Discovery of a potent deubiquitinase (DUB) small molecule activity‐based probe enables broad spectrum DUB activity profiling in living cells

Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates. This leads to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes

4′-Phosphopantetheine and long acyl chain-dependent interactions are integral to human mitochondrial acyl carrier protein function

The mitochondrial acyl carrier protein (human ACPM, yeast Acp1) is an essential mitochondrial protein. Through binding of nascent acyl chains on the serine (S112)-bound 4′-phosphopantetheine (4′-PP) cofactor, ACPM is involved in mitochondrial fatty acid synthesis and lipoic acid biogenesis. Recently, yeast Acp1 was found to interact with several mitochondrial complexes, including the iron–sulfur (Fe–S) cluster biosynthesis and respiratory complexes, via the binding to LYRM proteins, a family of proteins involved in assembly/stability of complexes. Importantly, the interaction of LYRM proteins with Acp1 was shown to be essential in maintaining integrity of mitochondrial complexes. In human, recent structures show that ACPM binding to LYRM proteins involves acyl chains attached to the 4′-PP cofactor. Here, we performed an detailed characterization of the mitochondrial interactome of human ACPM by mass spectrometry (MS) and demonstrate the crucial role of the 4′-PP cofactor in most of ACPM interactions. Specifically, we show that ACPM interacts with endogenous Fe–S cluster complex components through binding of the LYRM protein ISD11/LYRM4. Using knockdown experiments, we further determine that ACPM is essential for the stability of mitochondrial respiratory complexes I, II and III, as well as the Fe–S cluster biosynthesis complex. Finally, using native MS and a top-down MS approach, we show that C14, C16 and C18 3-keto-acyl chains on ACPM are implicated in binding to ISD11 through analysis of the recombinant ACPM–ISD11 complex. Taken together, our data provide novel understanding of the role of 4′-PP- and long acyl chains-dependent interactions in human ACPM function

4′-Phosphopantetheine and long acyl chain-dependent interactions are integral to human mitochondrial acyl carrier protein function

The mitochondrial acyl carrier protein (human ACPM, yeast Acp1) is an essential mitochondrial protein. Through binding of nascent acyl chains on the serine (S112)-bound 4′-phosphopantetheine (4′-PP) cofactor, ACPM is involved in mitochondrial fatty acid synthesis and lipoic acid biogenesis. Recently, yeast Acp1 was found to interact with several mitochondrial complexes, including the iron–sulfur (Fe–S) cluster biosynthesis and respiratory complexes, via the binding to LYRM proteins, a family of proteins involved in assembly/stability of complexes. Importantly, the interaction of LYRM proteins with Acp1 was shown to be essential in maintaining integrity of mitochondrial complexes. In human, recent structures show that ACPM binding to LYRM proteins involves acyl chains attached to the 4′-PP cofactor. Here, we performed an detailed characterization of the mitochondrial interactome of human ACPM by mass spectrometry (MS) and demonstrate the crucial role of the 4′-PP cofactor in most of ACPM interactions. Specifically, we show that ACPM interacts with endogenous Fe–S cluster complex components through binding of the LYRM protein ISD11/LYRM4. Using knockdown experiments, we further determine that ACPM is essential for the stability of mitochondrial respiratory complexes I, II and III, as well as the Fe–S cluster biosynthesis complex. Finally, using native MS and a top-down MS approach, we show that C14, C16 and C18 3-keto-acyl chains on ACPM are implicated in binding to ISD11 through analysis of the recombinant ACPM–ISD11 complex. Taken together, our data provide novel understanding of the role of 4′-PP- and long acyl chains-dependent interactions in human ACPM function

Možnosti a způsoby využití kalů a sedimentů z ČOV

Stav nakládání s čistírenskými kaly v ČR - aktualizace za období 2002-2004(5) s vazbou na trendy a vývoj legislativy. Vývoj produkce kalů z komunálních ČOV a nakládání s kaly. Charakteristika technického stavu kalového hospodářství v ČR. Hlavní provozně využitelné metody hygienizace kalu. Provozní zkušenosti s přímou aplikací kalů na zemědělskou půdu. Vliv technologie odstranění nutrientů z odpadních vod na produkci kalů. Prognóza vývoje následného využití/odstranění kalu z ČOV. Dosahované složení kalů a limity rizikových látek v kalech ve vztahu k platné legislativě. Nové trendy nakládání s kaly