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.

Quantitative structural proteomics in living cells by covalent protein painting.

The fold and conformation of proteins are key to successful cellular function, but all techniques for protein structure determination are performed in an artificial environment with highly purified proteins. While protein conformations have been solved to atomic resolution and modern protein structure prediction tools rapidly generate near accurate models of proteins, there is an unmet need to uncover the conformations of proteins in living cells. Here, we describe Covalent Protein Painting (CPP), a simple and fast method to infer structural information on protein conformation in cells with a quantitative protein footprinting technology. CPP monitors the conformational landscape of the 3D proteome in cells with high sensitivity and throughput. A key advantage of CPP is its' ability to quantitatively compare the 3D proteomes between different experimental conditions and to discover significant changes in the protein conformations. We detail how to perform a successful CPP experiment, the factors to consider before performing the experiment, and how to interpret the results.

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.

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.

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.

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.

Expression of Amyloidogenic Transthyretin Drives Hepatic Proteostasis Remodeling in an Induced Pluripotent Stem Cell Model of Systemic Amyloid Disease.

The systemic amyloidoses are diverse disorders in which misfolded proteins are secreted by effector organs and deposited as proteotoxic aggregates at downstream tissues. Although well described clinically, the contribution of synthesizing organs to amyloid disease pathogenesis is unknown. Here, we utilize hereditary transthyretin amyloidosis (ATTR amyloidosis) induced pluripotent stem cells (iPSCs) to define the contribution of hepatocyte-like cells (HLCs) to the proteotoxicity of secreted transthyretin (TTR). To this end, we generated isogenic, patient-specific iPSCs expressing either amyloidogenic or wild-type TTR. We combined this tool with single-cell RNA sequencing to identify hepatic proteostasis factors correlating with destabilized TTR production in iPSC-derived HLCs. By generating an ATF6 inducible patient-specific iPSC line, we demonstrated that enhancing hepatic ER proteostasis preferentially reduces the secretion of amyloidogenic TTR. These data highlight the liver's capacity to chaperone misfolded TTR prior to deposition, and moreover suggest the potential for unfolded protein response modulating therapeutics in the treatment of diverse systemic amyloidoses.

Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity.

Synaptotagmin-11 (Syt11) is a Synaptotagmin isoform that lacks an apparent ability to bind calcium, phospholipids, or SNARE proteins. While human genetic studies have linked mutations in the Syt11 gene to schizophrenia and Parkinson's disease, the localization or physiological role of Syt11 remain unclear. We found that in neurons, Syt11 resides on abundant vesicles that differ from synaptic vesicles and resemble trafficking endosomes. These vesicles recycle via the plasma membrane in an activity-dependent manner, but their exocytosis is slow and desynchronized. Constitutive knockout mice lacking Syt11 died shortly after birth, suggesting Syt11-mediated membrane transport is required for survival. In contrast, selective ablation of Syt11 in excitatory forebrain neurons using a conditional knockout did not affect life span but impaired synaptic plasticity and memory. Syt11-deficient neurons displayed normal secretion of fast neurotransmitters and peptides but exhibited a reduction of long-term synaptic potentiation. Hence, Syt11 is an essential component of a neuronal vesicular trafficking pathway that differs from the well-characterized synaptic vesicle trafficking pathway but is also essential for life.

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.

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.

∆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.

ERdj3 is an endoplasmic reticulum degradation factor for mutant glucocerebrosidase variants linked to Gaucher's disease.

Gaucher's disease (GD) is caused by mutations that compromise ß-glucocerebrosidase (GCase) folding in the endoplasmic reticulum (ER), leading to excessive degradation instead of trafficking, which results in insufficient lysosomal function. We hypothesized that ER GCase interacting proteins play critical roles in making quality control decisions, i.e., facilitating ER-associated degradation (ERAD) instead of folding and trafficking. Utilizing GCase immunoprecipitation followed by mass-spectrometry-based proteomics, we identified endogenous HeLa cell GCase protein interactors, including ERdj3, an ER resident Hsp40 not previously established to interact with GCase. Depleting ERdj3 reduced the rate of mutant GCase degradation in patient-derived fibroblasts, while increasing folding, trafficking, and function by directing GCase to the profolding ER calnexin pathway. Inhibiting ERdj3-mediated mutant GCase degradation while simultaneously enhancing calnexin-associated folding, by way of a diltiazem-mediated increase in ER Ca(2+) levels, yields a synergistic rescue of L444P GCase lysosomal function. Our findings suggest a combination therapeutic strategy for ameliorating GD.

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.

A chaperone trap contributes to the onset of cystic fibrosis.

Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a 'chaperone trap'. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.

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.

Regulation of NF-kappaB activity and keratinocyte differentiation by the RIP4 protein: implications for cutaneous wound repair.

Receptor-interacting proteins (RIPs) are important regulators of cell proliferation and differentiation. As RIP4 is a crucial modulator of epidermal differentiation, we analyzed the expression of different rip genes in healing skin wounds. Rip4 expression was strongly downregulated in keratinocytes of the hyperproliferative epithelium at the wound edge early after injury and only returned to basal levels after completion of wound repair. Rip3 expression was strongly induced as early as 1 day after wounding. In contrast, rip and rip2 expression remained unaltered. To determine the factors that regulate rip4 gene expression in keratinocytes, human HaCaT keratinocytes were used as a model system. We found that scratch wounding as well as treatment with whole serum, phorbol esters, the growth/differentiation factors epidermal growth factor, transforming growth factor-beta, and activin A, or the proinflammatory cytokines tumor necrosis factor-alpha and IL-1beta strongly suppressed rip4 expression in these cells. In contrast, the steroid dexamethasone and all-trans retinoic acid slightly stimulated rip4 expression. Suppression of rip4 expression in keratinocytes using small interfering RNA technology reduced the activation of NF-kappaB, and enhanced the expression of epidermal differentiation markers in these cells. These data suggest important and unique functions of different RIP proteins in keratinocytes of normal and wounded skin.

Regulation of epidermal homeostasis and repair by phosphoinositide 3-kinase.

The epidermis undergoes continuous self-renewal to maintain its protective function. Whereas growth factors are known to modulate overall skin homeostasis, the intracellular signaling pathways, which control the delicate balance between proliferation and differentiation in keratinocytes, are largely unknown. Here we show transient upregulation of the phosphoinositide 3-kinase (PI3K) catalytic subunits p110alpha and p110beta in differentiating keratinocytes in vitro, expression of these subunits in the epidermis of normal and wounded skin, and enhanced Akt phosphorylation in the hyperproliferative wound epidermis. Stimulation of PI3K activity in cultured keratinocytes by stable expression of an inducible, constitutively active PI3K mutant promoted cell proliferation and inhibited terminal differentiation in keratinocyte monocultures and induced the formation of a hyperplastic, disorganized and poorly differentiated epithelium in organotypic skin cultures. Activation of PI3K signaling also caused reorganization of the actin cytoskeleton and induced keratinocyte migration in vitro and in skin organ cultures. The identification of 122 genes, which are differentially expressed after induction of PI3K signaling provides insight into the molecular mechanisms underlying the observed effects of active PI3K on keratinocytes and indicates that hyperproliferation may be achieved at the expense of genome integrity. These results identify PI3K as an important intracellular regulator of epidermal homeostasis and repair.

Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes.

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.