Phosphorylation of a membrane curvature–sensing motif switches function of the HOPS subunit Vps41 in membrane tethering

An AP-3–binding site required for vesicle–vacuole fusion is masked when Vps41 is associated with highly curved membranes, such as endosomes, but is exposed at membranes with lower curvature, such as vacuoles, because of phosphorylation of the membrane-binding motif

Structural Studies of Proteins involved in Nucleotide Metabolism : Studies of a ribonucleotide reductase from A. aeolicus and NUDT15

This thesis is separated into two parts. The first part concerns ribonucleotide reductase from Aquifex aeolicus. A. aeolicus is a hyperthermophilic bacterium that thrives at extremely high temperatures of 80-95 °C. We present the X-ray crystal structures of both the R1 and R2 subunits of this protein, which represents the first structure of a class Ia ribonucleotide reductase from a hyperthermophile and the first structure of an R1 from the NrdAh phylogenetic subclass. Several novel features were seen in the R1 structure such as the simultaneous binding of two ATP molecules in the ATP-cone domain as well as a novel “β-hairpin hook” feature which spans the dimer interface of the R1 protein. The gene encoding the R2 protein contains a self-cleaving intein domain. We examined two constructs of this protein, one with the sequence of the intein removed at the DNA level and the wild-type construct. Both crystal structures were found to be identical, showing the efficient cleavage of the intein domain in the wild-type construct.  The second part of this thesis concerns the NUDIX hydrolase NUDT15. The physiological function of NUDT15 is still unknown, however certain mutations in this gene are associated with thiopurine intolerance in patients. Thiopurines are chemotherapeutic drugs used in the treatment of diseases such as acute lymphoblastic leukemia, the most common type of childhood leukemia, and inflammatory bowel disease. Thiopurine drugs are converted by the cell to the active metabolite 6-thio-dGTP which can then act as a substrate for DNA polymerase. Incorporation of these anti-metabolites into DNA produces the desired cytotoxic effects. We show that NUDT15 breaks down the active metabolites of these drugs which leads to a lowered effective dose. The absence of a functioning NUDT15 protein in patients that have inactivating mutations in the gene coding for NUDT15 results in a drastically increased effective dose of these compounds. A normal dose of a thiopurine drug can lead to severe and possibly life-threatening complications in these patients. The role of NUDT15 in thiopurine metabolism is established by in vitro and cellular data as well as the X-ray crystal structure of NUDT15 in complex with 6-thio-GMP. Acyclovir and ganciclovir are two antiviral drugs whose mechanism of action is similar to that of thiopurines. These drugs are also metabolized to their tri-phosphorylated forms and are then preferentially incorporated into viral DNA. Here again, we use in vitro, cellular and structural data to show that NUDT15 breaks down the active metabolites of these drugs. Two separate and structurally distinct lines of potent inhibitors for NUDT15 were developed with support of crystallographic studies. We show that cells are sensitized to both thiopurine and antiviral treatments in the presence of these inhibitors. Binding of our inhibitors to NUDT15 provided substantial thermal stabilization. The stabilizing effect of inhibitor binding enabled us to solve structures of the four most clinically relevant NUDT15 variants, thus elucidating the structural basis for the thiopurine sensitivity phenotype

Structural and Biochemical Investigation of Class i Ribonucleotide Reductase from the Hyperthermophile Aquifex aeolicus

Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found in nearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π-stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments

Smart Checkpots – Proof of concept for a mobile pest and climate monitoring system in greenhouses

In the research project "Smart Checkpots - Optimized Plant Protection for Ornamental Plant Production", an automated, mobile monitoring system for pest infestation and beneficial insects as well as for temperature and humidity measurement is being developed. The system consists of “swarms” (representing a variable number) of networked Checkpots placed like “artificial plants” directly in the crop during the entire production process. Each Checkpot carries a yellow sticky card, a camera and a climate sensor and records an image of the yellow sticky card along with the current temperature and relative humidity at a selectable interval. These data are used for visualization of pest occurrence and fungal infestation risk as well as input parameters for decision support models for plant protection measures. An integrated radiometric recording of the Checkpot positions, based on Bluetooth Low energy 5.1 technology, ensures the permanent allocation of the collected data to the monitored crop sets, and enables a set-accurate decision on measures. This conference paper presents the results of the implementation and review of this concept. It is focused on the accuracy of position data acquisition during a three-hour parallel operation of three Checkpot prototypes in two horticultural enterprises with errorfree data transmission to a central computer every quarter of an hour. For image and climate data, only the basic feasibility had to be proven. Furthermore, first concepts concerning data and user interfaces were developed

Smart Checkpots - Proof of Concept für ein mobiles Schädlings- und Klimamonitoring im Gewächshaus

In dem Verbundprojekt „Smart Checkpots - Optimierter Pflanzenschutz für die Zierpflanzenproduktion“ wird ein automatisiertes, mobiles Monitoringsystem zum Schädlingsbefall sowie zur Temperatur- und Luftfeuchtemessung entwickelt. Das System besteht aus Schwärmen von vernetzten Checkpots im Bestand, mit denen jeweils täglich ein Bild der mitgeführten Gelbtafel sowie stündlich die Temperatur und rel. Luftfeuchte erfasst werden. Diese Daten dienen zur Visualisierung von Schädlingsaufkommen und Pilzbefallrisiko sowie als Eingangsparameter für Entscheidungshilfemodelle zu Maßnahmen des Pflanzenschutzes. Eine integrierte radiometrische Erfassung der Checkpotpositionen gewährleistet die permanente Zuordnung der erhobenen Daten zu den überwachten Kultursätzen und ermöglicht eine satzgenaue Maßnahmenentscheidung. Dieser Tagungsbeitrag präsentiert den aktuellen Entwicklungsstand nach einem Projektjahr als „Proof of Concept“

NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir

Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

SUMMARY The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria during infection, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c acts after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen

NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir

Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002–2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen