Showing papers by "Bernhard Kuster published in 2022"

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target

Abstract: Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-β-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Linking post-translational modifications and protein turnover by site-resolved protein turnover profiling

Abstract: Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we speculate that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We show examples of how the turnover data may give insights into unknown functions of PTMs and provide a freely accessible online tool that allows interrogation and visualisation of all turnover data. The SPOT methodology is applicable to many cell types and modifications, offering the potential to prioritize PTMs for future functional investigations.

Plant Proteome Dynamics.

Abstract: Proteins are intimately involved in executing and controlling virtually all cellular processes. To understand the molecular mechanisms that underlie plant phenotypes, it is essential to investigate protein expression, interactions, and modifications, to name a few. The proteome is highly dynamic in time and space, and a plethora of protein modifications, protein interactions, and network constellations are at play under specific conditions and developmental stages. Analysis of proteomes aims to characterize the entire protein complement of a particular cell type, tissue, or organism-a challenging task, given the dynamic nature of the proteome. Modern mass spectrometry-based proteomics technology can be used to address this complexity at a system-wide scale by the global identification and quantification of thousands of proteins. In this review, we present current methods and technologies employed in mass spectrometry-based proteomics and provide examples of dynamic changes in the plant proteome elucidated by proteomic approaches. Expected final online publication date for the Annual Review of Plant Biology, Volume 73 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Linking post-translational modifications and protein turnover by site-resolved protein turnover profiling

Abstract: Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we speculate that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We show examples of how the turnover data may give insights into unknown functions of PTMs and provide a freely accessible online tool that allows interrogation and visualisation of all turnover data. The SPOT methodology is applicable to many cell types and modifications, offering the potential to prioritize PTMs for future functional investigations.

Loss of UCP1 function augments recruitment of futile lipid cycling for thermogenesis in murine brown fat

Abstract: Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing.In this study, we explore different potential sources of UCP1-independent thermogenesis and identify a futile ATP-consuming triglyceride/fatty acid cycle as the main contributor to cellular heat production in brown adipocytes lacking UCP1. We uncover the mechanism on a molecular level and pinpoint the key enzymes involved using pharmacological and genetic interference.ATGL is the most important lipase in terms of releasing fatty acids from lipid droplets, while DGAT1 accounts for the majority of fatty acid re-esterification in UCP1-ablated brown adipocytes. Furthermore, we demonstrate that chronic cold exposure causes a pronounced remodeling of adipose tissues and leads to the recruitment of lipid cycling capacity specifically in BAT of UCP1-knockout mice, possibly fueled by fatty acids from white fat. Quantification of triglyceride/fatty acid cycling clearly shows that UCP1-ablated animals significantly increase turnover rates at room temperature and below.Our results suggest an important role for futile lipid cycling in adaptive thermogenesis and total energy expenditure.

Lactobacillus supports Clostridiales to restrict gut colonization by multidrug-resistant Enterobacteriaceae

Abstract: Infections by multidrug-resistant Enterobacteriaceae (MRE) are life-threatening to patients. The intestinal microbiome protects against MRE colonization, but antibiotics cause collateral damage to commensals and open the way to colonization and subsequent infection. Despite the significance of this problem, the specific commensals and mechanisms that restrict MRE colonization remain largely unknown. Here, by performing a multi-omic prospective study of hospitalized patients combined with mice experiments, we find that Lactobacillus is key, though not sufficient, to restrict MRE gut colonization. Lactobacillus rhamnosus and murinus increase the levels of Clostridiales bacteria, which induces a hostile environment for MRE growth through increased butyrate levels and reduced nutrient sources. This mechanism of colonization resistance, an interaction between Lactobacillus spp. and Clostridiales involving cooperation between microbiota members, is conserved in mice and patients. These results stress the importance of exploiting microbiome interactions for developing effective probiotics that prevent infections in hospitalized patients.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates

Abstract: Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-β (Aβ) peptides and formation of Aβ deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aβ pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aβ1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aβ deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

