Enhanced feature matching in single-cell proteomics characterizes IFN-γ response and co-existence of cell states.

Proteome analysis by data-independent acquisition (DIA) has become a powerful approach to obtain deep proteome coverage, and has gained recent traction for label-free analysis of single cells. However, optimal experimental design for DIA-based single-cell proteomics has not been fully explored, and performance metrics of subsequent data analysis tools remain to be evaluated. Therefore, we here formalize and comprehensively evaluate a DIA data analysis strategy that exploits the co-analysis of low-input samples with a so-called matching enhancer (ME) of higher input, to increase sensitivity, proteome coverage, and data completeness. We assess the matching specificity of DIA-ME by a two-proteome model, and demonstrate that false discovery and false transfer are maintained at low levels when using DIA-NN software, while preserving quantification accuracy. We apply DIA-ME to investigate the proteome response of U-2 OS cells to interferon gamma (IFN-γ) in single cells, and recapitulate the time-resolved induction of IFN-γ response proteins as observed in bulk material. Moreover, we uncover co- and anti-correlating patterns of protein expression within the same cell, indicating mutually exclusive protein modules and the co-existence of different cell states. Collectively our data show that DIA-ME is a powerful, scalable, and easy-to-implement strategy for single-cell proteomics.

Lipid droplets and small extracellular vesicles: More than two independent entities.

Despite increasing knowledge about small extracellular vesicle (sEV) composition and functions in cell-cell communication, the mechanism behind their biogenesis remains unclear. Here, we reveal for the first time that sEV biogenesis and release into the microenvironment are tightly connected with another important organelle, Lipid Droplets (LDs). The correlation was observed in several human cancer cell lines as well as patient-derived colorectal cancer stem cells (CR-CSCs). Our results demonstrated that external stimuli such as radiation, pH, hypoxia or lipid-interfering drugs, known to affect the number of LDs/cell, similarly influenced sEV secretion. Importantly, through multiple omics data, at both mRNA and protein levels, we revealed RAB5C as a potential important molecular player behind this organelle connection. Altogether, the potential to fine-tune sEV biogenesis by targeting LDs could significantly impact the amount, cargos and properties of these sEVs, opening new clinical perspectives.

P53-dependent hypusination of eIF5A affects mitochondrial translation and senescence immune surveillance.

Cellular senescence is characterized by a permanent growth arrest and is associated with tissue aging and cancer. Senescent cells secrete a number of different cytokines referred to as the senescence-associated secretory phenotype (SASP), which impacts the surrounding tissue and immune response. Here, we find that senescent cells exhibit higher rates of protein synthesis compared to proliferating cells and identify eIF5A as a crucial regulator of this process. Polyamine metabolism and hypusination of eIF5A play a pivotal role in sustaining elevated levels of protein synthesis in senescent cells. Mechanistically, we identify a p53-dependent program in senescent cells that maintains hypusination levels of eIF5A. Finally, we demonstrate that functional eIF5A is required for synthesizing mitochondrial ribosomal proteins and monitoring the immune clearance of premalignant senescent cells in vivo. Our findings establish an important role of protein synthesis during cellular senescence and suggest a link between eIF5A, polyamine metabolism, and senescence immune surveillance.

A single-sample workflow for joint metabolomic and proteomic analysis of clinical specimens.

Understanding the interplay of the proteome and the metabolome helps to understand cellular regulation and response. To enable robust inferences from such multi-omics analyses, we introduced and evaluated a workflow for combined proteome and metabolome analysis starting from a single sample. Specifically, we integrated established and individually optimized protocols for metabolomic and proteomic profiling (EtOH/MTBE and autoSP3, respectively) into a unified workflow (termed MTBE-SP3), and took advantage of the fact that the protein residue of the metabolomic sample can be used as a direct input for proteome analysis. We particularly evaluated the performance of proteome analysis in MTBE-SP3, and demonstrated equivalence of proteome profiles irrespective of prior metabolite extraction. In addition, MTBE-SP3 combines the advantages of EtOH/MTBE and autoSP3 for semi-automated metabolite extraction and fully automated proteome sample preparation, respectively, thus advancing standardization and scalability for large-scale studies. We showed that MTBE-SP3 can be applied to various biological matrices (FFPE tissue, fresh-frozen tissue, plasma, serum and cells) to enable implementation in a variety of clinical settings. To demonstrate applicability, we applied MTBE-SP3 and autoSP3 to a lung adenocarcinoma cohort showing consistent proteomic alterations between tumour and non-tumour adjacent tissue independent of the method used. Integration with metabolomic data obtained from the same samples revealed mitochondrial dysfunction in tumour tissue through deregulation of OGDH, SDH family enzymes and PKM. In summary, MTBE-SP3 enables the facile and reliable parallel measurement of proteins and metabolites obtained from the same sample, benefiting from reduced sample variation and input amount. This workflow is particularly applicable for studies with limited sample availability and offers the potential to enhance the integration of metabolomic and proteomic datasets.

