Frankfurt, Germany), samples (ca. 300 µg) were fractionated by strong cation exchange chromatography and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS)27. Samples were analyzed on an Orbitrap XL (Thermo Scientific) mass spectrometer coupled to an Ultimate3000 micro pump (Thermo Scientific) as described previously27,28. Buffer A was 0.5% (v/v) acetic acid, buffer B 0.5% (v/v) acetic acid in 80% (v/v) acetonitrile (HPLC grade). Liquid phases were applied at a flow rate of 300 nL/min with an increasing gradient of organic solvent for peptide separation. Reprosil-Pur 120 ODS-3 (Dr. Maisch) was used to pack column tips of 75 µm inner diameter and 11 cm length. The MS was operated in data dependent mode and each MS scan was followed by a maximum of five MS/MS scans.

Dimethyl labelling data analysis and Protein Identification LC-MS/MS data was obtained in raw format and converted to the mzXMLformat29, using msconvert29,30with centroiding of MS1 and MS2 data, and deisotoping of MS2 data. For spectrum to sequence assignment X! Tandem (version 2013.09.01)30,31 was used (part of version 4.7 of the Trans Proteomic Pipeline, TPP). The proteome database consisted of Mycobacteriumsmegmatis reviewed canonical UniProt sequences (6,601 protein entries) downloaded from UniProt on August 20th 2014, combined with both a randomized and a reversed decoy database (DB toolkit32). Static modification for X! Tandem parameters included: precursor mass error of 10 ppm, fragment ion mass tolerance of 0.3 Da, tryptic cleavage specificity with no missed cleavage sites, residue modifications: cysteine carboxyamidomethylation

(+57.02

Da),

lysine

and

N-terminal

dimethylation

(light

formaldehyde 28.03 Da; heavy formaldehyde 34.06 Da); novariable modifications. X! Tandem results were further validated by PeptideProphet33at a confidence level of > 95%. Peptides were assembled to proteins using ProteinProphet34with a false discovery rate (FDR) < 1.0%. For relative peptide and protein quantification XPRESS35was used. Mass tolerance used for quantification was 20 ppm. Files obtained by TPP were further processed with an inhouse developed script and analyzed by R Studio v.0.99.446 as an IDE for R (R Foundation -7ACS Paragon Plus Environment


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for Statistical Computing, Vienna, Austria) as previously described36.Overlap of identified proteins was determined using R and BioVenn 2007. Ratios were log2 transformed, normalized by centering and a linear model was fitted using the limma package37 followed by a moderated-t-test. Proteins identified with at least two peptides were consideredwith an altered abundance by having a log2 fold change of more/less than 50% (Fc (log2) ≤ -0.58 or Fc (log2) ≥ 0.58)and a p-value

Comprehensive Proteomic Analysis of Nitrogen-Starved

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