OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localised energy production and calcium buffering requirements. Here we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover Miro1 turnover on damaged mitochondria is altered in Parkinson's disease (PD) patient derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analysing the kinetics of Miro1 ubiquitination we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent K27 type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower timescale, within 2-3 hours of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at serine 156 (S156), but is dependent on the recently identified serine S65 residue within Parkin that is phosphorylated by PINK1. Interestingly we find that Miro1 can stabilise phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that S65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in PD pathogenesis

Observation of the Isospin-Violating Decay Ds∗+→Ds+π0D_s^{*+}\to D_s^+\pi^0

Using data collected with the CLEO~II detector, we have observed the isospin-violating decay $D_s^{*+}\to D_s^+\pi^0$. The decay rate for this mode, relative to the dominant radiative decay, is found to be $\Gamma(D_s^{*+}\to D_s^+\pi^0)/\Gamma(D_s^{*+}\to D_s^+\gamma)= 0.062^{+0.020}_{-0.018}\pm0.022$.Comment: 8 page uuencoded postscript file, also available through http://w4.lns.cornell.edu/public/CLN

Measurements of the Ratios B(Ds+→ηℓ+ν)/B(Ds+→ϕℓ+ν){\cal B}(D_s^+\to \eta\ell^+\nu)/{\cal B}(D_s^+\to \phi\ell^+\nu) and B(Ds+→η′ℓ+ν)/B(Ds+→ϕℓ+ν){\cal B}(D_s^+\to \eta'\ell^+\nu)/{\cal B}(D_s^+\to \phi\ell^+\nu)

Using the CLEO~II detector we measure ${\cal B}(D_s^+\to \eta e^+\nu)/{\cal B}(D_s^+\to \phi e^+\nu) =1.24\pm0.12\pm0.15$, ${\cal B}(D_s^+\to \eta' e^+\nu)/{\cal B}(D_s^+\to \phi e^+\nu) =0.43\pm0.11\pm0.07$ and ${\cal B}(D_s^+\to \eta' e^+\nu)/{\cal B}(D_s^+\to \eta e^+\nu) =0.35\pm0.09\pm0.07$. We find the vector to pseudoscalar ratio, ${\cal B}(D_s^+\to \phi e^+\nu)/{\cal B}(D_s^+\to (\eta+\eta') e^+\nu) =0.60\pm0.06\pm0.06$, which is similar to the ratio found in non strange $D$ decays.Comment: 11 page uuencoded postscript file, postscript file also available through http://w4.lns.cornell.edu/public/CLN

Semileptonic Branching Fraction of Charged and Neutral B Mesons

An examination of leptons in ${\Upsilon (4S)}$ events tagged by reconstructed $B$ decays yields semileptonic branching fractions of $b_-=(10.1 \pm 1.8\pm 1.4)\%$ for charged and $b_0=(10.9 \pm 0.7\pm 1.1)\%$ for neutral $B$ mesons. This is the first measurement for charged $B$. Assuming equality of the charged and neutral semileptonic widths, the ratio $b_-/b_0=0.93 \pm 0.18 \pm 0.12$ is equivalent to the ratio of lifetimes. A postscript version is available through World-Wide-Web in http://w4.lns.cornell.edu/public/CLNS/1994Comment: 9 pages (in REVTEX format) Preprint CLNS94-1286, CLEO 94-1

Limit on the Two-Photon Production of the Glueball Candidate fJ(2220)f_{J}(2220) at CLEO

We use the CLEO detector at the Cornell electron-positron storage ring, CESR, to search for the two-photon production of the glueball candidate f_J(2220) in its decay to K_s K_s. We present a restrictive upper limit on the product of the two-photon partial width and the K_s K_s branching fraction. We use this limit to calculate a lower limit on the stickiness, which is a measure of the two-gluon coupling relative to the two-photon coupling. This limit on stickiness indicates that the f_J(2220) has substantial glueball content.Comment: 9 page postscript file, postscript file also available through http://w4.lns.cornell.edu/public/CLN

Measurement of the Bˉ→Dℓνˉ\bar{B}\to D\ell\bar{\nu} Partila Width and Form Factor Parameters

We have studied the decay $\bar{B} \to D\ell\bar{\nu}$, where $\ell=e or \mu$. From a fit to the differential decay rate $d\Gamma/dw$ we measure the rate normalization ${\cal F}_D(1)|V_{cb}|$ and form factor slope $\hat{\rho}^2_D$, and, using measured values of $\tau_B$, find $\Gamma(\bar{B} \to D\ell\bar{\nu}) = (12.0 \pm 0.9 \pm 2.1) ns^{-1}$. The resulting branching fractions are ${\cal B}(\bar{B}^0 \to D^+\ell^-\bar{\nu})=(1.87 \pm 0.15 \pm 0.32)$ and ${\cal B}(B^- \to D^0\ell^-\bar{\nu})=(1.94 \pm 0.15 \pm 0.34)$. The form factor parameters are in agreement with those measured in $\bar{B} \to D^*\ell\bar{\nu}$ decays, as predicted by heavy quark effective theory.Comment: 11 pages, postscript file also available through http://w4.lns.cornell.edu/public/CLN

Measurement of the Inclusive Semi-electronic D0D^0 Branching Fraction

Using the angular correlation between the $\pi^+$ emitted in a $D^{*+} \rightarrow D^0 \pi^+$ decay and the $e^+$ emitted in the subsequent $D^0 \rightarrow Xe^+\nu$ decay, we have measured the branching fraction for the inclusive semi-electronic decay of the $D^0$ meson to be: {\cal B}(D^0 \rightarrow X e^+ \nu) = [6.64 \pm 0.18 (stat.) \pm 0.29 (syst.)] \%. The result is based on 1.7 fb$^{-1}$ of $e^+e^-$ collisions recorded by the CLEO II detector located at the Cornell Electron Storage Ring (CESR). Combining the analysis presented in this paper with previous CLEO results we find, \frac{{\cal B} (D^0 \rightarrow X e^+ \nu)} {{\cal B} (D^0 \rightarrow K^- \pi^+)} = 1.684 \pm 0.056 (stat.) \pm 0.093(syst.) and \frac{{\cal B}(D\rightarrow K^-e^+\nu)} {{\cal B}(D\rightarrow Xe^+\nu)} = 0.581 \pm 0.023 (stat.) \pm 0.028(syst.). The difference between the inclusive rate and the sum of the measured exclusive branching fractions (measured at CLEO and other experiments) is $(3.3 \pm 7.2) \%$ of the inclusive rate.Comment: Latex file, 33pages, 4 figures Submitted to PR