Analysis of the Tropism of SARS-CoV-2 Based on the Host Interactome of the Spike Protein.

The ß-coronavirus SARS-CoV-2 causes severe acute respiratory syndrome (COVID-19) in humans. It enters and infects epithelial airway cells upon binding of the receptor binding domain (RBD) of the virus entry protein spike to the host receptor protein Angiotensin Converting Enzyme 2 (ACE2). Here, we used coimmunoprecipitation coupled with bottom-up mass spectrometry to identify host proteins that engaged with the spike protein in human bronchial epithelial cells (16HBEo-). We found that the spike protein bound to extracellular laminin and thrombospondin and endoplasmatic reticulum (ER)-resident DJB11 and FBX2 proteins. The ER-resident proteins UGGT1, CALX, HSP7A, and GRP78/BiP bound preferentially to the original Wuhan D614 over the mutated G614 spike protein in the more rapidly spreading Alpha SARS-CoV-2 strain. The increase in protein binding to the D614 spike might be explained by higher accessibility of cryptic sites in "RDB open" and "S2 only" D614 spike protein conformations and may enable SARS-CoV-2 to infect additional, ACE2-negative cell types. Moreover, a novel proteome-based cell type set enrichment analysis (pCtSEA) found that host factors like laminin might render additional cell types such as macrophages and epithelial cells in the nephron permissive to SARS-CoV-2 infection.

Nvp63 and nvPIWIL1 Suppress Retrotransposon Activation in the Sea Anemone Nematostella vectensis.

The transcription factors p63 and p73 have high similarity to the tumor suppressor protein p53. While the importance of p53 in DNA damage control is established, the functions of p63 or p73 remain elusive. Here, we analyzed nvp63, the cnidarian homologue of p63, that is expressed in the mesenteries of the starlet sea anemone Nematostella vectensis and that is activated in response to DNA damage. We used ultraviolet light (UV) to induce DNA damage and determined the chromatin-bound proteome with quantitative, bottom-up proteomics. We found that genotoxic stress or nvp63 knockdown recruited the protein nvPIWIL1, a homologue of the piRNA-binding PIWI protein family. Knockdown nvPIWIL1 increased protein expression from open reading frames (ORFs) that overlap with class I and II transposable element DNA sequences in the genome of N. vectensis. UV irradiation induced apoptosis, and apoptosis was reduced in the absence of nvp63 but increased with the loss of nvPIWIL1. Loss of nvp63 increased the presence of class I LTR and non-LTR retrotransposon but not of class II DNA transposon-associated protein products. These results suggest that an evolutionary early function of nvp63 might be to control genome stability in response to activation of transposable elements, which induce DNA damage during reintegration in the genome.

Cancer Conformational Landscape Shapes Tumorigenesis.

During tumorigenesis, DNA mutations in protein coding sequences can alter amino acid sequences which can change the structures of proteins. While the 3D structure of mutated proteins has been studied with atomic resolution, the precise impact of somatic mutations on the 3D proteome during malignant transformation remains unknown because methods to reveal in vivo protein structures in high throughput are limited. Here, we measured the accessibility of the lysine ε-amine for chemical modification across proteomes using covalent protein painting (CPP) to indirectly determine alterations in the 3D proteome. CPP is a novel, high-throughput quantitative mass spectrometric method that surveyed a total of 8052 lysine sites across the 60 cell lines of the well-studied anticancer cell line panel (NCI60). Overall, 5.2 structural alterations differentiated any cancer cell line from the other 59. Structural aberrations in 98 effector proteins correlated with the selected presence of 90 commonly mutated proteins in the NCI60 cell line panel, suggesting that different tumor genotypes reshape a limited set of effector proteins. We searched our dataset for druggable conformational aberrations and identified 49 changes in the cancer conformational landscape that correlated with the growth inhibition profiles of 300 drug candidates out of 50,000 small molecules. We found that alterations in heat shock proteins are key predictors of anticancer drug efficacy, which implies that the proteostasis network may have a general but hitherto unrecognized role in maintaining malignancy. Individual lysine sites may serve as biomarkers to guide drug selection or may be directly targeted for anticancer drug development.

SMG1 and CDK12 Link ΔNp63α Phosphorylation to RNA Surveillance in Keratinocytes.

The tumor suppressor p53-like protein p63 is required for self-renewal of epidermal tissues. Loss of p63 or exposure to ultraviolet (UV) irradiation triggers terminal differentiation in keratinocytes. However, it remains unclear how p63 diverts epidermal cells from proliferation to terminal differentiation, thereby contributing to successful tissue self-renewal. Here, we used bottom-up proteomics to identify the proteome at the chromatin in normal human epidermal keratinocytes following UV irradiation and p63 depletion. We found that loss of p63 increased DNA damage and that UV irradiation recruited the cyclin-dependent kinase CDK12 and the serine/threonine protein kinase SMG1 to chromatin only in the presence of p63. A post-translational modification analysis of ΔNp63α with mass spectrometry revealed that phosphorylation of T357/S358 and S368 was dependent on SMG1, whereas CDK12 increased the phosphorylation of ΔNp63α at S66/S68 and S301. Indirect phosphorylation of ΔNp63α in the presence of SMG1 enabled ΔNp63α to bind to the tumor suppressor p53-specific DNA recognition sequence, whereas CDK12 rendered ΔNp63α less responsive to UV irradiation and was not required for specific DNA binding. CDK12 and SMG1 are known to regulate the transcription and splicing of RNAs and the decay of nonsense RNAs, respectively, and a subset of p63-specific protein-protein interactions at the chromatin also linked p63 to RNA transcription and decay. We observed that in the absence of p63, UV irradiation resulted in more ORF1p. ORF1p is the first protein product of the intronless non-LTR retrotransposon LINE-1, indicating a derailed surveillance of RNA processing and/or translation. Our results suggest that p63 phosphorylation and transcriptional activation might correspond to altered RNA processing and/or translation to protect proliferating keratinocytes from increased genotoxic stress.

Temporal Quantitative Profiling of Newly Synthesized Proteins during Aß Accumulation.

Accumulation of aggregated amyloid beta (Aß) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimer's disease pathology. However, how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cell's response to stimuli, but to date, there have been few reliable methods to do this. To identify the protein regulatory network during the development of Aß deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2-, 5-, and 9-month-old APP/PS1 AD transgenic mice. This bio-orthogonal noncanonical amino acid tagging analysis combined PALM (pulse azidohomoalanine labeling in mammals) and HILAQ (heavy isotope labeled AHA quantitation) to reveal a comprehensive dataset of NSPs prior to and post Aß deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI), and mitochondrial respiratory chain throughout all time points and two brain regions. These results point to a pathological dysregulation of vesicle transportation which occurs prior to Aß accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes in protein synthesis and provides unique insights into the mechanisms that underlie AD.

Protein Footprinting via Covalent Protein Painting Reveals Structural Changes of the Proteome in Alzheimer's Disease.

Misfolding and aggregation of amyloid-ß peptide and hyperphosphorylated tau are molecular markers of Alzheimer's disease (AD), and although the 3D structures of these aberrantly folded proteins have been visualized in exquisite detail, no method has been able to survey protein folding across the proteome in AD. Here, we present covalent protein painting (CPP), a mass spectrometry-based protein footprinting approach to quantify the accessibility of lysine ε-amines for covalent modification at the surface of natively folded proteins. We used CPP to survey the reactivity of 2645 lysine residues and therewith the structural proteome of HEK293T cells and found that reactivity increased upon mild heat shock. CPP revealed that the accessibility of lysine residues for covalent modification in tubulin-ß (TUBB), in succinate dehydrogenase (SHDB), and in amyloid-ß peptide (Aß) is altered in human postmortem brain samples of patients with neurodegenerative diseases. The structural alterations of TUBB and SHDB in patients with AD, dementia with Lewy bodies (DLB), or both point to broader perturbations of the 3D proteome beyond Aß and hyperphosphorylated tau.

∆F508 CFTR interactome remodelling promotes rescue of cystic fibrosis.

Deletion of phenylalanine 508 of the cystic fibrosis transmembrane conductance regulator (∆F508 CFTR) is the major cause of cystic fibrosis, one of the most common inherited childhood diseases. The mutated CFTR anion channel is not fully glycosylated and shows minimal activity in bronchial epithelial cells of patients with cystic fibrosis. Low temperature or inhibition of histone deacetylases can partly rescue ∆F508 CFTR cellular processing defects and function. A favourable change of ∆F508 CFTR protein-protein interactions was proposed as a mechanism of rescue; however, CFTR interactome dynamics during temperature shift and inhibition of histone deacetylases are unknown. Here we report the first comprehensive analysis of the CFTR and ∆F508 CFTR interactome and its dynamics during temperature shift and inhibition of histone deacetylases. By using a novel deep proteomic analysis method, we identify 638 individual high-confidence CFTR interactors and discover a ∆F508 deletion-specific interactome, which is extensively remodelled upon rescue. Detailed analysis of the interactome remodelling identifies key novel interactors, whose loss promote ∆F508 CFTR channel function in primary cystic fibrosis epithelia or which are critical for CFTR biogenesis. Our results demonstrate that global remodelling of ∆F508 CFTR interactions is crucial for rescue, and provide comprehensive insight into the molecular disease mechanisms of cystic fibrosis caused by deletion of F508.

Interference-free proteome quantification with MS/MS-based isobaric isotopologue detection.

Chemical labeling of peptides prior to shotgun proteomics allows relative quantification of proteins in biological samples independent of sample origin. Current strategies utilize isobaric labels that fragment into reporter ions. However, quantification of reporter ions results in distorted ratio measurements due to contaminating peptides that are co-selected in the same precursor isolation window. Here, we show that quantitation of isobaric peptide fragment isotopologues in tandem mass spectra reduces precursor interference. The method is based on the relative quantitation of isobaric isotopologues of dimethylated peptide fragments in tandem mass spectra following higher energy collisional dissociation (HCD). The approach enables precise quantification of a proteome down to single spectra per protein and quantifies >90% of proteins in a MudPIT experiment and accurately measures proteins in a model cell line for cystic fibrosis.

In vivo Protein Footprinting Reveals the Dynamic Conformational Changes of Proteome of Multiple Tissues in Progressing Alzheimer's Disease.

Numerous studies have investigated changes in protein expression at the system level using proteomic mass spectrometry, but only recently have studies explored the structure of proteins at the proteome level. We developed covalent protein painting (CPP), a protein footprinting method that quantitatively labels exposed lysine, and have now extended the method to whole intact animals to measure surface accessibility as a surrogate of in vivo protein conformations. We investigated how protein structure and protein expression change as Alzheimer's disease (AD) progresses by conducting in vivo whole animal labeling of AD mice. This allowed us to analyze broadly protein accessibility in various organs over the course of AD. We observed that structural changes of proteins related to 'energy generation,' 'carbon metabolism,' and 'metal ion homeostasis' preceded expression changes in the brain. We found that proteins in certain pathways undergoing structural changes were significantly co-regulated in the brain, kidney, muscle, and spleen.

p63 protects the female germ line during meiotic arrest.

Meiosis in the female germ line of mammals is distinguished by a prolonged arrest in prophase of meiosis I between homologous chromosome recombination and ovulation. How DNA damage is detected in these arrested oocytes is poorly understood, but it is variably thought to involve p53, a central tumour suppressor in mammals. While the function of p53 in monitoring the genome of somatic cells is clear, a consensus for the importance of p53 for germ line integrity has yet to emerge. Here we show that the p53 homologue p63 (refs 5, 6), and specifically the TAp63 isoform, is constitutively expressed in female germ cells during meiotic arrest and is essential in a process of DNA damage-induced oocyte death not involving p53. We also show that DNA damage induces both the phosphorylation of p63 and its binding to p53 cognate DNA sites and that these events are linked to oocyte death. Our data support a model whereby p63 is the primordial member of the p53 family and acts in a conserved process of monitoring the integrity of the female germ line, whereas the functions of p53 are restricted to vertebrate somatic cells for tumour suppression. These findings have implications for understanding female germ line fidelity, the regulation of fertility and the evolution of tumour suppressor mechanisms.

The Host Interactome of Spike Expands the Tropism of SARS-CoV-2.

The SARS-CoV-2 virus causes severe acute respiratory syndrome (COVID-19) and has rapidly created a global pandemic. Patients that survive may face a slow recovery with long lasting side effects that can afflict different organs. SARS-CoV-2 primarily infects epithelial airway cells that express the host entry receptor Angiotensin Converting Enzyme 2 (ACE2) which binds to spike protein trimers on the surface of SARS-CoV-2 virions. However, SARS-CoV-2 can spread to other tissues even though they are negative for ACE2. To gain insight into the molecular constituents that might influence SARS-CoV-2 tropism, we determined which additional host factors engage with the viral spike protein in disease-relevant human bronchial epithelial cells (16HBEo-). We found that spike recruited the extracellular proteins laminin and thrombospondin and was retained in the endoplasmatic reticulum (ER) by the proteins DJB11 and FBX2 which support re-folding or degradation of nascent proteins in the ER. Because emerging mutations of the spike protein potentially impact the virus tropism, we compared the interactome of D614 spike with that of the rapidly spreading G614 mutated spike. More D614 than G614 spike associated with the proteins UGGT1, calnexin, HSP7A and GRP78/BiP which ensure glycosylation and folding of proteins in the ER. In contrast to G614 spike, D614 spike was endoproteolytically cleaved, and the N-terminal S1 domain was degraded in the ER even though C-terminal 'S2 only' proteoforms remained present. D614 spike also bound more laminin than G614 spike, which suggested that extracellular laminins may function as co-factor for an alternative, 'S2 only' dependent virus entry. Because the host interactome determines whether an infection is productive, we developed a novel proteome-based cell type set enrichment analysis (pCtSEA). With pCtSEA we determined that the host interactome of the spike protein may extend the tropism of SARS-CoV-2 beyond mucous epithelia to several different cell types, including macrophages and epithelial cells in the nephron. An 'S2 only' dependent, alternative infection of additional cell types with SARS-CoV-2 may impact vaccination strategies and may provide a molecular explanation for a severe or prolonged progression of disease in select COVID-19 patients.

A posttranslational modification code for CFTR maturation is altered in cystic fibrosis.

The multistep process regulating the maturation of membrane proteins in the endoplasmic reticulum (ER) and the secretory pathway is disrupted in many protein misfolding disorders. Mutations in the ion channel CFTR that impair its folding and subsequent localization to the plasma membrane cause cystic fibrosis (CF), an inherited and eventually lethal disease that impairs the function of multiple organs, mostly the lungs. Here, we found that proper maturation of CFTR is dependent on cross-talk between phosphorylation and methylation events in the regulatory insertion (RI) element of the protein. Manipulating these posttranslational modifications (PTMs) prevented the maturation of wild-type CFTR and instead induced its degradation by ER quality control systems. Deletion of Phe508 (ΔF508), the most prevalent mutation in CF, and other mutations in CFTR that impair its trafficking, such as N1303K, also led to quantitative and qualitative PTM changes that prevented the maturation of misfolded CFTR. Further analysis revealed that a wild-type CFTR-like PTM pattern and function was restored in ΔF508 CFTR when cells were cultured at 28°C but only in the presence of the kinase CK2α. Furthermore, the ability to replicate this PTM pattern predicted the efficacy of treatments in restoring ΔF508 CFTR activity. Accordingly, evaluation of patient information revealed that point mutations of several of the modification sites are associated with clinical CF. These findings identify a minimal quantitative and qualitative PTM code for CFTR maturation that distinguishes correctly folded from misfolded CFTR.

Understanding molecular mechanisms of disease through spatial proteomics.

Mammalian cells are organized into different compartments that separate and facilitate physiological processes by providing specialized local environments and allowing different, otherwise incompatible biological processes to be carried out simultaneously. Proteins are targeted to these subcellular locations where they fulfill specialized, compartment-specific functions. Spatial proteomics aims to localize and quantify proteins within subcellular structures.

Increased proteomic complexity in Drosophila hybrids during development.

Cellular proteomes are thought to be optimized for function, leaving no room for proteome plasticity and, thus, evolution. However, hybrid animals that result from a viable cross of two different species harbor hybrid proteomes of unknown complexity. We charted the hybrid proteome of a viable cross between Drosophila melanogaster females and Drosophila simulans males with bottom-up proteomics. Developing hybrids harbored 20% novel proteins in addition to proteins that were also present in either parental species. In contrast, adult hybrids and developmentally failing embryos of the reciprocal cross showed less additional proteins (5 and 6%, respectively). High levels of heat shock proteins, proteasome-associated proteins, and proteasomal subunits indicated that proteostasis sustains the expanded complexity of the proteome in developing hybrids. We conclude that increased proteostasis gives way to proteomic plasticity and thus opens up additional space for rapid phenotypic variation during embryonic development.

Deducing the presence of proteins and proteoforms in quantitative proteomics.

The human genome harbors just 20,000 genes suggesting that the variety of possible protein products per gene plays a significant role in generating functional diversity. In bottom-up proteomics peptides are mapped back to proteins and proteoforms to describe a proteome; however, accurate quantitation of proteoforms is challenging due to incomplete protein sequence coverage and mapping ambiguities. Here, we demonstrate that a new software tool called ProteinClusterQuant (PCQ) can be used to deduce the presence of proteoforms that would have otherwise been missed, as exemplified in a proteomic comparison of two fly species, Drosophila melanogaster and D. virilis. PCQ was used to identify reduced levels of serine/threonine protein kinases PKN1 and PKN4 in CFBE41o- cells compared to HBE41o- cells and to elucidate that shorter proteoforms of full-length caspase-4 and ephrin B receptor are differentially expressed. Thus, PCQ extends current analyses in quantitative proteomics and facilitates finding differentially regulated proteins and proteoforms.

Deep interactome profiling of membrane proteins by co-interacting protein identification technology.

Affinity purification coupled to mass spectrometry (AP-MS) is the method of choice for analyzing protein-protein interactions, but common protocols frequently recover only the most stable interactions and tend to result in low bait yield for membrane proteins. Here, we present a novel, deep interactome sequencing approach called CoPIT (co-interacting protein identification technology), which allows comprehensive identification and analysis of membrane protein interactomes and their dynamics. CoPIT integrates experimental and computational methods for a coimmunoprecipitation (Co-IP)-based workflow from sample preparation for mass spectrometric analysis to visualization of protein-protein interaction networks. The approach particularly improves the results for membrane protein interactomes, which have proven to be difficult to identify and analyze. CoPIT was used successfully to identify the interactome of the cystic fibrosis transmembrane conductance regulator (CFTR), demonstrating its validity and performance. The experimental step in this case achieved up to 100-fold-higher bait yield than previous methods by optimizing lysis, elution, sample clean-up and detection of interacting proteins by multidimensional protein identification technology (MudPIT). Here, we further provide evidence that CoPIT is applicable to other types of proteins as well, and that it can be successfully used as a general Co-IP method. The protocol describes all steps, ranging from considerations for experimental design, Co-IP, preparation of the sample for mass spectrometric analysis, and data analysis steps, to the final visualization of interaction networks. Although the experimental part can be performed in <3 d, data analysis may take up to a few weeks.

Retinoic acid inhibits downregulation of DeltaNp63alpha expression during terminal differentiation of human primary keratinocytes.

Recently, the p53 homolog p63 has been implicated in sustaining the epidermal stem cell population. The p63 gene encodes six major products with transactivating or dominant-negative properties. The expression pattern of these isoforms in keratinocytes was investigated here. Northern blot, ribonuclease protection assay, reverse transcription-polymerase chain reaction, and western blot techniques sensitive for all six p63 isotypes verified the predominant expression of the truncated and potentially dominant-negative isotype DeltaNp63alpha in human keratinocytes. The expression of this isoform is downregulated when proliferating human primary keratinocytes begin to differentiate after growth factor withdrawal. The onset of differentiation does not change the ratio of two other weakly expressed isotypes DeltaNp63gamma and TAp63alpha relative to DeltaNp63alpha. Treatment of primary human keratinocytes with all-trans retinoic acid does not alter the expression pattern of p63 isotypes but prevents its downregulation as observed in control cell cultures. These data suggest that p63 expression in human keratinocytes is affected by all-trans retinoic acid and this influence might contribute to the fine tuned keratinocyte proliferation and differentiation equilibrium in the mammalian epidermis.

Differential regulation of transcription and induction of programmed cell death by human p53-family members p63 and p73.

The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. With p63 and p73 two genes coding for proteins homologous to p53 have been identified. We describe the properties of seven human p63 and p73 proteins as transcriptional activators of p21WAF1/CIP1 expression and apoptotic inducers in direct comparison to p53 in the same assay systems employing DLD-1-tet-off colon cells. Programmed cell death is detected in cells expressing high levels of p53 and p73alpha. Cells overexpressing TAp63alpha, TAp63gamma, TA*p63alpha, TA*p63gamma, DeltaNp63alpha, and DeltaNp63gamma display low or no detectable apoptosis.

Digital imaging mass spectrometry.

Methods to visualize the two-dimensional (2D) distribution of molecules by mass spectrometric imaging evolve rapidly and yield novel applications in biology, medicine, and material surface sciences. Most mass spectrometric imagers acquire high mass resolution spectra spot-by-spot and thereby scan the object's surface. Thus, imaging is slow and image reconstruction remains cumbersome. Here we describe an imaging mass spectrometer that exploits the true imaging capabilities by ion optical means for the time of flight mass separation. The mass spectrometer is equipped with the ASIC Timepix chip as an array detector to acquire the position, mass, and intensity of ions that are imaged by matrix-assisted laser desorption/ionization (MALDI) directly from the target sample onto the detector. This imaging mass spectrometer has a spatial resolving power at the specimen of (84 ± 35) µm with a mass resolution of 45 and locates atoms or organic compounds on a surface area up to ~2 cm(2). Extended laser spots of ~5 mm(2) on structured specimens allows parallel imaging of selected masses. The digital imaging mass spectrometer proves high hit-multiplicity, straightforward image reconstruction, and potential for high-speed readout at 4 kHz or more. This device demonstrates a simple way of true image acquisition like a digital photographic camera. The technology may enable a fast analysis of biomolecular samples in near future.

The p53 tumor suppressor-like protein nvp63 mediates selective germ cell death in the sea anemone Nematostella vectensis.

Here we report the identification and molecular function of the p53 tumor suppressor-like protein nvp63 in a non-bilaterian animal, the starlet sea anemone Nematostella vectensis. So far, p53-like proteins had been found in bilaterians only. The evolutionary origin of p53-like proteins is highly disputed and primordial p53-like proteins are variably thought to protect somatic cells from genotoxic stress. Here we show that ultraviolet (UV) irradiation at low levels selectively induces programmed cell death in early gametes but not somatic cells of adult N. vectensis polyps. We demonstrate with RNA interference that nvp63 mediates this cell death in vivo. Nvp63 is the most archaic member of three p53-like proteins found in N. vectensis and in congruence with all known p53-like proteins, nvp63 binds to the vertebrate p53 DNA recognition sequence and activates target gene transcription in vitro. A transactivation inhibitory domain at its C-terminus with high homology to the vertebrate p63 may regulate nvp63 on a molecular level. The genotoxic stress induced and nvp63 mediated apoptosis in N. vectensis gametes reveals an evolutionary ancient germ cell protective pathway which relies on p63-like proteins and is conserved from cnidarians to vertebrates.