Ubiquitin Ligases of the N-End Rule Pathway: Assessment of Mutations in UBR1 That Cause the Johanson-Blizzard Syndrome

Background: Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates. Methodology/Principal Findings: Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients. Conclusions/Significance: These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS

Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome.

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder

Rudolf Slavicek und die Gnathologie in Österreich

Mit der Entwicklung der Prothetik und den prothetischen Instrumenten rückte die Okklusion in den Fokus der zahnmedizinischen Forschung. Diese Forschungsbemühungen fanden ihren Niederschlag in der Gnathologie als einem Teilbereich der Zahnheilkunde. Insbesondere die vom Zweiten Weltkrieg verschonten USA waren das Zentrum der gnathologischen Entwicklung. Rudolf Slavicek zeigt schon am Anfang seiner Karriere Interesse für die Prothetik und die Anwendung der theoretischen Lehren in diesem Bereich der Zahnheilkunde. Diese Tatsache führte ihn dazu, sehr früh Kontakte mit den Forschern in den USA zu knüpfen und sich am Wissenstransfer zu beteiligen. Seine Kompetenz wurde von den Kollegen an der Universität Wien erkannt, weshalb er als Professor an diese Einrichtung berufen wurde. Das ermöglichte ihm, sich mehr der Forschung zu widmen. Fachlich konzentrierte er sich auf die Förderung der Multi- und Interdisziplinarität in der Zahnheilkunde sowie auf die Entwicklung der Diagnoseinstrumente und -methoden wie Axiographie und SAM II Artikulator. Seine theoretischen Lehren in der Gnathologie wurden im Wiener Konzept der Okklusion formuliert. Die Bereiche der internationalen Zusammenarbeit und der postgradualen Bildung waren Slavicek ebenfalls sehr wichtig. Seine Tätigkeit in diesem Bereich fand ihren Höhepunkt der Gründung und Leitung des Zahnärztlichen Fortbildungsinstitutes (ZAFI) sowie der Vienna School of Interdisciplinary Dentistry (VieSID), die der Verbreitung und Förderung der gnathologischen Lehre dienen sollten.With the development of prosthetics and prosthetic instruments, occlusion moves in focus of the dental research. These research efforts were reflected in the Gnathologie as a segment of dentistry. In particular, from the Second World War spared USA were the center of the gnathological developement. Rudolf Slavicek showed at the beginning of his career interest in prosthetics and the application of theoretical teaching in this area of dentistry. This fact led him very early to establish contacts with researchers in the US and to participate in the knowledge transfer from the US to Europe. His colleagues at the University of Vienna acknowledged his competencies in this area of dentistry, which is why he was appointed as a professor at this institution. This enabled him to devote himself to research even more. He focused on the promotion of multi- and interdisciplinary action in dentistry and the development of diagnostic tools and new methods such axiography and SAM II articulator. His theoretical gnathological teachings were formulated in the Vienna concept of occlusion. The areas of international cooperation and post-graduate education were also important to Slavicek. His activities in this area culminated in his chairmanship and establishment of Dental Training Institute (ZAFI) and the Vienna School of Interdisciplinary Dentistry (VieSID), which should serve to disseminate and promote his gnathological teachings.eingereicht von Maja SukaloZusammenfassung in englischer SpracheParalleltitel laut Übersetzung der VerfasserinMedizinische Universität Wien, Diplomarbeit, 2016(VLID)243703

Johanson-Blizzard syndrome

Johanson-Blizzard syndrome (JBS) is a rare autosomal recessive disease characterized by exocrine pancreatic insufficiency, hypoplastic or aplastic nasal alae, cutis aplasia on the scalp, and other features including developmental delay, failure to thrive, hearing loss, mental retardation, hypothyroidism, dental abnormalities, and anomalies in cardiac and genitourinary systems. More than 60 cases of this syndrome have been reported to date. We describe the case of a male infant with typical symptoms of JBS. In addition, a new clinical feature which has not previously been documented, that is anemia requiring frequent blood transfusions and mild to moderate thrombocytopenia was observed. A molecular study was performed which revealed a novel homozygous UBR1 mutation. Possible explanations for this new association are discussed

Adams-Oliver syndrome caused by mutations of the EOGT gene

Adams-Oliver syndrome (AOS) is a rare congenital disease characterized by aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It shows significant genetic heterogeneity and can be transmitted by autosomal dominant or recessive inheritance. Recessive inheritance is associated with mutations of DOCK6 or EOGT; however, only few cases have been published so far. We present two families with EOGT-associated AOS. Due to pseudodominance in one family, the recognition of the recessive inheritance pattern was difficult. We identified two novel AOS-causing mutations (c.404G>A/p.Cys135Tyr and c.311+1G>T). The phenotype in the presented families was dominated by large ACC, whereas TTLD were mostly subtle or even absent and no major malformations occured. Our observations along with the previously published cases indicate that the two types of recessive AOS (EOGT- vs. DOCK6-associated) differ significanty regarding the frequency of neurologic or ocular deficits

Clinical features in JBS patients.

a<p>Ref. 1;</p>b<p>Ref. 6 and unpublished data.</p

Functional activity of yeast Ubr1 mimics of missense JBS-UBR1 mutants.

<p>(A) Relative enzymatic activity of βgal in extracts from <i>S. cerevisiae</i> JD55 (<i>ubr1Δ</i>) that expressed His-βgal or Tyr-βgal, and also carried an empty vector, or an otherwise identical plasmid expressing wild-type <i>S. cerevisiae</i> Ubr1, or (separately) its three missense mutants Ubr1^V146L, Ubr1^H160R, or Ubr1^Q1224E. The activity of βgal was measured in triplicates, with standard deviations shown. (B) Relative levels of induction of the peptide transporter Ptr2 were assayed by measuring the activity of a plasmid-borne <i>lacZ</i> (βgal-encoding) reporter that was expressed from the P<i>_PTR2</i> promoter in <i>ubr1Δ S. cerevisiae</i> that carried either an empty vector or otherwise identical plasmids that expressed either wild-type Ubr1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xia1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a> or its indicated mutants. Cells were grown to A₆₀₀ of ∼0.8 in SC(-Ura, -Leu) medium at 30°C, followed by measurements, in triplicate, of βgal activity in cell extracts, with standard deviations shown. (C) The lysine-requiring JD55 (<i>ubr1Δ</i>) <i>S. cerevisiae</i> strain was grown on plates containing 110 µM lysine (Lys) or 66 µM Lys-Ala dipeptide as the sole source of Lys in the medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a>. JD52 (<i>ubr1Δ</i>) cells carried a vector plasmid or otherwise identical plasmids expressing wild-type Ubr1 or its missense mutants Ubr1^H160R, Ubr1^V146L and Ubr1^Q1224E. Cells were grown to A₆₀₀ of ∼1 in SC(-Leu) medium at 30°C, washed in sterile water, serially diluted 5-fold, spotted on SC(-Leu, -Lys) plates containing 110 µM Lys or 66 µM Lys-Ala, and incubated at 30°C for 3 days. (D) Cell extracts (equal total protein levels) from experiments described in panels A and B were subjected to SDS-PAGE, followed by immunoblotting with affinity-purified anti-Ubr1 antibody (upper panel) and anti-tubulin antibody (a loading control; lower panel). Asterisk indicates a protein that crossreacts with anti-Ubr1 antibody. (E) Extracts from human lymphocytes (equal amounts of total protein) were subjected to SDS-PAGE, followed by immunoblotting with antibody to human UBR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s4" target="_blank">Materials and Methods</a>). Lane 1, wild-type lymphocytes. Lane 2, same as lane 1 but from lymphocytes of patient #2 (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g002" target="_blank">2</a>). Lane 3, same as lane 1 but with lymphocytes from patient #3. Lane 4, same as lane 1, but with lymphocytes from a JBS patient with a homozygous nonsense mutation in <i>UBR1</i>, previously shown to have no detectable UBR1 (null UBR1 control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Varshavsky3" target="_blank">[17]</a>. Lane 5, same as a lane 1.</p

<i>S. cerevisiae</i> Ubr1 N-recognin.

<p>(A) A diagram of the 225 kDa <i>S. cerevisiae</i> Ubr1. The indicated evolutionarily conserved regions of Ubr1 are the UBR box, the BRR (basic residues-rich) domain, the Cys/His-rich RING-H2 domain, and the AI (<u>a</u>uto<u>in</u>hibitory) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Tasaki1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xie1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>. Three missense mutations in patients #1-3 of the present work are indicated as well (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B, C</a>). (B) Ribbon diagram of the <i>S. cerevisiae</i> UBR domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Choi1" target="_blank">[48]</a> in a complex with RLGES, the N-terminal region of the separase-produced fragment of Scc1, a subunit of cohesin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Rao1" target="_blank">[75]</a>. The bound RLGES peptide is shown as a stick model, with carbon atoms colored yellow. Several residues are marked with a black sphere and numbered to facilitate the tracing of the polypeptide chain. The names of residues of the RLGES peptide are in red, with the letter ‘s’ (substrate) appended to their position numbers. Side-chains of residues in the UBR domain that are present near JBS mutations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B, C</a>) are shown in a stick form, with carbon atoms colored green. Three coordinated zinc ions of the UBR domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Choi1" target="_blank">[48]</a> are shown as red spheres. (C) Close-up view of the UBR region near the V146L mutation (patient #1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B</a>). In panel B, this region of UBR is boxed and labeled as ‘C’. The residues of UBR that accommodate the position-2 Leu residue (‘Leu2s’) of the RLGES peptide substrate are shown and labeled. The van der Waals sphere of the mutant Leu residue, in the UBR1^V146L mutant, is shown as purple dots. (D) Close-up view of the UBR region near the H160R mutation (patient #2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Fig. 1B</a>). In panel B, this region of UBR is boxed and labeled as ‘D’. The residues of UBR coordinating Zn3 atom are shown and labeled. The van der Waals sphere of the mutant Arg residue, in the UBR1^H160R mutant, is shown as purple dots. The views in (C) and (D) are oriented to maximize visibility of mutation-proximal residues.</p

The mammalian Arg/N-end rule pathway and missense mutations in human <i>UBR1</i> that underlie specific cases of the Johanson-Blizzard syndrome (JBS).

<p>(A) The mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. ‘Primary’, ‘secondary’ and ‘tertiary’ denote mechanistically distinct subsets of destabilizing N-terminal residues (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s1" target="_blank">Introduction</a>). C* denotes oxidized Cys, either Cys-sulfinate or Cys-sulfonate. MetAPs, Met-aminopeptidases. (B) Single-residue mutations in the UBR1 proteins of JBS patients #1 and #2. The positions of mutant residues are indicated both for the original mutations in human UBR1 and for their mimics in <i>S. cerevisiae</i>. (C) Same as in B but the mutation in UBR1 of patient #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s2" target="_blank">Results</a>).</p