Generation of hydrophobic patch variants of ClpP1 and ClpP2.

<p>A. Cartoon representation of the LGF-loops (dark blue) of the chaperone binding to hydrophobic surface patches (green) on the protease core (grey). B. Top view of a heptameric protease ring. The hydrophobic patches (green) are formed by residues of two adjacent protease subunits (grey). C. Mutations introduced in ClpP1 and ClpP2 to create the hydrophobic patch variants hpClpP1 (ClpP1^{S61A, Y63V, L83A, Y91V}) and hpClpP2 (ClpP2^{Y75V, Y95V}). D. Alignment of Mtb ClpP1 and ClpP2 with EcClpP. Conservation is colored from white (not conserved) to black (identical). The identity between ClpP1/ClpP2 is 39.5%, between EcClpP/ClpP1 46% and EcClpP/ClpP2 44.4%. Red arrows highlight the residue positions of the EcClpP hydrophobic patch residues. Residues depicted in green in panel E are marked with a green box. E. Surface representation of ClpP1, ClpP2 (4U0G.pdb) and EcClpP (3MT6.pdb) ring faces. Individual subunits are colored alternatingly in light and dark grey. Hydrophobic patch residues are colored in green and labelled accordingly. For ClpP1 and ClpP2 the hydrophobic patch residues used for mutation are shown, for EcClpP reference residues are shown as described in the literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125345#pone.0125345.ref014" target="_blank">14</a>]. F. Creation of a set of mature mixed wild-type ClpP1 and ClpP2 (wtP1, wtP2) and hydrophobic patch variants (hpP1, hpP2). Large-scale processing of ClpP1 and ClpP2 (70 μM protomer each) containing the N-terminal propeptide (proClpP1, proClpP2) to mature ClpP1 and ClpP2 (mClpP1, mClpP2) in the presence of 1 mM activator. The reaction was performed in Buffer A. All samples were run on the same gel. The lane containing the size marker was removed for better visual representation (white line). G. Analytical gel filtration was performed with the mature ClpP1P2 complexes created in panel F. The peak at 11 ml shows that all complexes have assembled into double-rings.</p

The <i>Mycobacterium tuberculosis</i> ClpP1P2 Protease Interacts Asymmetrically with Its ATPase Partners ClpX and ClpC1

<div><p>Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperone rings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation. Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to <i>E</i>. <i>coli</i>, <i>Mycobacterium tuberculosis</i> harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2, and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently, this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobic chaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both <i>Mycobacterium tuberculosis</i> Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clp proteases of <i>Mycobacterium tuberculosis</i>, and probably other Actinobacteria, apart from the well-studied <i>E</i>. <i>coli</i> system, where chaperones bind to both sides of the protease core, and it frees the ClpP1 interaction interface for putative new binding partners.</p></div

Chaperone-mediated degradation of ssrA-tagged substrates by ClpP1P2 requires the hydrophobic patch on ClpP2.

<p>ClpX and ClpC1-dependent degradation of model substrates was assayed with a set of mature ClpP1P2 particles, created from mixed wild-type (wtP1, wtP2) and hydrophobic patch variants (hpP1, hpP2) of ClpP1 and ClpP2. A. Degradation of MDH-ssrA (2 μM) mediated by ClpX (1 μM hexamer) and wt, hp or mixed mature ClpP1P2 particles (0.5 μM double-ring particle), was followed by the disappearance of the MDH-ssrA band in SDS-PAGE. The band just below MDH-ssrA that is not degraded (*) was confirmed by MS/MS to be composed of MDH, most probably lacking the ssrA tag. B. Degradation of GFP-ssrA (2 μM) mediated by ClpC1 (1 μM hexamer) and by wt, hp and mixed mature ClpP1P2 particles (0.5 μM double-ring particle) was monitored by the loss of the intrinsic GFP fluorescence signal. The signal was globally normalized. Additionally, time points were taken at the beginning and the end of the reaction and degradation of GFP-ssrA was confirmed by SDS-PAGE.</p

Crystal Structure of the Complex between Prokaryotic Ubiquitin-like Protein and Its Ligase PafA

Prokaryotic ubiquitin-like protein (Pup) is covalently attached to target proteins by the ligase PafA, tagging substrates for proteasomal degradation. The crystal structure of Pup in complex with PafA, reported here, reveals that a long groove wrapping around the enzyme serves as a docking site for Pup. Upon binding, the C-terminal region of the intrinsically disordered Pup becomes ordered to form two helices connected by a linker, positioning the C-terminal glutamate in the active site of PafA

Additional file 2: Figure S2. of Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins

Secondary shifts of Mtb Pup ~  Mtb FabD and Mtb Pup ~ lysine. Chemical shifts of the native protein ( Mtb Pup ~ Lys in red, Mtb Pup ~  Mtb FabD-3KR in green) minus shifts for Mtb Pup ~  Mtb PanB unfolded in Urea (data from [3]). The region with helical propensity in free Mtb Pup is indicated in grey. (PNG 213 kb

ANALISIS TINGKAT KEMANDIRIAN DAERAH PADA KABUPATEN/KOTA DI PROVINSI BENGKULU

Kemandirian keuangan merupakan hal yang sangat penting dalam pelaksanaan otonomi daerah, karena dengan kemandirian berarti suatu daerah itu sudah berdaya dalam pelaksanaan otonomi daerah, dengan begitu daerah yang sudah mandiri atau berdaya, sama artinya dengan daerah tersebut sudah bisa menghidupi sendiri kebutuhan dalam pelaksanaan otonomi daerah. Hal ini menimbulkan pertanyaan apa penyebab Provinsi Bengkulu belum bisa mewujudkan kemandirian daerahnya? Apakah pemerintah pusat dengan sengaja membiarkan kondisi seperti ini agar terus terjadi ketergantungan pemerintah daerah terhadap dana transfer atau disebabkan ketidakmampuan Pemerintah Provinsi Bengkulu sendiri dalam mewujudkan kemandirian daerah. Tujuan penelitian ini adalah: (1) Untuk mengetahui tingkat kemandirian daerah kabupaten/kota di Provinsi Bengkulu dan (2) Untuk mengetahui perbedaan kemandirian daerah kabupaten/kota induk dengan kabupaten/kota pemekaran. Penelitian ini merupakan jenis penelitian deskriptif. Jenis data yang digunakan adalah data sekunder. Metode analisis data digunakan adalah analisis deskriptif. Berdasarkan hasil analisis data diperoleh hasil bahwa: (1)Tingkat kemandirian daerah kabupaten/kota di Provinsi Bengkulu mayoritas berada pada kategori rendah, karena berada pada skala interval di bawah 25%, yang berarti bahwa memiliki kemampuan keuangan daerah yang rendah; (2) Kabupaten/kota di Provinsi Bengkulu memiliki dua klasifikasi daerah, yakni daerah cepat maju dan cepat tumbuh dan daerah relative tertinggal; (3) Pola hubungan antara pemerintah daerah kabupaten/kota di Provinsi Bengkulu dengan pemerintah provinsi/pusat mayoritas adalah instruktif; (4) Berdasarkan hasil perhitungandi atas, diketahui bahwa nilai t hitung yang diperoleh adalah sebesar 3,290 > t sebesar 1,96. Hal ini berarti bahwa Ho ditolak dan Ha diterima. Hasil tersebut membuktikan bahwa terdapat perbedaan kemandirian daerah antara kabupaten/kota induk dengan kabupaten/kota pemekaran di Provinsi Bengkulu. Perbedaan rata-rata keduanya adalah kabupaten induk memiliki rata-rata kemandirian sebesar 5,78%, sedangkan kabupaten pemekaran memiliki rata-rata kemandirian sebesar 3,92%; dan (5) Hasil pengujian perbedaan kemandirian antara Kabupaten Bengkulu Selatan dengan Kabupaten Seluma dan Kabupaten Kaur tidak terjadi perbedaan. Selanjutnya, Kabupaten Bengkulu Utara dengan Kabupaten Mukomuko tidak terjadi perbedaan kemandirian, sedangkan dengan Kabupaten Bengkulu Tengah terdapat perbedaan. Hasil pengujian tabel(0,025) perbedaan kemandirian antara Kabupaten Rejang Lebong dengan Kabupaten Kepahiang dan Kabupaten Lebong tidak terjadi perbedaan. Berdasarkan hasil analisis terhadap tingkat kemandirian daerah kabupaten/kota di Provinsi Bengkulu terlihat dengan jelas bahwa kemandirian daerah berada pada kategori sangat rendah. Berdasarkan hasil tersebut, dapat diberikan beberapa saran berkaitan dengan peningkatan kemandirian dan kemampuan keuangan daerah, yakni: (1) Dalam kerangka otonomi daerah pemerintah daerah kabupaten/kota di Provinsi Bengkulu harus dapat meningkatkan pendapatan asli daerahnya dengan mencari sumber-sumber pendapatan yang sah, sehingga mendongkrak pendapatan asli daerah. Hal ini dimaksudkan untuk meningkatkan kemandirian daerah tersebut dan (2) Pemerintah daerah kabupaten/kota di Propinsi Bengkulu dalam menyusun dan realisasi pendapatan dan belanja daerah perlu juga memperhatikan arah perkembangan pola hubungan dan kemampuan keuangan daerahnya agar menunjukkan kondisi yang lebih baik

Bpa interacts with the 20S proteasome through its C-terminal HbYX motif.

