Posttranslational modification of histone 1.4 by SET7/9 and ARTD1

Proteins are important for the regulation of biochemical processes and are crucial for the structure and function of cells. Proteins are post-translationally often chemically modified, which regulates their function and enzymatic activities. The work presented in this thesis shows that the mono-methyltransferase SET7/9 modifies the linker histone H1.4 only at the C-terminal domain. Several lysine residues (K121, K129, K159, K171, K177 and K192) were identified to be methylated. SET7/9 only modified lysine residues at the terminal position of a given KAK motif. Also, the addition of DNA to the methylation reaction inhibited the modification of H1.4 by SET7/9. Expression analysis of GFP-tagged full length H1.4 or its C-terminal or N- terminal fragments in U2OS cells suggested that all fusion proteins localize to either hetero- or euchromatin in the nucleus. Furthermore, the ADP-ribosyltransferase ARTD1 modified several amino acid residues in the C-terminal domain of H1.4, a process that inhibited SET7/9-dependent methylation. This mutual exclusion of the two modifications suggests that post- translational modification of structurally close amino acids can influence each other and thereby regulate the functionality of proteins. Keywords: H1.4, SET7/9, ARTD

Colder ambient temperatures influence acute onset canine intervertebral disc extrusion.

Canine intervertebral disc disease is one of the most common neurologic conditions in veterinary medicine but the influence of environmental factors thereon has not been fully investigated. Subjectively, there has been the impression of increased admissions of acute spinal cord injuries due to intervertebral disc extrusion during periods of colder temperatures. In the present retrospective study, the different weather conditions (temperature, precipitation, sunshine, humidity, and atmospheric pressure) during the acute onset of clinical signs and a lag period of 3 days prior to the occurrence of symptoms were analyzed. One-hundred-and-one client owned dogs from the meteorological region of the Lake Geneva were presented to two referral centers during the 6-year (2007–2012) study period. Chondrodystrophic dogs represented 65.3% of our population, with the French Bulldog (19.8%) and Dachshund (17.8%) being the most common breeds. Multivariable logistic regression analysis identified an increased occurrence of intervertebral disc disease during colder temperatures. Our results are congruent with those of human studies which have shown that lower ambient temperatures are associated with more pain and increased risk for muscle injuries. The interplay of endogenous (breed, anatomical characteristics) and exogenous (environmental) factors should be addressed in a larger cohort study

Crosstalk between SET7/9-dependent methylation and ARTD1-mediated ADP-ribosylation of histone H1.4

BACKGROUND: Different histone post-translational modifications (PTMs) fine-tune and integrate different cellular signaling pathways at the chromatin level. ADP-ribose modification of histones by cellular ADP-ribosyltransferases such as ARTD1 (PARP1) is one of the many elements of the histone code. All 5 histone proteins were described to be ADP-ribosylated in vitro and in vivo. However, the crosstalk between ADP-ribosylation and other modifications is little understood. RESULTS: In experiments with isolated histones, it was found that ADP-ribosylation of H3 by ARTD1 prevents H3 methylation by SET7/9. However, poly(ADP-ribosyl)ation (PARylation) of histone H3 surprisingly allowed subsequent methylation of H1 by SET7/9. Histone H1 was thus identified as a new target for SET7/9. The SET7/9 methylation sites in H1.4 were pinpointed to the last lysine residues of the six KAK motifs in the C-terminal domain (K121, K129, K159, K171, K177 and K192). Interestingly, H1 and the known SET7/9 target protein H3 competed with each other for SET7/9-dependent methylation. CONCLUSIONS: The results presented here identify H1.4 as a novel SET7/9 target protein, and document an intricate crosstalk between H3 and H1 methylation and PARylation, thus implying substrate competition as a regulatory mechanism. Thereby, these results underline the role of ADP-ribosylation as an element of the histone code

Billroth II procedure for the treatment of spontaneous gastrointestinal perforation in two cats.

Case description: A 9-year-old castrated male domestic shorthair cat (cat 1) and a 10-year-old castrated male Maine Coon cat (cat 2) were presented for recurrent feline lower urinary tract disease after receiving outpatient care from their primary veterinarians. Clinical findings: Physical examination findings for both cats were initially within reference limits. After a short period of hospitalization, both cats developed peritoneal effusion; results of cytologic analysis of a sample of the fluid were consistent with septic peritonitis. During exploratory laparotomy, perforation of the pylorus or proximal portion of the duodenum secondary to ulceration was identified. Treatment and outcome: Both cats underwent partial duodenectomy, partial gastrectomy (pylorectomy), and gastrojejunostomy (Billroth II procedure). The cats recovered from surgery and returned to a normal quality of life; however, each had mild episodes of anorexia but maintained a stable body weight. Cat 2 required additional surgery for trichobezoar removal 7 weeks later but recovered quickly. At 7 months after trichobezoar removal, cat 2 developed intermittent vomiting, but clinicopathologic, abdominal ultrasonographic, and upper gastrointestinal tract endoscopic findings were within reference limits. At 9 (cat 2) and 13 (cat 1) months after the Billroth II procedure, both cats were reported to be in good general health and without gastrointestinal signs. Clinical relevance: In both cats, the Billroth II procedure was technically straightforward and associated with a full recovery and good medium- to long-term quality of life. A Billroth II procedure could be considered for treatment of cats with large mural lesions in the pyloroduodenal region

Non-iatrogenic traumatic isolated bilothorax in a cat

Case summary A 6-month-old spayed female domestic shorthair cat presented for evaluation of suspected bite wounds over the right caudal thorax and left cranial flank. Thoracic radiographs identified a mild right-sided pneumothorax, a small volume of right-sided pleural effusion, with increased soft tissue opacity in the right cranial and middle lung lobes. Abdominal ultrasound identified a very small gall bladder and several small pockets of free peritoneal fluid. Cytological analysis of peritoneal fluid was consistent with a modified transudate. Following initial diagnostic investigations, yellow–orange fluid began to emanate from the right-sided thoracic wound. Biochemical analysis of this fluid was consistent with bile. Exploratory coeliotomy revealed a right-sided radial diaphragmatic tear, with herniation of the quadrate liver lobe and a portion of the gall bladder into the right pleural space. The gall bladder was bi-lobed and avulsion of a single herniated lobe resulted in leakage of bile into the right pleural cavity, without concurrent bile peritonitis (biloabdomen). The cat underwent total cholecystectomy and diaphragmatic defect repair and recovered uneventfully. Relevance and novel information To our knowledge, at the time of writing non-iatrogenic isolated bilothorax without concurrent biloabdomen has not been previously reported in the cat. This case highlights the importance of thorough assessment of cats with seemingly innocuous thoracic bite wounds. Despite the rarity of its occurrence, bilothorax should be considered a differential in cats with pleural effusion, even in the absence of bile peritonitis. We believe that the optimal treatment of cases of bilothorax is multifactorial and should be determined on a case-by-case basis

Light-mediated discovery of surfaceome nanoscale organization and intercellular receptor interaction networks

The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8+ T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.ISSN:2041-172

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed