Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

The 6^{6}H states studied in the d(8He,α)d(^8\text{He},\alpha) reaction and evidence of extremely correlated character of the 5^{5}H ground state

The extremely neutron-rich system $^{6}$H was studied in the direct $^2\text{H}(^8\text{He},{^4\text{He}})^{6}$H transfer reaction with a 26 $A$ MeV secondary $^{8}$He beam. The measured missing mass spectrum shows a resonant state in $^{6}$H at $6.8(3)$ MeV relative to the $^3$H+$3n$ threshold. The population cross section of the presumably $p$-wave states in the energy range from 4 to 8 MeV is $d\sigma/d\Omega_{\text{c.m.}} \simeq 190(40)$ $\mu$b/sr in the angular range $5^{\circ}<\theta_{\text{c.m.}}<16^{\circ}$. The obtained missing mass spectrum is free of the $^{6}$H events below 3.5 MeV ($d\sigma/d\Omega_{\text{c.m.}} \lesssim 3$ $\mu$b/sr in the same angular range). The steep rise of the $^{6}$H missing mass spectrum at 3 MeV allows to show that $4.5(3)$ MeV is the lower limit for the possible resonant state energy in $^{6}$H tolerated by our data. According to paring energy estimates, such a $4.5(3)$ MeV resonance is a realistic candidate for the $^{6}$H ground state (g.s.). The obtained results confirm that the decay mechanism of the $^{7}$H g.s.\ (located at 2.2 MeV above the $^{3}$H+$4n$ threshold) is the ``true'' (or simultaneous) $4n$ emission. The resonance energy profiles and the momentum distributions of the sequential $^{6}$H \,\rightarrow \, ^5H(g.s.)+n\, \rightarrow \, ^3H+$3n$ decay fragments were analyzed by the theoretically-updated direct four-body-decay and sequential-emission mechanisms. The measured momentum distributions of the $^{3}$H fragments in the $^{6}$H rest frame indicate very strong ``dineutron-type'' correlations in the $^{5}$H ground state decay.Comment: 9 pages, 11 figure

Study of Proton and Deuteron Pickup Reactions 2H(10Be,3He)9Li an 2H(10Be,4He)8Li with 44 A MeV 10Be Radioactive Beam at ACCULINNA-2 Fragment Separator

The proton and deuteron pickup reactions 2H(10Be,3He)9Li and\\ 2H(10Be,4He)8Li radioactive beam produced by the new fragment separator ACCULINNA-2 at FLNR, JINR\@. These measurements were initially motivated as test reactions intended for the elucidation of results obtained in the study of the extremely neutron-rich 7H and 6H systems created in the 2H(10Be,3He)9Li and 2H(10Be,4He)8Li reactions using the same setup. In the 2H(10Be,3He)9Li reaction the 9Li ground-state ($3/2^-$) and its first excited state (2.69~MeV, $1/2^-$) were identified in the low-energy region of its excitation spectrum. The differential cross sections for the 9Li g.~s.) population were extracted at forward center-of-mass angles ($3^\circ-13^\circ$) and compared with the FRESCO calculations. Spectroscopic factor of $\sim 1.7$, derived by a model for the 10Be$= p +$9Li(g.s.) clustering was found in accord with the experimental data. The energy spectrum of 8Li populated in the 2H(10Be,4He)8Li reaction shows the strong peak which corresponds to excitation of the second excited state of 8Li (2.25 MeV, $3^+$). The fact that the ground and the first excited states of 8Li were not observed is fully consistent with Shell-Model calculations carried out for the 10Be g.\,s. and 8Li level structure applying momentum selection rules

Common Genetic Denominators for Ca++-Based Skeleton in Metazoa: Role of Osteoclast-Stimulating Factor and of Carbonic Anhydrase in a Calcareous Sponge

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl2) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca2+-depletion condition (1 mM CaCl2). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast)

Osteoclast stimulation factor 1 (Ostf1) KNOCKOUT increases trabecular bone mass in mice

Osteoclast stimulation factor 1 (OSTF1) is an SH3-domain containing protein that was initially identified as a factor involved in the indirect activation of osteoclasts. It has been linked to spinal muscular atrophy in humans through its interaction with SMN1, and is one of six genes deleted in a human developmental microdeletion syndrome. To investigate the function of OSTF1, we generated an Ostf1 knockout mouse model, with exons 3 and 4 of Ostf1 replaced by a LacZ orf. Extensive X-Gal staining was performed to examine the developmental and adult expression pattern, followed by phenotyping. We show widespread expression of the gene in the vasculature of most organs and in a number of cell types in adult and embryonic mouse tissues. Furthermore, whilst SHIRPA testing revealed no behavioural defects, we demonstrate increased trabecular mass in the long bones, confirming a role for OSTF1 in bone development

SH3P2 in complex with Cbl and Src

International audienc

Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation

Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome

Application of fluidized bed combustion ashes for enhancement of mining waste management

W górnictwie węgla kamiennego znaczącym problemem ekologicznym i ekonomicznym są odpady wydobywcze. Szczególnie kłopotliwymi są muły węglowe ze względu na ich konsystencję. W tej postaci są trudne do zagospodarowania, jak również do bezpiecznego ich składowania. Coraz częściej muły węglowe poddawane są granulowaniu z zastosowaniem wapna palonego lub wapna hydratyzowanego. Przeprowadzone badania i próby na instalacjach przemysłowych wykazały, że tradycyjne spoiwa wapniowe można zastępować ubocznymi produktami spalania zawierającymi aktywne związki wapnia. Zastosowanie popiołów fluidalnych umożliwia otrzymywanie granulowanych mułów o właściwościach fizykochemicznych spełniających wymagania na paliwa niskokaloryczne jak również jako składnika mieszanek paliwowych. Najwyższą wytrzymałość mechaniczną na warunki atmosferyczne, transportu i składowania wykazały muły granulowane z domielonym popiołem dennym z kotłów fluidalnych. Poprzez wyselekcjonowanie odpadów średnioziarnistych wydzielanych w osadzarkach i odpowiednie ich wymieszanie z dobranymi popiołami wapniowymi (fluidalne popioły lotne i denne) uzyskano mieszanki kruszywowo-spoiwowe spełniające wymagania obowiązujące na materiały stosowane w budowie dróg, autostrad i budowli hydrotechnicznych. Dokonane wdrożenia i zrealizowane inwestycję pozwoliły na zagospodarowanie odpadów do produkcji paliw granulowanych i mieszanek kruszynowo-spoiwowych, co wpłynęło na zmniejszenie ilości składowanych odpadów wydobywczych i popiołów oraz uzyskanie dodatkowych efektów ekologicznych i ekonomicznych.In coal-mining industry a major environmental and economical problem are mining wastes. Particularly troublesome, due to their consistency, are the coal slimes. In this form they are difficult to be managed, as well as to be safely stored. More often coal slimes are granulated using quicklime or hydrated lime. The studies and tests conducted on industrial installations have shown that the traditional lime binders can be replaced by combustion by-products containing active compounds of calcium. Application of fluidized bed combustion ash (FBC ash) allows receiving granulated slimes of physicochemical properties that meet the requirements of low calorific fuels, as well as a component of fuel blends. The granulated slimes with grinded bottom ash from fluidized bed boilers (FCB bottom ash) have shown the highest mechanical resistance to the weather conditions, transport and storage. Through the selection of medium-grained waste produced settling lagoons and appropriate mixing with selected calcium ashes (FBC fly ash and FBC bottom ash) the aggregate-binder blends have been obtained that meet the requirements applicable to materials used in construction of roads, highways and hydrotechnical engineering. The implementation and investments made allowed for the management of waste to produce granulated fuels and aggregate-binder blends, what resulted in reducing the amount of stored mining waste and ash, as well as in obtaining the additional environmental and economic benefits