<p>A, Vector constructs for the bacterial two-hybrid screen. PrcA and Δ7PrcA were fused to the C-terminus of the catalytic T18 domain, Bpa and BpaΔHbYX to the C-terminus of the catalytic T25 domain of adenylate cyclase. B, MacConkey agar matrix of all pairwise combinations of the pUT18C and pKT25 constructs in triplicates. Successful interaction between T18 and T25 switches the colony from lac− to lac+ and the resulting acidification of the agar is visualized by the pH indicator turning red. C, A quantitative β-galactosidase assay of the same hybrid experiments as shown in B. The assay was performed on chloroform-treated <i>E. coli</i> cells grown overnight in liquid LB medium containing 0.5 mM IPTG. The background activity is indicated by the negative control (no insert, corresponding to pKT25 and pUT18C carrying only the adenylate cyclase domains without fusion). Bars represent averages ± SEM of at least three replicates. D, Mpa-mediated proteasomal PanB-Pup degradation is inhibited in presence of association-competent Bpa but not in presence of BpaΔHbYX. Concentrations: Mpa (0.2 µM), 20S proteasome (0.1 µM), Bpa or BpaΔHbYX (14 µM protomer).</p

Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

<div><p>The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen <i>Mycobacterium tuberculosis</i> (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of <i>Mycobacterium tuberculosis</i> we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates <i>in vitro</i> by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins.</p></div

Bpa is conserved in actinobacteria encoding the proteasomal core particle genes.

<p>A, Multiple sequence alignment of Bpa orthologs (Rv3780 in Mtb) from different actinobacteria. The predominant residues in positions with an identity score above 0.5 are shaded in blue where increasing similarity is indicated by a gradient from light to dark blue. The completely conserved penultimate tyrosine of the HbYX motif is colored in red. B, Occurrence and location of the Pup-proteasome gene locus and the bpa gene. Each line represents the location of all open reading frames (orfs) of a bacterium in relation to the Pup proteasome gene locus. The position and orientation (if a corresponding homolog exists) of the proteasomal genes <i>prcB</i> and <i>prcA</i> are indicated by turquoise and <i>bpa</i> by a red arrow, respectively. The pupylation genes are given in shades of grey. Organisms are abbreviated as follows: <i>M. tuberculosis</i> (Mtb), <i>M. leprae</i> (Mlep), <i>M. smegmatis</i> (Msm), <i>N. farcinica</i> (Nfar), <i>R. erythropolis</i> (Re<i>), S. coelicolor</i> (Scoe), <i>T. fusca</i> (Tfus), <i>K. radiotolerans</i> (Krad), <i>Janibacter sp.</i> (Jani), <i>A. cellulolyticus</i> (Acel), <i>S. erythraea</i> (Ser), <i>Nocardioides sp.</i> (Noc), <i>S. tropica</i> (Strop), <i>Frankia sp.</i> (Frankia), <i>A. aurescens</i> (Aaur), <i>R. salmoninarum</i> (Rsal), <i>A. ferrooxidans</i> (Afer), <i>Brevibacterium sp.</i> (Brevi), <i>B. mcbrellneri</i> (Bmcb), <i>F. alni</i> (Fal), <i>L. ferrooxidans</i> (Lfer), <i>P. acnes</i> (Pac), <i>A. odontolyticus</i> (Aodo), <i>B. adolescentis</i> (Bado), <i>C. diphtheria</i> (Cdip), <i>C. glutamicum</i> (Cglu), <i>K. rhizophila</i> (Krhi), <i>M. luteus</i> (Mlut).</p

Bpa can be retained by immobilized Δ7PrcAB or half-proteasomes.

<p>A, Elution fractions of the pull-downs of Bpa or BpaΔHbYX with different Strep-immobilized proteasome particles. Strep-tagged proteasome particles were eluted from Strep-Tactin Sepharose with 2.5 mM desthiobiotin and the elution fractions were visualized on Coomassie stained SDS-PAGE. Bpa binds to Δ7PrcAB, while BpaΔHbYX does not. PrcAB interaction with Bpa is not detectable, while half-proteasomes (proPrcAB) retain a small amount of Bpa. B, Maturation of half-proteasomes at 37°C for 24 hours followed by SDS-PAGE. No difference in processing speed can be observed in the reactions supplemented with Bpa compared to PrcAB alone within this time frame.</p