DEAD box RNA helicases are pervasive protein kinase interactors and activators.

DEAD box (DDX) RNA helicases are a large family of ATPases, many of which have unknown functions. There is emerging evidence that besides their role in RNA biology, DDX proteins may stimulate protein kinases. To investigate if protein kinase-DDX interaction is a more widespread phenomenon, we conducted three orthogonal large-scale screens, including proteomics analysis with 32 RNA helicases, protein array profiling, and kinome-wide in vitro kinase assays. We retrieved Ser/Thr protein kinases as prominent interactors of RNA helicases and report hundreds of binary interactions. We identified members of ten protein kinase families, which bind to, and are stimulated by, DDX proteins, including CDK, CK1, CK2, DYRK, MARK, NEK, PRKC, SRPK, STE7/MAP2K, and STE20/PAK family members. We identified MARK1 in all screens and validated that DDX proteins accelerate the MARK1 catalytic rate. These findings indicate pervasive interactions between protein kinases and DEAD box RNA helicases, and provide a rich resource to explore their regulatory relationships.

Eukaryotic Initiation Translation Factor 2A activation by cannabidiolic acid alters the protein homeostasis balance in glioblastoma cells.

Eukaryotic Initiation Translation Factor 2A (EIF2A) is considered to be primarily responsible for the initiation of translation when a cell is subjected to stressful conditions. However, information regarding this protein is still incomplete. Using a combination of proteomic approaches, we demonstrated that EIF2A is the molecular target of the naturally occurring bioactive compound cannabidiolic acid (CBDA) within human glioblastoma cells. This finding allowed us to undertake a study aimed at obtaining further information on the functions that EIF2A plays in tumor cells. Indeed, our data showed that CBDA is able to activate EIF2A when the cells are in no-stress conditions. It induces conformational changes in the protein structure, thus increasing EIF2A affinity towards the proteins participating in the Eukaryotic Translation Machinery. Consequently, following glioblastoma cells incubation with CBDA we observed an enhanced neosynthesis of proteins involved in the stress response, nucleic acid translation and organization, and protein catabolism. These changes in gene expression resulted in increased levels of ubiquitinated proteins and accumulation of the autophagosome. Our results, in addition to shedding light on the molecular mechanism underlying the biological effect of a phytocannabinoid in cancer cells, demonstrated that EIF2A plays a critical role in regulation of protein homeostasis.

ARID1A regulates DNA repair through chromatin organization and its deficiency triggers DNA damage-mediated anti-tumor immune response.

AT-rich interaction domain protein 1A (ARID1A), a SWI/SNF chromatin remodeling complex subunit, is frequently mutated across various cancer entities. Loss of ARID1A leads to DNA repair defects. Here, we show that ARID1A plays epigenetic roles to promote both DNA double-strand breaks (DSBs) repair pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). ARID1A is accumulated at DSBs after DNA damage and regulates chromatin loops formation by recruiting RAD21 and CTCF to DSBs. Simultaneously, ARID1A facilitates transcription silencing at DSBs in transcriptionally active chromatin by recruiting HDAC1 and RSF1 to control the distribution of activating histone marks, chromatin accessibility, and eviction of RNAPII. ARID1A depletion resulted in enhanced accumulation of micronuclei, activation of cGAS-STING pathway, and an increased expression of immunomodulatory cytokines upon ionizing radiation. Furthermore, low ARID1A expression in cancer patients receiving radiotherapy was associated with higher infiltration of several immune cells. The high mutation rate of ARID1A in various cancer types highlights its clinical relevance as a promising biomarker that correlates with the level of immune regulatory cytokines and estimates the levels of tumor-infiltrating immune cells, which can predict the response to the combination of radio- and immunotherapy.

Deposition of onco-histone H3.3-G34W leads to DNA repair deficiency and activates cGAS/STING-mediated immune responses.

Mutations in histone H3.3-encoding genes causing mutant histone tails are associated with specific cancers such as pediatric glioblastomas (H3.3-G34R/V) and giant cell tumor of the bone (H3.3-G34W). The mechanisms by which these mutations promote malignancy are not completely understood. Here we show that cells expressing H3.3-G34W exhibit DNA double-strand breaks (DSBs) repair defects and increased cellular sensitivity to ionizing radiation (IR). Mechanistically, H3.3-G34W can be deposited to damaged chromatin, but in contrast to wild-type H3.3, does not interact with non-homologous end-joining (NHEJ) key effectors KU70/80 and XRCC4 leading to NHEJ deficiency. Together with defective cell cycle checkpoints reported previously, this DNA repair deficiency in H3.3-G34W cells led to accumulation of micronuclei and cytosolic DNA following IR, which subsequently led to activation of the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, thereby inducing release of immune-stimulatory cytokines. These findings suggest a potential for radiotherapy for tumors expressing H3.3-G34W, which can be further improved by combination with STING agonists to induce immune-mediated therapeutic efficacy.

Secretome Analysis: Reading Cellular Sign Language to Understand Intercellular Communication.

A significant portion of mammalian proteomes is secreted to the extracellular space to fulfill crucial roles in cell-to-cell communication. To best recapitulate the intricate and multi-faceted crosstalk between cells in a live organism, there is an ever-increasing need for methods to study protein secretion in model systems that include multiple cell types. In addition, posttranslational modifications further expand the complexity and versatility of cellular communication. This review aims to summarize recent strategies and model systems that employ cellular coculture, chemical biology tools, protein enrichment, and proteomic methods to characterize the composition and function of cellular secretomes. This is all geared towards gaining better understanding of organismal biology in vivo mediated by secretory signaling.

The impact of cycling hypoxia on the phenotype of HPV-positive cervical cancer cells.

Cycling hypoxia (cycH) is a prevalent form of tumor hypoxia that is characterized by exposure of tumor cells to recurrent phases of hypoxia and reoxygenation. CycH has been associated with a particularly aggressive cellular phenotype of tumor cells and increased therapy resistance. By performing comparative analyses under normoxia, physoxia, chronic hypoxia, and cycH, we here uncover distinct effects of cycH on the phenotype of human papillomavirus (HPV)-positive cervical cancer cells. We show that-other than under chronic hypoxia-viral E6/E7 oncogene expression is largely maintained under cycH as is the E6/E7-dependent regulation of p53 and retinoblastoma protein. Further, cycH enables HPV-positive cancer cells to evade prosenescent chemotherapy, similar to chronic hypoxia. Moreover, cells under cycH exhibit a particularly pronounced resistance to the proapoptotic effects of Cisplatin. Quantitative proteome analyses reveal that cycH induces a unique proteomic signature in cervical cancer cells, which includes a significant downregulation of luminal lysosomal proteins. These encompass the potentially proapoptotic cathepsins B and cathepsin L, which, however, appear not to affect the response to Cisplatin under any of the O2 conditions tested. Rather, we show that the proapoptotic Caspase 8/BH3-interacting domain death agonist (BID) cascade plays a pivotal role for the efficiency of Cisplatin-induced apoptosis in HPV-positive cancer cells under all investigated O2 conditions. In addition, we provide evidence that BID activation by Cisplatin is impaired under cycH, which could contribute to the high resistance to the proapoptotic effects of Cisplatin. Collectively, this study provides the first insights into the profound phenotypic alterations induced by cycH in HPV-positive cancer cells, with implications for their therapeutic susceptibility.

An integrated workflow for quantitative analysis of the newly synthesized proteome.

The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific cues. This can be investigated by pulse-labeling of cells with clickable and stable-isotope-coded amino acids for the enrichment and mass spectrometric characterization of newly synthesized proteins (NSPs), however convoluted protocols prohibit their routine application. Here we report the optimization of multiple steps in sample preparation, mass spectrometry and data analysis, and we integrate them into a semi-automated workflow for the quantitative analysis of the newly synthesized proteome (QuaNPA). Reduced input requirements and data-independent acquisition (DIA) enable the analysis of triple-SILAC-labeled NSP samples, with enhanced throughput while featuring high quantitative accuracy. We apply QuaNPA to investigate the time-resolved cellular response to interferon-gamma (IFNg), observing rapid induction of targets 2 h after IFNg treatment. QuaNPA provides a powerful approach for large-scale investigation of NSPs to gain insight into complex cellular processes.

DNMT and HDAC inhibition induces immunogenic neoantigens from human endogenous retroviral element-derived transcripts.

Immunotherapies targeting cancer-specific neoantigens have revolutionized the treatment of cancer patients. Recent evidence suggests that epigenetic therapies synergize with immunotherapies, mediated by the de-repression of endogenous retroviral element (ERV)-encoded promoters, and the initiation of transcription. Here, we use deep RNA sequencing from cancer cell lines treated with DNA methyltransferase inhibitor (DNMTi) and/or Histone deacetylase inhibitor (HDACi), to assemble a de novo transcriptome and identify several thousand ERV-derived, treatment-induced novel polyadenylated transcripts (TINPATs). Using immunopeptidomics, we demonstrate the human leukocyte antigen (HLA) presentation of 45 spectra-validated treatment-induced neopeptides (t-neopeptides) arising from TINPATs. We illustrate the potential of the identified t-neopeptides to elicit a T-cell response to effectively target cancer cells. We further verify the presence of t-neopeptides in AML patient samples after in vivo treatment with the DNMT inhibitor Decitabine. Our findings highlight the potential of ERV-derived neoantigens in epigenetic and immune therapies.

Automated Sample Preparation for Mass Spectrometry-Based Clinical Proteomics.

Mass spectrometry (MS)-based proteomics is a rapidly maturing discipline, thus gaining momentum for routine molecular profiling of clinical specimens to improve disease classification, diagnostics, and therapy development. Yet, hurdles need to be overcome to enhance reproducibility in preanalytical sample processing, especially in large, quantity-limited sample cohorts. Therefore, automated sonication and single-pot solid-phase-enhanced sample preparation (autoSP3) was developed as a streamlined workflow that integrates all tasks from tissue lysis and protein extraction, protein cleanup, and proteolysis. It enables the concurrent processing of 96 clinical samples of any type (fresh-frozen or FFPE tissue, liquid biopsies, or cells) on an automated liquid handling platform, which can be directly interfaced to LC-MS for proteome analysis of clinical specimens with high sensitivity, high reproducibility, and short turn-around times.

The ribosomal protein S6 kinase alpha-1 (RPS6KA1) induces resistance to venetoclax/azacitidine in acute myeloid leukemia.

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

PEPseq quantifies transcriptome-wide changes in protein occupancy and reveals selective translational repression after translational stress.

Post-transcriptional gene regulation is accomplished by the interplay of the transcriptome with RNA-binding proteins, which occurs in a dynamic manner in response to altered cellular conditions. Recording the combined occupancy of all proteins binding to the transcriptome offers the opportunity to interrogate if a particular treatment leads to any interaction changes, pointing to sites in RNA that undergo post-transcriptional regulation. Here, we establish a method to monitor protein occupancy in a transcriptome-wide fashion by RNA sequencing. To this end, peptide-enhanced pull-down for RNA sequencing (or PEPseq) uses metabolic RNA labelling with 4-thiouridine (4SU) for light-induced protein-RNA crosslinking, and N-hydroxysuccinimide (NHS) chemistry to isolate protein-crosslinked RNA fragments across all long RNA biotypes. We use PEPseq to investigate changes in protein occupancy during the onset of arsenite-induced translational stress in human cells and reveal an increase of protein interactions in the coding region of a distinct set of mRNAs, including mRNAs coding for the majority of cytosolic ribosomal proteins. We use quantitative proteomics to demonstrate that translation of these mRNAs remains repressed during the initial hours of recovery after arsenite stress. Thus, we present PEPseq as a discovery platform for the unbiased investigation of post-transcriptional regulation.

Spatial tissue proteomics reveals distinct landscapes of heterogeneity in cutaneous papillomavirus-induced keratinocyte carcinomas.

