Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24.

Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or non-specific binding to unintended membrane protein targets. However, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here, we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored the proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs, but also highlight new approaches for documenting off-target drug binding

The Ursinus Weekly, February 27, 1975

Women\u27s open dorms suspended for Friday P.M. • Forum: Savitch reports • Festival of Arts includes music, crafts and dance • Union creates Hell\u27s Half Acre • 1975 interview schedule • Bloodmobile will visit U.C. campus • Football team donates gift to Varsity Club • Focus: Dean Ruth R. Harris; George P. Kinek • Editorial: Time for all or time for none • Can the Flyers repeat? • Overtime jinx • Yes we can in 1975 • Badminton: Good • Hoopla • U.C. grapplers end seasonhttps://digitalcommons.ursinus.edu/weekly/1030/thumbnail.jp

The Ursinus Weekly, January 8, 1976

U.C. German prof. dies • Three named to Board • New Myrin hours official • ProTheatre plans • Excerpts from S.F.A.R.C. minutes • U.C. refund policy • Course changes • Pennsylvania Germans and the Indians • Editorial: Inflation urges increase • Alumni corner: Dr. John H. Westerhoff, III, \u2755 • Big Al: Everyone\u27s favorite • Phils trade for pennant • A.N.S. lists varied programs • Jobs offered abroad • M.A.C. Southern • Too tall Stubits • Ursinus hoopla! • Sixers in \u2776? • Gymnastics • Much more to come in \u2776 • Our other team • Our Bowl reviewhttps://digitalcommons.ursinus.edu/weekly/1049/thumbnail.jp

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6.

JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.Commonwealth Scholarship Commission, U

A preparative mass spectrometer to deposit intact large native protein complexes

Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase, where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information on well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific Q Exactive UHMR mass spectrometer). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential

The Ursinus Weekly, May 1, 1975

S.F.A.R.C. update • Meistersingers: More than music • USGA questionnaire encourages response • New Yorker critic graduation speaker • Medical school entrances • How to succeed debuts tomorrow in Bearpit • Editorial: Disgust: By the students, of the students! • Letters to the editor: Meekness? • Alumni meet • Feminism: Where? • Inexpensive or just plain cheap • Actors comment • Conflict simulation activities • 2 games, 2 losses • Tennis time • Intramurals • Focus: Steve Fisher • Flyers go for cup! • Lacrosse lookout • Requesthttps://digitalcommons.ursinus.edu/weekly/1037/thumbnail.jp

The Ursinus Weekly, February 26, 1976

WRUC is on the air • Dr. Parsons tells of visit to Germany • Election results • Student interns join WPAZ radio staff • Student-Board Comm. meets • Loyalty fund is thriving • Spiropoulos examines J.F.K. assassination • Free Library • Editorial: Food for thought or? • U.C. speakers • Editorial: Energy woes are for real and jobs are at stake • Careers vs. liberal arts • Viewpoint: Mr. Bloom\u27s point is worth repeating • Bearpit action • Seminars for women offered: Women and the law; Women and finance • JV Bears play .500 • Bears do swim! • Our bouncing Bears • Focus: Greg Thren • Spring feverhttps://digitalcommons.ursinus.edu/weekly/1052/thumbnail.jp

A mass spectrometry-based approach to distinguish annular and specific lipid binding to membrane proteins

Membrane proteins engage in a variety of contacts with theirsurrounding lipids, but distinguishing between specifically boundlipids, and non-specific annular interactionsis a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the Presenilin Homologue protease are subject to constant exchange with detergent. Bycontrast,detergent-resistantlipids bound at the dimer interface in the Leucine transportershowdecreased koffrates in molecular dynamics simulations.Turning tothe lipid flippase MurJ, we findthat addition of the natural substrate lipid-II results in the formation of a 1:1 protein-lipid complex, where the lipid cannot be displaced by detergentfromthe highly protected active site.In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution. [Abstract copyright: © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The role of lipids in mechanosensation

Acknowledgements: This work was supported by Wellcome Trust grants WT092552MA (J.H.N. and I.R.B.), Senior Investigator Award WT100209MA (J.H.N.), 093228 (T.K.S.) and 092970 (M.S.P.S.), and Biotechnology and Biological Sciences Research Council grants BB/I019855/1 (M.S.P.S.), BB/H017917/1 (J.H.N. and I.R.B.) and BB/J009784/1 (H.B.). We acknowledge the Diamond Light Source for beam time. I.R.B. is supported as a Leverhulme Emeritus Fellow. J.H.N. is supported as a Royal Society Wolfson Merit Award holder and as a 1000 Talent Scholar at Sichuan University. A.C.E.D. was supported by an Engineering and Physical Sciences Research Council Systems Biology Doctoral Training Centre student fellowship. We thank R. Phillips, A. Lee and S. Conway for helpful discussions.Peer reviewedPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprintPostprin