Pupylated proteins are subject to broad proteasomal degradation specificity and differential depupylation

In actinobacteria, post-translational modification of proteins with prokaryotic ubiquitin-like protein Pup targets them for degradation by a bacterial proteasome assembly consisting of the 20S core particle (CP) and the mycobacterial proteasomal ATPase (Mpa). Modification of hundreds of cellular proteins with Pup at specific surface lysines is carried out by a single Pup-ligase (PafA, proteasome accessory factor A). Pupylated substrates are recruited to the degradative pathway by binding of Pup to the N-terminal coiled-coil domains of Mpa. Alternatively, pupylation can be reversed by the enzyme Dop (deamidase of Pup). Although pupylated substrates outcompete free Pup in proteasomal degradation, potential discrimination of the degradation complex between the various pupylated substrates has not been investigated. Here we show that Mpa binds stably to an open-gate variant of the proteasome (oCP) and associates with bona fide substrates with highly similar affinities. The proteasomal degradation of substrates differing in size, structure and assembly state was recorded in real-time, showing that the pupylated substrates are processed by the Mpa-oCP complex with comparable kinetic parameters. Furthermore, the members of a complex, pupylated proteome (pupylome) purified from Mycobacterium smegmatis are degraded evenly as followed by western blotting. In contrast, analysis of the depupylation behavior of several pupylome members suggests substrate-specific differences in enzymatic turnover, leading to the conclusion that largely indiscriminate degradation competes with differentiated depupylation to control the ultimate fate of pupylated substrates.ISSN:1932-620

Structural and mechanistic basis of proton-coupled metal ion transport in the SLC11/NRAMP family

Secondary active transporters of the SLC11/NRAMP family catalyse the uptake of iron and manganese into cells. These proteins are highly conserved across all kingdoms of life and thus likely share a common transport mechanism. Here we describe the structural and functional properties of the prokaryotic SLC11 transporter EcoDMT. Its crystal structure reveals a previously unknown outward-facing state of the protein family. In proteoliposomes EcoDMT mediates proton-coupled uptake of manganese at low micromolar concentrations. Mutants of residues in the transition-metal ion-binding site severely affect transport, whereas a mutation of a conserved histidine located near this site results in metal ion transport that appears uncoupled to proton transport. Combined with previous results, our study defines the conformational changes underlying transition-metal ion transport in the SLC11 family and it provides molecular insight to its coupling to protons

Prokaryotic ubiquitin-like protein remains intrinsically disordered when covalently attached to proteasomal target proteins

Background The post-translational modification pathway referred to as pupylation marks proteins for proteasomal degradation in Mycobacterium tuberculosis and other actinobacteria by covalently attaching the small protein Pup (prokaryotic ubiquitin-like protein) to target lysine residues. In contrast to the functionally analogous eukaryotic ubiquitin, Pup is intrinsically disordered in its free form. Its unfolded state allows Pup to adopt different structures upon interaction with different binding partners like the Pup ligase PafA and the proteasomal ATPase Mpa. While the disordered behavior of free Pup has been well characterized, it remained unknown whether Pup adopts a distinct structure when attached to a substrate. Results Using a combination of NMR experiments and biochemical analysis we demonstrate that Pup remains unstructured when ligated to two well-established pupylation substrates targeted for proteasomal degradation in Mycobacterium tuberculosis, malonyl transacylase (FabD) and ketopantoyl hydroxylmethyltransferase (PanB). Isotopically labeled Pup was linked to FabD and PanB by in vitro pupylation to generate homogeneously pupylated substrates suitable for NMR analysis. The single target lysine of PanB was identified by a combination of mass spectroscopy and mutational analysis. Chemical shift comparison between Pup in its free form and ligated to substrate reveals intrinsic disorder of Pup in the conjugate. Conclusion When linked to the proteasomal substrates FabD and PanB, Pup is unstructured and retains the ability to interact with its different binding partners. This suggests that it is not the conformation of Pup attached to these two substrates which determines their delivery to the proteasome, but the availability of the degradation complex and the depupylase.ISSN:1472-680

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

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

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 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

Bpa acts as a proteasomal activator.

<p>A, Peptidase activity measured by fluorescence detection of AMC molecules (λ_ex: 360 nm, λ_em: 460 nm) released from the fluorogenic peptide L-Suc-LLVY-AMC (150 µM). PrcAB exhibits a turnover number (TON) of 0.057±0.004 s⁻¹, which is more than three times less active than Δ7PrcAB with a TON of 0.185±0.014 s⁻¹. Addition of an excess of Bpa to the reaction increases peptidase activity of PrcAB about 1.8 fold to a TON of 0.105±0.008 s⁻¹. B, Degradation of β-casein (27 µM) at 37°C by Δ7PrcAB or PrcAB (0.2 µM) in presence or absence of Bpa or BpaΔHbYX (6 µM protomer) is sampled at different time points and analyzed by Coomassie-stained SDS-PAGE.</p

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