Infection with certain cutaneous human papillomaviruses (HPV), in conjunction with chronic ultraviolet (UV) exposure, are the major cofactors of non-melanoma skin cancer (NMSC), the most frequent cancer type worldwide. Cutaneous squamous cell carcinomas (SCCs) as well as tumors in general represent three-dimensional entities determined by both temporal and spatial constraints. Whole tissue proteomics is a straightforward approach to understand tumorigenesis in better detail, but studies focusing on different progression states toward a dedifferentiated SCC phenotype on a spatial level are rare. Here, we applied an innovative proteomic workflow on formalin-fixed, paraffin-embedded (FFPE) epithelial tumors derived from the preclinical animal model Mastomys coucha. This rodent is naturally infected with its genuine cutaneous papillomavirus and closely mimics skin carcinogenesis in the context of cutaneous HPV infections in humans. We deciphered cellular networks by comparing diverse epithelial tissues with respect to their differentiation level and infection status. Our study reveals novel regulatory proteins and pathways associated with virus-induced tumor initiation and progression of SCCs. This approach provides the basis to better comprehend the multistep process of skin carcinogenesis.

DEAD box RNA helicases act as nucleotide exchange factors for casein kinase 2.

DDX RNA helicases promote RNA processing, but DDX3X also activates casein kinase 1 (CK1ε). We show that other DDX proteins also stimulate the protein kinase activity of CK1ε and that this extends to casein kinase 2 (CK2). CK2 enzymatic activity was stimulated by various DDX proteins at high substrate concentrations. DDX1, DDX24, DDX41, and DDX54 were required for full kinase activity in vitro and in Xenopus embryos. Mutational analysis of DDX3X indicated that CK1 and CK2 kinase stimulation engages its RNA binding but not catalytic motifs. Mathematical modeling of enzyme kinetics and stopped-flow spectroscopy showed that DDX proteins function as nucleotide exchange factors toward CK2 and reduce unproductive reaction intermediates and substrate inhibition. Our study reveals protein kinase stimulation by nucleotide exchange as important for kinase regulation and as a generic function of DDX proteins.

MYT1L haploinsufficiency in human neurons and mice causes autism-associated phenotypes that can be reversed by genetic and pharmacologic intervention.

MYT1L is an autism spectrum disorder (ASD)-associated transcription factor that is expressed in virtually all neurons throughout life. How MYT1L mutations cause neurological phenotypes and whether they can be targeted remains enigmatic. Here, we examine the effects of MYT1L deficiency in human neurons and mice. Mutant mice exhibit neurodevelopmental delays with thinner cortices, behavioural phenotypes, and gene expression changes that resemble those of ASD patients. MYT1L target genes, including WNT and NOTCH, are activated upon MYT1L depletion and their chemical inhibition can rescue delayed neurogenesis in vitro. MYT1L deficiency also causes upregulation of the main cardiac sodium channel, SCN5A, and neuronal hyperactivity, which could be restored by shRNA-mediated knockdown of SCN5A or MYT1L overexpression in postmitotic neurons. Acute application of the sodium channel blocker, lamotrigine, also rescued electrophysiological defects in vitro and behaviour phenotypes in vivo. Hence, MYT1L mutation causes both developmental and postmitotic neurological defects. However, acute intervention can normalise resulting electrophysiological and behavioural phenotypes in adulthood.

Multi-layered chromatin proteomics identifies cell vulnerabilities in DNA repair.

The DNA damage response (DDR) is essential to maintain genome stability, and its deregulation predisposes to carcinogenesis while encompassing attractive targets for cancer therapy. Chromatin governs the DDR via the concerted interplay among different layers, including DNA, histone post-translational modifications (hPTMs) and chromatin-associated proteins. Here, we employ multi-layered proteomics to characterize chromatin-mediated functional interactions of repair proteins, signatures of hPTMs and the DNA-bound proteome during DNA double-strand break (DSB) repair at high temporal resolution. Our data illuminate the dynamics of known and novel DDR-associated factors both at chromatin and at DSBs. We functionally attribute novel chromatin-associated proteins to repair by non-homologous end-joining (NHEJ), homologous recombination (HR) and DSB repair pathway choice. We reveal histone reader ATAD2, microtubule organizer TPX2 and histone methyltransferase G9A as regulators of HR and involved in poly-ADP-ribose polymerase-inhibitor sensitivity. Furthermore, we distinguish hPTMs that are globally induced by DNA damage from those specifically acquired at sites flanking DSBs (γH2AX foci-specific) and profiled their dynamics during the DDR. Integration of complementary chromatin layers implicates G9A-mediated monomethylation of H3K56 in DSBs repair via HR. Our data provide a dynamic chromatin-centered view of the DDR that can be further mined to identify novel mechanistic links and cell vulnerabilities in DSB repair.