Enzymatic characterization of protein lysine methyltransferases

Histone lysine methylation is an epigenetic mechanism that is involved in the regulation of many biological processes. Over the last decade, the global interest in protein lysine methylation events increased drastically, because several protein lysine methyltransferases (PKMTs) and lysine methylation sites were identified in the genomes and proteomes of many organisms also including non-histone proteins functioning as substrates for PKMTs. The fast development of this field has moved the understanding of the biological outcome of lysine methylation into the center of research. Most urgently, it is necessary to improve our knowledge about lysine methylation by connecting specific target sites with the responsible PKMT and identifying the full substrate spectrum of PKMTs. In this thesis substrate specificity analysis was performed to tackle this challenge. It was shown that methylation of substrate specificity arrays is a good approach to analyze the substrate preference of PKMTs and identify subtle differences between related enzymes with same overall specificity. Furthermore, substrate specificity analysis was shown to be useful for the identification of novel substrates, which was successfully demonstrated for SUV4-20H1, SUV4-20H2, MLL1 and MLL3 in the present study. In vitro methylation experiments indicated that SUV4-20H1 and SUV4-20H2 introduce dimethylation on H4K20 using monomethylated H4K20 as substrate. SUV4-20H1 and SUV4-20H2 have an overlapping sequence motif, but SUV4-20H2 is less specific. This result was supported by the identification of one novel non-histone substrate for SUV4-20H1 and three non-histone targets for SUV4-20H2. MLL1 and MLL3 are H3K4 methyltransferases, but they belong to different MLL subfamilies. MLL1 catalyzes H3K4 trimethylation at promotors of developmental genes, whereas MLL3 introduces H3K4 monomethylation at enhancers. MLL1 and MLL3 are parts of related multi protein complexes also containing WDR5, RBBP5 and ASH2L. Substrate specificity analysis of MLL1 showed that it accepts several other residues at many positions of the target sequence, in addition to the residues in the original sequences of H3. At the protein level two novel substrates (TICRR and ZNF862) were methylated by MLL1. Comparison of the relative activity showed that the H3 protein was the best target in the absence of complex partners, but ZNF862 was preferred in presence of WRA. Finally, my data indicate that the substrate specificity of MLL3-WRA differed slightly from MLL1, suggesting that they may have different non-histone substrates. In several publications, assignments between PKMTs and methylated histone or non-histone target sites have been reported, but in some cases the data are questionable. This could lead to wrong interpretation of biological processes and misleading of follow-up studies. It has been shown for two examples in this study, that substrate specificity analysis can be used to identify problematic assignments between PKMT and methylation events, which need to be studied experimentally to confirm the published findings. Vougiouklakis et al. (2015) reported that SUV4-20H1 methylates ERK1 at K302 and K361, but these target sites do not fit to the specificity profile of SUV4-20H1. Indeed, I could not detect methylation of ERK1 by SUV4-20H1 or SUV4-20H2 at peptide and protein level although positive controls showed the expected methylation. Dhami et al. (2013) reported that Numb protein is methylated by SET8 at K158 and K163, which was not in agreement with the specificity data of SET8. In this thesis, Numb peptide and protein methylation was studied using recombinant SET8 purified from E.coli or HEK293 cells. In both cases, no methylation of Numb could be observed. These data suggest that these assignments of methylation substrates and PKMTs are likely not correct. Whole genome and whole transcriptome sequencing projects have frequently found somatic mutations in epigenetic enzymes in cancers. Somatic cancer mutations can have loss-of-function or gain-of-function effects on the enzymatic properties of PKMTs. Especially gain-of-function effects are a challenge in understanding their role in carcinogenesis. In this study, the effects of somatic cancer mutations found in the SET domain of MLL1 and MLL3 were analyzed. Four somatic cancer mutations of MLL1 and three of MLL3 were selected for analysis on the basis of their location close to binding sites of AdoMet, peptide or the interaction partners. The investigation of somatic cancer mutations in MLL1 and MLL3 indicated that each specific mutation has its unique effect on the enzymatic activity, product or substrate specificity and principle regulatory mechanism indicating that each mutant needs specific in depth experimental investigation in order to understand its carcinogenic effect. Moreover, inhibitor studies demonstrated that each mutant needs to be experimentally studied to allow for the development of mutation specific therapeutic strategies.Die Lysin-Methylierung von Histonen ist ein epigenetischer Mechanismus, der an der Regulation vieler biologischer Prozesse beteiligt ist. In den letzten Jahren hat das Interesse an der Lysin-Methylierung von Proteinen deutlich zugenommen, da eine Vielzahl von Protein-Lysin-Methyltransferasen (PKMTs) und Lysin-Methylierungsstellen in den Genomen und Proteomen von verschiedenen Organismen identifiziert wurden. Zusätzlich wurden auch Nicht-Histon-Proteine als mögliche Substrate für PKMTs entdeckt. Diese rasante Entwicklung hat die Lysin-Methylierung und deren Verständnis in den Mittelpunkt der Forschung gerückt. Um unser Wissen über die Lysin-Methylierung weiter zu verbessern, ist es wichtig die spezifischen Lysin Methylierungsstellen mit den verantwortlichen PKMTs in Verbindung zu bringen und das gesamte Substrat-Spektrum von PKMTs zu untersuchen. In dieser Arbeit wurde eine Substratspezifitäts-Analyse durchgeführt, um dieser Herausforderung gerecht zu werden. Es wurde gezeigt, dass die Methylierung von Peptidarrays ein guter Ansatz ist, um die Substratpräferenz von PKMTs zu analysieren und um geringfügige Unterschiede zwischen verwandten Enzymen mit der gleichen Gesamtspezifität zu identifizieren. Des Weiteren wurde gezeigt, dass die Substratspezifitäts-Analyse für die Identifizierung neuer Substrate von Nutzen ist, was in der vorliegenden Arbeit für SUV4-20H1, SUV4-20H2, MLL1 und MLL3 erfolgreich nachgewiesen wurde. In vitro Methylierungsexperimente haben gezeigt, dass SUV4-20H1 und SUV4-20H2 unter der Verwendung von monomethyliertem H4K20 als Substrat eine Dimethylierung von H4K20 katalysieren. SUV4-20H1 und SUV4-20H2 haben ein überlappendes Sequenzmotiv, wobei SUV4-20H2 eine geringere Spezifität aufweist. Dieses Ergebnis wurde durch die Identifizierung eines neuartigen Nicht-Histon-Substrats von SUV4-20H1 und von drei Nicht-Histon-Substraten für SUV4-20H2 bestätigt. MLL1 und MLL3 sind H3K4-Methyltransferasen, aber sie gehören zu verschiedenen MLL-Unterfamilien. MLL1 katalysiert die H3K4-Trimethylierung an Promotoren von Entwicklungsgenen, während MLL3 die Monomethylierung von H3K4 an Enhancern katalysiert. MLL1 und MLL3 sind beide Teil eines Multiproteinkomplexes, der unter anderem aus WDR5, RBBP5 und ASH2L besteht. Die Substratspezifitäts-Analyse von MLL1 hat gezeigt, dass MLL1 neben den Aminosäuren der ursprünglichen Sequenzen von H3 auch mehrere andere Aminosäuren an zahlreichen Stellen der Zielsequenz toleriert. Auf Proteinebene wurden zwei neue Substrate (TICRR und ZNF862) von MLL1 methyliert. Der Vergleich der relativen Aktivität hat gezeigt, dass das H3 Protein das bevorzugte Substrat in Abwesenheit der Komplexpartner war, wobei ZNF862 in Gegenwart von WRA bevorzugt wurde. Letztendlich haben die Ergebnisse gezeigt, dass sich die Substratspezifität von MLL3-WRA geringfügig von MLL1 unterscheidet, was darauf hindeutet, dass MLL3-WRA und MLL1 unterschiedliche Nicht-Histon-Substrate erkennen können. In mehreren Publikationen wurde über die Zuordung von PKMTs und methylierte Histon- und Nicht-Histon-Substratproteine berichtet, deren Ergebnisse jedoch fragwürdig sind. Dies könnte zu einer falschen Interpretation biologischer Prozesse und zur Irreführung von Folgeuntersuchungen führen. In dieser Arbeit wurde anhand von zwei Beispielen gezeigt, dass die Substratspezifitäts-Analyse verwendet werden kann, um problematische Zuordnungen zwischen PKMT und Methylierungsereignis zu identifizieren, welche experimentell untersucht werden müssen, um die veröffentlichten Ergebnisse zu überprüfen. Vougiouklakis und Mitarbeiter berichteten 2015, dass K302 und K361 von ERK1 durch SUV4-20H1 methyliert würden. Allerdings passen diese Methylierungsstellen nicht zum Spezifitätsprofil von SUV4-20H1. Tatsächlich konnte keine Methylierung von ERK1 durch SUV4-20H1 oder SUV4-20H2 auf Peptid- und Protein-Ebene nachgewiesen werden, obwohl Positivkontrollen die erwartete Methylierung aufweisen. Die Arbeitsgruppe um Dhami berichtete 2013, dass das Numb Protein durch SET8 an K158 und K163 methyliert wird, was wiederum nicht mit den Spezifitätsergebnissen von SET8 übereinstimmt. In dieser Arbeit wurde die Methylierung von Numb auf Peptid- und Proteinebene unter der Verwendung von rekombinantem SET8 (aufgereinigt aus E.coli oder HEK293-Zellen) untersucht. In beiden Fällen konnte keine Methylierung von Numb beobachtet werden. Diese Ergebnisse deuten darauf hin, dass diese Zuordnungen zwischen Methylierungssubstraten und PKMTs wahrscheinlich nicht richtig sind. In Genom- und Transkriptom Sequenzierungsprojekten wurden häufig auftretende somatische Mutationen in epigenetischen Enzymen gefunden, die zur Krebsentwicklung führen. Somatische Krebsmutationen können „loss-of-function“ oder „gain-of-function“ Effekte auf die enzymatischen Eigenschaften von PKMTs haben. Insbesondere die „gain-of-function“ Effekte stellen für das Verständnis ihrer Rolle bei der Krebsentstehung eine Herausforderung dar. In dieser Arbeit wurden die Effekte von somatischen Krebsmutationen analysiert, die in der SET-Domäne von MLL1 und MLL3 gefunden wurden. Vier somatische Krebsmutationen von MLL1 und drei von MLL3 wurden aufgrund ihrer Position in der Nähe der Bindungsstellen von AdoMet, Peptid oder den Interaktionspartnern ausgewählt. Die Untersuchung der somatischen Krebsmutationen in MLL1 und MLL3 hat gezeigt, dass jede spezifische Mutation einen speziellen Effekt auf die enzymatische Aktivität, die Produkt- oder Substratspezifität und den prinzipiellen regulatorischen Mechanismus hat. Das bedeutet, dass für jede Mutante spezifische und detaillierte experimentelle Untersuchungen erforderlich sind, um ihre karzinogene Wirkung zu verstehen. Darüber hinaus haben Inhibitorstudien gezeigt, dass jede Mutante experimentell untersucht werden muss, um die Entwicklung von mutationsspezifischen therapeutischen Strategien zu ermöglichen

The Ursinus Weekly, October 11, 1948

Bishop Corson to speak at exercises honoring college\u27s 79th academic year • Weekly staff sees numerous changes • Haverford trounces bears 26-12 as Ted Test scores four touchdowns, kicks extra point • Alterations cause campus new look • MacQueen elected to council office • Warner-Haines is chosen to play for old timers • Dr. McClure gives address at opening chapel service • IRC names representatives for mid-Atlantic parley • Joe Bechtle views University of Tulsa • Frosh beat sophs in battle of brawn • Spanish Club plans year • Young to receive top football award • MacWilliams heads coed hockey team • Mules to be foe of soccer team • Grizzlies to tackle Dickinson Saturday • Y conducts rally; retreat to be held • Faculty promotes fivehttps://digitalcommons.ursinus.edu/weekly/1597/thumbnail.jp

Somatic cancer mutations in the MLL1 histone methyltransferase modulate its enzymatic activity and dependence on the WDR5/RBBP5/ASH2L complex

Somatic missense mutations in the mixed lineage leukemia 1 (MLL1) histone H3K4 methyltransferase are often observed in cancers. MLL1 forms a complex with WDR5, RBBP5, and ASH2L (WRA) which stimulates its activity. The MM-102 compound prevents the interaction between MLL1 and WDR5 and functions as an MLL1 inhibitor. We have studied the effects of four cancer mutations in the catalytic SET domain of MLL1 on the enzymatic activity of MLL1 and MLL1–WRA complexes. In addition, we studied the interaction of the MLL1 mutants with the WRA proteins and inhibition of MLL1–WRA complexes by MM-102. All four investigated mutations had strong effects on the activity of MLL1. R3903H was inactive and S3865F showed reduced activity both alone and in complex with WRA, but its activity was stimulated by the WRA complex. By contrast, R3864C and R3841W were both more active than wild-type MLL1, but still less active than the wild-type MLL1–WRA complex. Both mutants were not stimulated by complex formation with WRA, although no differences in the interaction with the complex proteins were observed. These results indicate that both mutants are in an active conformation even in the absence of the WRA complex and their normal control of activity by the WRA complex is altered. In agreement with this observation, the activity of R3864C and R3841W was not reduced by addition of the MM-102 inhibitor. We show that different cancer mutations in MLL1 lead to a loss or increase in activity, illustrating the complex and tumor-specific role of MLL1 in carcinogenesis. Our data exemplify that biochemical investigations of somatic tumor mutations are required to decipher their pathological role. Moreover, our data indicate that MM-102 may not be used as an MLL1 inhibitor if the R3864C and R3841W mutations are present. More generally, the efficacy of any enzyme inhibitor must be experimentally confirmed for mutant enzymes before an application can be considered

Somatic cancer mutations in the MLL3-SET domain alter the catalytic properties of the enzyme

BACKGROUND: Somatic mutations in epigenetic enzymes are frequently found in cancer tissues. The MLL3 H3K4-specific protein lysine monomethyltransferase is an important epigenetic enzyme, and it is among the most recurrently mutated enzymes in cancers. MLL3 mainly introduces H3K4me1 at enhancers. RESULTS: We investigated the enzymatic properties of MLL3 variants that carry somatic cancer mutations. Asn4848 is located at the cofactor binding sites, and the N4848S exchange renders the enzyme inactive. Tyr4884 is part of an aromatic pocket at the active center of the enzyme, and Y4884C converts MLL3 from a monomethyltransferase with substrate preference for H3K4me0 to a trimethyltransferase with H3K4me1 as preferred substrate. Expression of Y4884C leads to aberrant H3K4me3 formation in cells. CONCLUSIONS: Our data show that different somatic cancer mutations of MLL3 affect the enzyme activity in distinct and opposing manner highlighting the importance of experimentally studying the effects of somatic cancer mutations in key regulatory enzymes in order to develop and apply targeted tumor therapy

Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K_d of 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with a K_d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells

Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K_d of 16 μM, whereas the otherwise identical Nt-Pro–bearing sequence PGLW binds to GID4 more tightly, with a K_d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW–bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells

The Ursinus Weekly, February 14, 1949

Enrollment hits 1009 as 28 new students begin college life • Richardson Dilworth to speak tonight: former mayoralty nominee to expose corruption in Philadelphia government • Floy Lewis named queen • Frats begin rushing week • Lauterbach outlines proposed US policy in mid-week forum • Frosh girls slated to receive colors at annual program • Local NSA leaders choose Philadelphia for convention site • How could your college life be improved? • B-listers take lead by tripping imbeciles • Retreat held by Y; letters to Congress sent by commission • Student life of faculty members uncovered in old yearbooks • Popular couple wins laurels in print for countless services to student body • Ingber paces Cadets as bears yield 59-40 • Coeds score second win as Rosemont bows 34-23 • Beaver meet looms as local mermaids improve technique • Unbeaten record slashed as bruinettes lose 32-24 • Initial match fatal as Ford grapplers wallop bruins 23-11 • Bears drop fifth tilt as Drexel wins 75-61 • Jay Vees drop two; one point decides as PMC wins 45-44 • Pettit tops offense as junior varsity captures two wins • Three tilts listed for opening night of inter-dorm loophttps://digitalcommons.ursinus.edu/weekly/1607/thumbnail.jp

The Ursinus Weekly, October 18, 1948

Sophs apparently foiled as freshmen prexy, Lee Trimble, is safely hidden • WAA introduces \u2752 to Ursinus sports • Dale White elected editor of Lantern as Wentzel resigns • Football, fun, light fantastic promise successful old timers\u27 day celebration • Forum to feature election discussion • Grads get degrees on Founders\u27 Day • Five men appointed to act with faculty committees • Dressner, Buchanan picked as council representatives • German club plans dinner; to make Philadelphia trip • Thespians greet applicants at first meeting of year • Former student to return in concert with soprano • Frosh show ends customs for men • NSA head requests college democracy • A happy thought for hapless frosh • Freshman reviews first two weeks • Frosh live again after customs end • Soph ruler reveals innermost thoughts • Junior looks back on freshman year • Frosh views hist.1 with heavy heart • Modern miss visits ancient Latin lands • Subs work all year but get no credit • Dickinson romps to 24-0 victory over bear; Gerry Miller features with 85 yard runback • Bears seek victory on old timers\u27 day • Coeds triumph 5-2 in season\u27s opener • Mules trip bruins in soccer opener • Church colleges hit by Lafayette prexy • Footlighters start ambitious season • Staiger writes article for organic chemistry journal • Pre-meds plan activities; members need high gradeshttps://digitalcommons.ursinus.edu/weekly/1598/thumbnail.jp

IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis–associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the subnuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions