Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy.

Proteolysis-targeting chimeras (PROTACs) represent a new therapeutic modality involving selectively directing disease-causing proteins for degradation through proteolytic systems. Our ability to exploit targeted protein degradation (TPD) for antibiotic development remains nascent due to our limited understanding of which bacterial proteins are amenable to a TPD strategy. Here, we use a genetic system to model chemically-induced proximity and degradation to screen essential proteins in Mycobacterium smegmatis (Msm), a model for the human pathogen M. tuberculosis (Mtb). By integrating experimental screening of 72 protein candidates and machine learning, we find that drug-induced proximity to the bacterial ClpC1P1P2 proteolytic complex leads to the degradation of many endogenous proteins, especially those with disordered termini. Additionally, TPD of essential Msm proteins inhibits bacterial growth and potentiates the effects of existing antimicrobial compounds. Together, our results provide biological principles to select and evaluate attractive targets for future Mtb PROTAC development, as both standalone antibiotics and potentiators of existing antibiotic efficacy.

Predictomes: A classifier-curated database of AlphaFold-modeled protein-protein interactions.

Protein-protein interactions (PPIs) are ubiquitous in biology, yet a comprehensive structural characterization of the PPIs underlying biochemical processes is lacking. Although AlphaFold-Multimer (AF-M) has the potential to fill this knowledge gap, standard AF-M confidence metrics do not reliably separate relevant PPIs from an abundance of false positive predictions. To address this limitation, we used machine learning on well curated datasets to train a Structure Prediction and Omics informed Classifier called SPOC that shows excellent performance in separating true and false PPIs, including in proteome-wide screens. We applied SPOC to an all-by-all matrix of nearly 300 human genome maintenance proteins, generating ~40,000 predictions that can be viewed at predictomes.org, where users can also score their own predictions with SPOC. High confidence PPIs discovered using our approach suggest novel hypotheses in genome maintenance. Our results provide a framework for interpreting large scale AF-M screens and help lay the foundation for a proteome-wide structural interactome.

Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

In silico protein interaction screening uncovers DONSON's role in replication initiation.

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.

NOP16 is a histone mimetic that regulates Histone H3K27 methylation and gene repression.

Post-translational modifications of histone tails alter chromatin accessibility to regulate gene expression. Some viruses exploit the importance of histone modifications by expressing histone mimetic proteins that contain histone-like sequences to sequester complexes that recognize modified histones. Here we identify an evolutionarily conserved and ubiquitously expressed, endogenous mammalian protein Nucleolar protein 16 (NOP16) that functions as a H3K27 mimic. NOP16 binds to EED in the H3K27 trimethylation PRC2 complex and to the H3K27 demethylase JMJD3. NOP16 knockout selectively globally increases H3K27me3, a heterochromatin mark, without altering methylation of H3K4, H3K9, or H3K36 or acetylation of H3K27. NOP16 is overexpressed and linked to poor prognosis in breast cancer. Depletion of NOP16 in breast cancer cell lines causes cell cycle arrest, decreases cell proliferation and selectively decreases expression of E2F target genes and of genes involved in cell cycle, growth and apoptosis. Conversely, ectopic NOP16 expression in triple negative breast cancer cell lines increases cell proliferation, cell migration and invasivity in vitro and tumor growth in vivo , while NOP16 knockout or knockdown has the opposite effect. Thus, NOP16 is a histone mimic that competes with Histone H3 for H3K27 methylation and demethylation. When it is overexpressed in cancer, it derepresses genes that promote cell cycle progression to augment breast cancer growth.

Sample multiplexing-based targeted pathway proteomics with real-time analytics reveals the impact of genetic variation on protein expression.

Targeted proteomics enables hypothesis-driven research by measuring the cellular expression of protein cohorts related by function, disease, or class after perturbation. Here, we present a pathway-centric approach and an assay builder resource for targeting entire pathways of up to 200 proteins selected from >10,000 expressed proteins to directly measure their abundances, exploiting sample multiplexing to increase throughput by 16-fold. The strategy, termed GoDig, requires only a single-shot LC-MS analysis, ~1 µg combined peptide material, a list of up to 200 proteins, and real-time analytics to trigger simultaneous quantification of up to 16 samples for hundreds of analytes. We apply GoDig to quantify the impact of genetic variation on protein expression in mice fed a high-fat diet. We create several GoDig assays to quantify the expression of multiple protein families (kinases, lipid metabolism- and lipid droplet-associated proteins) across 480 fully-genotyped Diversity Outbred mice, revealing protein quantitative trait loci and establishing potential linkages between specific proteins and lipid homeostasis.

SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.

Asparagine couples mitochondrial respiration to ATF4 activity and tumor growth.

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP, respiration generates biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Here, we show that in addition to depleting intracellular aspartate, electron transport chain (ETC) inhibition depletes aspartate-derived asparagine, increases ATF4 levels, and impairs mTOR complex I (mTORC1) activity. Exogenous asparagine restores proliferation, ATF4 and mTORC1 activities, and mTORC1-dependent nucleotide synthesis in the context of ETC inhibition, suggesting that asparagine communicates active respiration to ATF4 and mTORC1. Finally, we show that combination of the ETC inhibitor metformin, which limits tumor asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumor asparagine consumption, effectively impairs tumor growth in multiple mouse models of cancer. Because environmental asparagine is sufficient to restore tumor growth in the context of respiration impairment, our findings suggest that asparagine synthesis is a fundamental purpose of tumor mitochondrial respiration, which can be harnessed for therapeutic benefit to cancer patients.

GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery.

Canonical Wnt signaling is emerging as a major regulator of endocytosis. Here, we report that Wnt-induced macropinocytosis is regulated through glycogen synthase kinase 3 (GSK3) and the ß-catenin destruction complex. We find that mutation of Axin1, a tumor suppressor and component of the destruction complex, results in the activation of macropinocytosis. Surprisingly, inhibition of GSK3 by lithium chloride (LiCl), CHIR99021, or dominant-negative GSK3 triggers macropinocytosis. GSK3 inhibition causes a rapid increase in acidic endolysosomes that is independent of new protein synthesis. GSK3 inhibition or Axin1 mutation increases lysosomal activity, which can be followed with tracers of active cathepsin D, ß-glucosidase, and ovalbumin degradation. Microinjection of LiCl into the blastula cavity of Xenopus embryos causes a striking increase in dextran macropinocytosis. The effects of GSK3 inhibition on protein degradation in endolysosomes are blocked by the macropinocytosis inhibitors EIPA or IPA-3, suggesting that increases in membrane trafficking drive lysosomal activity.

Publisher Correction: In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer.

Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.

Differential Metabolic Reprogramming by Zika Virus Promotes Cell Death in Human versus Mosquito Cells.

Zika virus is a pathogen that poses serious consequences, including congenital microcephaly. Although many viruses reprogram host cell metabolism, whether Zika virus alters cellular metabolism and the functional consequences of Zika-induced metabolic changes remain unknown. Here, we show that Zika virus infection differentially reprograms glucose metabolism in human versus C6/36 mosquito cells by increasing glucose use in the tricarboxylic acid cycle in human cells versus increasing glucose use in the pentose phosphate pathway in mosquito cells. Infection of human cells selectively depletes nucleotide triphosphate levels, leading to elevated AMP/ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and caspase-mediated cell death. AMPK is also phosphorylated in Zika virus-infected mouse brain. Inhibiting AMPK in human cells decreases Zika virus-mediated cell death, whereas activating AMPK in mosquito cells promotes Zika virus-mediated cell death. These findings suggest that the differential metabolic reprogramming during Zika virus infection of human versus mosquito cells determines whether cell death occurs.

High vigabatrin dosage is associated with lower risk of infantile spasms relapse among children with tuberous sclerosis complex.

After initially successful treatment of infantile spasms, the long-term cumulative risk of relapse approaches 50%, and there is no established protocol to mitigate this risk. Although vigabatrin may be an effective means to prevent relapse, there is little guidance as to ideal duration and dosage. Using a cohort of children with infantile spasms and tuberous sclerosis complex (TSC), we evaluated the potential association of post-response VGB treatment and the rate of infantile spasms relapse. Patients with infantile spasms and clinical response to vigabatrin were identified among a multicenter prospective observational cohort of children with TSC. For each patient we recorded dates of infantile spasms onset, response to vigabatrin, relapse (if any), and quantified duration and dosage of vigabatrin after response. Time to relapse as a function of vigabatrin exposure was evaluated using survival analyses. We identified 50 children who responded to VGB. During a median follow-up of 16.6 months (IQR 10.3-22.9), 12 (24%) patients subsequently relapsed after a median of 7.8 months (IQR 3.1-9.6). Relapse occurred after VGB discontinuation in four patients, and during continued VGB treatment in the remaining eight cases. In survival analyses, risk of relapse was unaffected by the presence or absence of VGB treatment (HR 0.31, 95%CI 0.01-28.4, P = 0.61), but weighted-average dosage was associated with marked reduction in relapse risk: Each 50 mg/kg/d increment in dosage was associated with 61% reduction in risk (HR 0.39, 95%CI 0.17 - 0.90, P = 0.026). This study suggests that the risk of infantile spasms relapse in TSC may be reduced by high-dose vigabatrin treatment.

Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization.

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Increased cell survival and cytogenetic integrity by spatial dose redistribution at a compact synchrotron X-ray source.

X-ray microbeam radiotherapy can potentially widen the therapeutic window due to a geometrical redistribution of the dose. However, high requirements on photon flux, beam collimation, and system stability restrict its application mainly to large-scale, cost-intensive synchrotron facilities. With a unique laser-based Compact Light Source using inverse Compton scattering, we investigated the translation of this promising radiotherapy technique to a machine of future clinical relevance. We performed in vitro colony-forming assays and chromosome aberration tests in normal tissue cells after microbeam irradiation compared to homogeneous irradiation at the same mean dose using 25 keV X-rays. The microplanar pattern was achieved with a tungsten slit array of 50 µm slit size and a spacing of 350 µm. Applying microbeams significantly increased cell survival for a mean dose above 2 Gy, which indicates fewer normal tissue complications. The observation of significantly less chromosome aberrations suggests a lower risk of second cancer development. Our findings provide valuable insight into the mechanisms of microbeam radiotherapy and prove its applicability at a compact synchrotron, which contributes to its future clinical translation.

Linking Gamma-H2AX Foci and Cancer in Rat Skin Exposed to Heavy Ions and Electron Radiation.

This study uses acute doses of three test radiations, [Ar ions (L = 125 keVµ), Ne ions (L = 25 keVµ) and electron radiation] to examine a potential quantitative link between rat skin cancer induction and gamma-H2AX foci in rat keratinocytes exposed in vitro to radiations with comparable L values. Theory provided a testable link between cancer yield and gamma-H2AX foci yields: YCa(D,L)rat = (NF)2YAX(D,L)keratinocyte (eqn 1), where YCa(D,L) is cancers(rat) at 1.0 y, YAX(D,L) is in vitro gamma-H2AX foci(keratinocyte) , D is radiation dose, L is linear energy transfer, N is irradiated keratinocytes in vivo, and F is the error rate of end joining. An explicit expression for cancer yield was derived based on cancers arising in the ion track region in proportion to D and L (first term) and independently in proportion to D in the delta ray region in between the ion tracks (second term): YCa(D,L) = CCaLD + BCaD (eqn 1a). Parameters quantified include: CCa = 0.000589 ± 0.000150 cancers-micron[rat(kev)Gy]; BCa = 0.0088 ± 0.0035 cancers(ratGy), F = (8.18 ± 0.91) × 10; N = (8.8 ± 1.2) × 10 and (NF)2 = 0.036 ± 0.006 cancer keratinocyte(rat H2AX foci). Verification of eqns (1) and (1a) and the constancy of F support the hypothesis that end-rejoining errors play a major role in radiation carcinogenesis in rat skin. Cancer yields per rat were consistently predictable based on gamma-H2AX foci yields in keratinocytes in vitro such that 27.8 H2AXfoci(keratinocyte) predicted 1.0 cancer(rat) at 1 y.

Evaluation of the Essen Rotary as a new technique for bacterial swabs: results of a prospective controlled clinical investigation in 50 patients with chronic leg ulcers.

Most chronic wounds are colonised with different microorganisms, especially problematic bacteria like methicillin-resistant Staphylococcus aureus (MRSA), which represent an increasing therapeutic challenge in the modern wound therapy regimen. Therefore, it is essential to specify the bacteria in wounds for an individual-specific treatment. In most patients, an exemplary bacterial swab is taken from the centre of the wound surface. This so-called Levine technique is propagated currently as the gold standard. The aim of our clinical investigation was to compare the results of different swab techniques to the new established Essen Rotary. In this monocentric prospective investigation, 50 patients with chronic leg ulcers were examined consecutively. The results of our clinical study show that bacteria are heterogeneously spread on wound surfaces. The analysis of the semiquantitative measured results showed that the Essen Rotary could detect significant more bacteria with a total amount of 111 bacteria (P = 0·049) compared to usual swab techniques. Considerably, only the Essen Rotary identified five compared to three MRSA-patients detected by other techniques. The Essen Rotary is an efficient, economic and uncomplicated modification of bacteriological swab techniques which detects significant more bacteria compared to other conventional swab techniques. Therefore, the Essen Rotary may become the new gold standard in routinely taken bacteriological swabs especially for MRSA screenings in patients with chronic leg ulcers.

Spindle disturbances in human-hamster hybrid (A(L) ) cells induced by the electrical component of the mobile communication frequency range signal.

The production of spindle disturbances in a human-hamster hybrid (A(L) ) cell line by an electromagnetic field (EMF) with field strength of 90 V/m at a frequency of 900 MHz was studied in greater detail. The experimental setup presented allows investigating whether either the electrical (E) and/or the magnetic (H) field component of EMF can be associated with the effectiveness of the spindle-disturbing potential. Therefore, both field components of a transversal electromagnetic field (TEM) wave have been separated during exposure of the biological system. This procedure should give more insight on understanding the underlying mechanisms of non-thermal effects of EMF. A statistical comparison of the proportions of the fractions of ana- and telophases with spindle disturbances, obtained for five different exposure conditions with respect to unexposed controls (sham condition), showed that only cells exposed to the H-field component of the EMF were not different from the control. Therefore, the results of the present study indicate that an exposure of cells to EMF at E-field strengths of 45 and 90 V/m, as well as to the separated E component of the EMF, induces significant spindle disturbances in ana- and telophases of the cell cycle.

Chromosome analysis of the differential radiosensitivity of an Epstein-Barr virus (EBV)-transformed B cell line and B and T lymphocytes from the same blood donor.

PURPOSE: To date, simultaneously performed investigations on the differential radiosensitivity of an Epstein-Barr virus (EBV)-transformed B cell line as well as B and T lymphocytes of human peripheral blood are not available. Thus the aim of the present study was to fill this gap by directly comparing the corresponding dose-response relationships of dicentrics obtained in blood samples from the same donor. MATERIAL AND METHODS: Cell samples of whole blood or low passage cells of an EBV-transformed B cell line were irradiated by 120 kV X-rays in chambers tightly embedded in a polymethylmethacrylate phantom. Chromosome analysis was performed in phytohemagglutinin-stimulated T lymphocytes, in pokeweed mitogen-stimulated B lymphocytes and in the EBV-transformed B cell line. RESULTS: Based on dose-response relationships of dicentrics, different radiosensitivity values relative to T lymphocytes were found from 1.53-1.46 for the EBV-transformed cell line, from 0.76-0.80 for resting B lymphocytes and from 2.36-2.20 for cycling B lymphocytes within the dose range from 0.25-4 Gy. CONCLUSIONS: Owing to these different radiosensitivity values, care has to be taken when dose-response relationships of dicentrics determined in B cell lines are used in biological dosimetry to estimate any dose levels for radiation protection purposes.

Dicentric chromosomes and gamma-H2AX foci formation in lymphocytes of human blood samples exposed to a CT scanner: a direct comparison of dose response relationships.

Experiments using the induction of dicentric chromosomes (dicentrics) as well as the gamma-H2AX foci formation in lymphocytes of blood samples from a healthy donor were performed to directly evaluate the radiation sensitivity of both biological endpoints. For computed tomography scans at dose levels from 0.025 to 1 Gy, a linear-quadratic dose-response relationship for dicentrics and a linear dose-response relationship for gamma-H2AX foci were obtained. The coefficients of the dose-response relationship for dicentrics are alpha = (3.76 +/- 0.29) x 10(-2) Gy(-1) and beta = (5.54 +/- 0.45) x 10(-2) Gy(-2), the linear coefficient for gamma-H2AX foci is (7.38 +/- 0.11) Gy(-1). The findings indicate that scoring of dicentrics as well as microscopic analysis of gamma-H2AX foci are sensitive methods to quantify a radiation-induced biological damage at low doses. However, since gamma-H2AX foci can be partially repaired within a few hours, biological damages present for days or even months, which constitute the clinically relevant endpoints, can only be quantified reliably by scoring of chromosome aberrations. Thus currently the quantification of dicentrics or reciprocal translocations remains the recommended method for estimating the effect of exposures to low dose levels of radiation ('biological dosimetry'). However, owing to the high radiation sensitivity of the gamma-H2AX foci assay observed in the present study, further investigations on the effectiveness of low-linear energy transfer radiation qualities in producing gamma-H2AX foci in lymphocytes from healthy donors should be performed.