On the potential of micro-flow LC-MS/MS in proteomics

Abstract: ABSTRACT Introduction Due to its excellent sensitivity, nano-flow liquid chromatography tandem mass spectrometry (LC-MS/MS) is the mainstay in proteome research; however, this comes at the expense of limited throughput and robustness. In contrast, micro-flow LC-MS/MS enables high-throughput, robustness, quantitative reproducibility, and precision while retaining a moderate degree of sensitivity. Such features make it an attractive technology for a wide range of proteomic applications. In particular, large-scale projects involving the analysis of hundreds to thousands of samples. Areas covered This review summarizes the history of chromatographic separation in discovery proteomics with a focus on micro-flow LC-MS/MS, discusses the current state-of-the-art, highlights advances in column development and instrumentation, and provides guidance on which LC flow best supports different types of proteomic applications. Expert opinion Micro-flow LC-MS/MS will replace nano-flow LC-MS/MS in many proteomic applications, particularly when sample quantities are not limited and sample cohorts are large. Examples include clinical analyses of body fluids, tissues, drug discovery and chemical biology investigations, plus systems biology projects across all kingdoms of life. When combined with rapid and sensitive MS, intelligent data acquisition, and informatics approaches, it will soon become possible to analyze large cohorts of more than 10,000 samples in a comprehensive and fully quantitative fashion.

Prosit-TMT: Deep Learning Boosts Identification of TMT-Labeled Peptides.

Abstract: The prediction of fragment ion intensities and retention time of peptides has gained significant attention over the past few years. However, the progress shown in the accurate prediction of such properties focused primarily on unlabeled peptides. Tandem mass tags (TMT) are chemical peptide labels that are coupled to free amine groups usually after protein digestion to enable the multiplexed analysis of multiple samples in bottom-up mass spectrometry. It is a standard workflow in proteomics ranging from single-cell to high-throughput proteomics. Particularly for TMT, increasing the number of confidently identified spectra is highly desirable as it provides identification and quantification information with every spectrum. Here, we report on the generation of an extensive resource of synthetic TMT-labeled peptides as part of the ProteomeTools project and present the extension of the deep learning model Prosit to accurately predict the retention time and fragment ion intensities of TMT-labeled peptides with high accuracy. Prosit-TMT supports CID and HCD fragmentation and ion trap and Orbitrap mass analyzers in a single model. Reanalysis of published TMT data sets show that this single model extracts substantial additional information. Applying Prosit-TMT, we discovered that the expression of many proteins in human breast milk follows a distinct daily cycle which may prime the newborn for nutritional or environmental cues.

Proteomic profiling in cerebral amyloid angiopathy reveals an overlap with CADASIL highlighting accumulation of HTRA1 and its substrates

Abstract: Cerebral amyloid angiopathy (CAA) is an age-related condition and a major cause of intracerebral hemorrhage and cognitive decline that shows close links with Alzheimer's disease (AD). CAA is characterized by the aggregation of amyloid-β (Aβ) peptides and formation of Aβ deposits in the brain vasculature resulting in a disruption of the angioarchitecture. Capillaries are a critical site of Aβ pathology in CAA type 1 and become dysfunctional during disease progression. Here, applying an advanced protocol for the isolation of parenchymal microvessels from post-mortem brain tissue combined with liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the proteomes of CAA type 1 cases (n = 12) including a patient with hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D), and of AD cases without microvascular amyloid pathology (n = 13) in comparison to neurologically healthy controls (n = 12). ELISA measurements revealed microvascular Aβ1-40 levels to be exclusively enriched in CAA samples (mean: > 3000-fold compared to controls). The proteomic profile of CAA type 1 was characterized by massive enrichment of multiple predominantly secreted proteins and showed significant overlap with the recently reported brain microvascular proteome of patients with cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a hereditary cerebral small vessel disease (SVD) characterized by the aggregation of the Notch3 extracellular domain. We found this overlap to be largely attributable to the accumulation of high-temperature requirement protein A1 (HTRA1), a serine protease with an established role in the brain vasculature, and several of its substrates. Notably, this signature was not present in AD cases. We further show that HTRA1 co-localizes with Aβ deposits in brain capillaries from CAA type 1 patients indicating a pathologic recruitment process. Together, these findings suggest a central role of HTRA1-dependent protein homeostasis in the CAA microvasculature and a molecular connection between multiple types of brain microvascular disease.

Novel, highly potent PROTACs targeting AURORA-A kinase

Abstract: The family of AURORA kinases is essential for cell cycle progression and dysregulation of AURORA-A in cancer led to a large number of clinical and pre-clinical inhibitors. However, ATP competitive AURORA-A inhibitors usually do not target non-catalytic functions that have also been identified as mechanisms promoting tumorigenesis. To target non-catalytic as well as catalytic functions, we developed a series of PROTACs (PROteolysis TArgeting Chimeras) based on the selective AURORA-A kinase inhibitor MK-5108 (VX-689) and the CEREBLON E3-ligase ligand thalidomide. The most potent PROTAC, JB301, had good physicochemical properties and cell penetration resulting in degradation of AURORA-A in leukemic cells at single digit nM concentration.

Epigenetic drug screening defines a PRMT5 inhibitor–sensitive pancreatic cancer subtype

Abstract: Systemic therapies for pancreatic ductal adenocarcinoma (PDAC) remain unsatisfactory. Clinical prognosis is particularly poor for tumor subtypes with activating aberrations in the MYC pathway, creating an urgent need for novel therapeutic targets. To unbiasedly find MYC-associated epigenetic dependencies, we conducted a drug screen in pancreatic cancer cell lines. Here, we found that protein arginine N-methyltransferase 5 (PRMT5) inhibitors triggered an MYC-associated dependency. In human and murine PDACs, a robust connection of MYC and PRMT5 was detected. By the use of gain- and loss-of-function models, we confirmed the increased efficacy of PRMT5 inhibitors in MYC-deregulated PDACs. Although inhibition of PRMT5 was inducing DNA damage and arresting PDAC cells in the G2/M phase of the cell cycle, apoptotic cell death was executed predominantly in cells with high MYC expression. Experiments in primary patient-derived PDAC models demonstrated the existence of a highly PRMT5 inhibitor–sensitive subtype. Our work suggests developing PRMT5 inhibitor–based therapies for PDAC.

Development of selective inhibitors of phosphatidylinositol 3-kinase C2α

Abstract: Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.

Proteogenomic analysis reveals RNA as an important source for tumor-agnostic neoantigen identification correlating with T-cell infiltration

Abstract: Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS- based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates. Statement of significance The significance of this study lies not only in the potential of our optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) for clinical application, but sheds light on the entity-agnostic prevalence of HLA class I peptide presentation of RNA processing events to be used for tumor targeting.

The OTUD6B‐LIN28B‐MYC axis determines the proliferative state in multiple myeloma

Abstract: Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S‐transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle‐specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S‐phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post‐transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS‐multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B‐LIN28B axis.

Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes.

Abstract: LIMKs are important regulators of actin and microtubule dynamics, and they play essential roles in many cellular processes. Deregulation of LIMKs has been linked to the development of diverse diseases, including cancers and cognitive disabilities, but well-characterized inhibitors known as chemical probes are still lacking. Here, we report the characterization of three highly selective LIMK1/2 inhibitors covering all canonical binding modes (type I/II/III) and the structure-based design of the type II/III inhibitors. Characterization of these chemical probes revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed excellent selectivity in a comprehensive scanMAX kinase selectivity panel. Phosphoproteomics revealed remarkable differences between type I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models of fragile X-chromosome, 15 showed promising activity, suggesting the potential application of allosteric LIMK inhibitors treating this orphan disease.

Merits of Diazirine Photo-Immobilization for Target Profiling of Natural Products and Cofactors.

Abstract: Finding the targets of natural products is of key importance in both chemical biology and drug discovery, and deconvolution of cofactor interactomes contributes to the functional annotation of the proteome. Identifying the proteins that underlie natural compound activity in phenotypic screens helps to validate the respective targets and, potentially, expand the druggable proteome. Here, we present a generally applicable protocol for the photoactivated immobilization of unmodified and microgram quantities of natural products on diazirine-decorated beads and their use for systematic affinity-based proteome profiling. We show that among 31 molecules of very diverse reported activity and biosynthetic origin, 25 could indeed be immobilized. Dose-response competition binding experiments using lysates of human or bacterial cells followed by quantitative mass spectrometry recapitulated targets of 9 molecules with <100 μM affinity. Among them, immobilization of coenzyme A produced a tool to interrogate proteins containing a HotDog domain. Surprisingly, immobilization of the cofactor flavin adenine dinucleotide (FAD) led to the identification of nanomolar interactions with dozens of RNA-binding proteins.