Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage

Intramembrane metalloproteases regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σ K during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σ K cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired Pro-σ K interaction with SpoIVFB in co-purification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the Proregion of Pro-σ K . Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σ K cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis . We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined interaction of the IMMP SpoIVFB with its substrate Pro-σ K , whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σ K binding and cleavage. Corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

Phosphatidylinositol-4,5-Bisphosphate Binding to Amphiphysin-II Modulates T-Tubule Remodeling: Implications for Heart Failure

BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function.

Upscaling of Permanent Magnet Dismantling and Recycling through VALOMAG Project

Neodymium-Iron-Boron (NdFeB) based permanent magnets are indispensable in today’s technology-driven society. Moreover, their use is likely to increase since they are key in clean energy applications such as wind turbines, hybrid/electric vehicles, and electric bikes. They contain critical raw materials as rare earth elements are used. Indeed, permanent magnets are considered strategic materials by the EU, and their recycling represents a potential secondary supply to decrease the import dependence. The VALOMAG project is developing a technical solution to recover rare earth (RE) based permanent magnets by dismantling end-of-life (EoL) products such as computer hard disc drives, electric motors, and generators from electric vehicles and wind turbines. It also assesses two short loop recycling technologies: Hydrogen Decrepitation (HD) or Hydrogenation–Disproportionation–Desorption–Recombination (HDDR) and strip-casting for high and medium quality magnet wastes; and hydrometallurgical processes for EoL low-quality magnets. Moreover, Life Cycle Assessment (LCA) and Process Integration with a Flowsheet simulation tool will integrate the whole recycling value chain (collection, dismantling, physical and chemical treatment options, and re-manufacturing) and assess the environmental impact and processes efficiency. A market study on the types and expected future quantities for the scrap magnets and the characterisation of the EoL magnets from hard disc drives (HDD) will be presented as preliminary results. Pre-treatment and sorting of 2.5 tons of NdFeB magnets scraps were carried out, and the two short loop recycling routes and the hydrometallurgical route are under investigation at the lab and pilot scale. The results will be used to develop a process integration and to assess the three routes through LCA.

Systematic Characterization of MicroRNA Processing Modes in Plants With Parallel Amplification of RNA Ends

In plants, the RNase III-type enzyme Dicer-like 1 (DCL1) processes most microRNAs (miRNAs) from their primary transcripts called pri-miRNAs. Four distinct processing modes (i.e., short base to loop, sequential base to loop, short loop to base, and sequential loop to base) have been characterized in Arabidopsis, mainly by the Specific Parallel Amplification of RNA Ends (SPARE) approach. However, SPARE is a targeted cloning method which requires optimization of cloning efficiency and specificity for each target. PARE (Parallel Amplification of RNA Ends) is an untargeted method per se and is widely used to identify miRNA mediated target slicing events. A major concern with PARE in characterizing miRNA processing modes is the potential contamination of mature miRNAs. Here, we provide a method to estimate miRNA contamination levels and showed that most publicly available PARE libraries have negligible miRNA contamination. Both the numbers and processing modes detected by PARE were similar to those identified by SPARE in Arabidopsis. PARE also determined the processing modes of 36 Arabidopsis miRNAs that were unexplored by SPARE, suggesting that it can complement the SPARE approach. Using publicly available PARE datasets, we identified the processing modes of 36, 91, 90, and 54 miRNAs in maize, rice, soybean, and tomato, respectively, and demonstrated that the processing mode was conserved overall within each miRNA family. Through its power of tracking miRNA processing remnants, PARE also facilitated miRNA characterization and annotation.

TP1.2.7The stuck colonoscope

Background With the increased awareness and push for earlier diagnosis of colorectal cancer, the number of patients undergoing colonoscopy is increasing. Being a common condition, a number of these patients will have herniae. We describe a rare complication during a Sigmoidoscopy in a patient with an Inguinal hernia. The case A 75-year-old man was booked for a flexible sigmoidoscopy for rectal bleeding. The scope was successfully inserted to the Splenic flexure. During withdrawal, the scope stopped moving. It was still possible to advance the scope, but not to withdraw. The patient confirmed the presence of a left inguinal hernia. Physical examination and the position on magnetic scope imager confirmed the loop in the hernia. Various manoeuvres to withdraw the scope were unsuccessful. A colleague was called for a second opinion. We came up with a plan to maintain a ‘long loop‘ position in the hernia, by holding the scope through the scrotum and allowing it to slide on withdrawal. This was successful and the patient was discharged. Discussion Incarceration of the scope in the hernia occurs when the hernial defect permits entry and exit of the scope, leaving a loop in the hernia, in a long loop position. During withdrawal, the configuration changes to a short loop, crowding the hernial defect, preventing the scope from sliding. On searching literature, we found that this technique has been described by Koltun et al and is known as the “Pulley” technique. We suggest that colonoscopists are familiar with this technique.

Genome-wide analysis of DNA G-quadruplex motifs across 37 species provides insights into G4 evolution

G-quadruplex (G4) structures have been predicted in the genomes of many organisms and proven to play regulatory roles in diverse cellular activities. However, there is little information on the evolutionary history and distribution characteristics of G4s. Here, whole-genome characteristics of potential G4s were studied in 37 evolutionarily representative species. During evolution, the number, length, and density of G4s generally increased. Immunofluorescence in seven species confirmed G4s’ presence and evolutionary pattern. G4s tended to cluster in chromosomes and were enriched in genetic regions. Short-loop G4s were conserved in most species, while loop-length diversity also existed, especially in mammals. The proportion of G4-bearing genes and orthologue genes, which appeared to be increasingly enriched in transcription factors, gradually increased. The antagonistic relationship between G4s and DNA methylation sites was detected. These findings imply that organisms may have evolutionarily developed G4 into a novel reversible and elaborate transcriptional regulatory mechanism benefiting multiple physiological activities of higher organisms.

Short loop functional commonality identified in leukaemia proteome highlights crucial protein sub-networks

Direct drug targeting of mutated proteins in cancer is not always possible and efficacy can be nullified by compensating protein–protein interactions (PPIs). Here, we establish an in silico pipeline to identify specific PPI sub-networks containing mutated proteins as potential targets, which we apply to mutation data of four different leukaemias. Our method is based on extracting cyclic interactions of a small number of proteins topologically and functionally linked in the Protein–Protein Interaction Network (PPIN), which we call short loop network motifs (SLM). We uncover a new property of PPINs named ‘short loop commonality’ to measure indirect PPIs occurring via common SLM interactions. This detects ‘modules’ of PPI networks enriched with annotated biological functions of proteins containing mutation hotspots, exemplified by FLT3 and other receptor tyrosine kinase proteins. We further identify functional dependency or mutual exclusivity of short loop commonality pairs in large-scale cellular CRISPR–Cas9 knockout screening data. Our pipeline provides a new strategy for identifying new therapeutic targets for drug discovery.