Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins

Genetic profiling of protein burden and nuclear export overload

Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload

Pallidal Hyperdopaminergic Innervation Underlying D2 Receptor-Dependent Behavioral Deficits in the Schizophrenia Animal Model Established by EGF

Epidermal growth factor (EGF) is one of the ErbB receptor ligands implicated in schizophrenia neuropathology as well as in dopaminergic development. Based on the immune inflammatory hypothesis for schizophrenia, neonatal rats are exposed to this cytokine and later develop neurobehavioral abnormality such as prepulse inhibition (PPI) deficit. Here we found that the EGF-treated rats exhibited persistent increases in tyrosine hydroxylase levels and dopamine content in the globus pallidus. Furthermore, pallidal dopamine release was elevated in EGF-treated rats, but normalized by subchronic treatment with risperidone concomitant with amelioration of their PPI deficits. To evaluate pathophysiologic roles of the dopamine abnormality, we administered reserpine bilaterally to the globus pallidus to reduce the local dopamine pool. Reserpine infusion ameliorated PPI deficits of EGF-treated rats without apparent aversive effects on locomotor activity in these rats. We also administered dopamine D1-like and D2-like receptor antagonists (SCH23390 and raclopride) and a D2-like receptor agonist (quinpirole) to the globus pallidus and measured PPI and bar-hang latencies. Raclopride (0.5 and 2.0 µg/site) significantly elevated PPI levels of EGF-treated rats, but SCH23390 (0.5 and 2.0 µg/site) had no effect. The higher dose of raclopride induced catalepsy-like changes in control animals but not in EGF-treated rats. Conversely, local quinpirole administration to EGF-untreated control rats induced PPI deficits and anti-cataleptic behaviors, confirming the pathophysiologic role of the pallidal hyperdopaminergic state. These findings suggest that the pallidal dopaminergic innervation is vulnerable to circulating EGF at perinatal and/or neonatal stages and has strong impact on the D2-like receptor-dependent behavioral deficits relevant to schizophrenia

Perinatal Epidermal Growth Factor Signal Perturbation Results in the Series of Abnormal Auditory Oscillations and Responses Relevant to Schizophrenia

Kai R., Namba H., Sotoyama H., et al. Perinatal Epidermal Growth Factor Signal Perturbation Results in the Series of Abnormal Auditory Oscillations and Responses Relevant to Schizophrenia. Schizophrenia Bulletin Open 2, sgaa070 (2021); https://doi.org/10.1093/schizbullopen/sgaa070.Auditory neurophysiological responses, such as steady-state responses, event-related potential P300/P3, and phase-Amplitude coupling, are promising translational biomarkers for schizophrenia, but their molecular underpinning is poorly understood. Focusing on ErbB receptor signals that are implicated in both schizophrenia and auditory processing/cognition, we explored the causal biological links between ErbB signals and these auditory traits with an experimental intervention into rats. We peripherally challenged rat pups with one of the amniotic ErbB ligands, epidermal growth factor (EGF), and characterized its consequence on the series of these auditory electrocorticographic measures. Auditory brainstem responses (ABRs) and cortical ON responses were also assessed under anesthesia to estimate the influence of higher brain regions. An auditory steady-state paradigm revealed attenuation of spectral power and phase synchrony to 40-Hz stimuli in EGF-challenged rats. We observed a reduction in duration mismatch negativity-like potentials and a delay of P3a responses, all of which are relevant to the reported auditory pathophysiological traits of patients with schizophrenia. Moreover, the perinatal EGF challenges resulted in enhanced theta-Alpha/beta and theta-gamma coupling within the auditory cortex and changes in ABRs. However, the EGF challenges retained the normal ranges of cortical ON responses, potentially ruling out their fundamental auditory deficits. Perinatal exposure of an ErbB ligand to rats strikingly reproduced the whole series of aberrant auditory responses and oscillations previously reported in patients with schizophrenia. Accordingly, these findings suggest that developmental deficits in ErbB/EGF signaling might be involved in the auditory pathophysiology associated with schizophrenia

Schizophrenia Animal Modeling with Epidermal Growth Factor and Its Homologs: Their Connections to the Inflammatory Pathway and the Dopamine System

Epidermal growth factor (EGF) and its homologs, such as neuregulins, bind to ErbB (Her) receptor kinases and regulate glial differentiation and dopaminergic/GABAergic maturation in the brain and are therefore implicated in schizophrenia neuropathology involving these cell abnormalities. In this review, we summarize the biological activities of the EGF family and its neuropathologic association with schizophrenia, mainly overviewing our previous model studies and the related articles. Transgenic mice as well as the rat/monkey models established by perinatal challenges of EGF or its homologs consistently exhibit various behavioral endophenotypes relevant to schizophrenia. In particular, post-pubertal elevation in baseline dopaminergic activity may illustrate the abnormal behaviors relevant to positive and negative symptoms as well as to the timing of this behavioral onset. With the given molecular interaction and transactivation of ErbB receptor kinases with Toll-like receptors (TLRs), EGF/ErbB signals are recruited by viral infection and inflammatory diseases such as COVID-19-mediated pneumonia and poxvirus-mediated fibroma and implicated in the immune–inflammatory hypothesis of schizophrenia. Finally, we also discuss the interaction of clozapine with ErbB receptor kinases as well as new antipsychotic development targeting these receptors

Neuropathologic Implication of Peripheral Neuregulin-1 and EGF Signals in Dopaminergic Dysfunction and Behavioral Deficits Relevant to Schizophrenia: Their Target Cells and Time Window

Neuregulin-1 and epidermal growth factor (EGF) are implicated in the pathogenesis of schizophrenia. To test the developmental hypothesis for schizophrenia, we administered these factors to rodent pups, juveniles, and adults and characterized neurobiological and behavioral consequences. These factors were also provided from their transgenes or infused into the adult brain. Here we summarize previous results from these experiments and discuss those from neuropathological aspects. In the neonatal stage but not the juvenile and adult stages, subcutaneously injected factors penetrated the blood-brain barrier and acted on brain neurons, which later resulted in persistent behavioral and dopaminergic impairments associated with schizophrenia. Neonatally EGF-treated animals exhibited persistent hyperdopaminergic abnormalities in the nigro-pallido-striatal system while neuregulin-1 treatment resulted in dopaminergic deficits in the corticolimbic dopamine system. Effects on GABAergic and glutamatergic systems were transient or limited. Even in the adult stage, intracerebral administration and transgenic expression of these factors produced similar but not identical behavioral impairments, although the effects of intracerebral administration were reversible. These findings suggest that dopaminergic development is highly vulnerable to circulating ErbB ligands in the pre- and perinatal stages. Once maldevelopment of the dopaminergic system is established during early development, dopamine-associating behavioral deficits become irreversible and manifest at postpubertal stages

Pallidal dopamine release enhanced in EGF-treated rats and normalized with risperidone.

<p>Neonatal rats were treated with EGF or cytochrome c (control) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025831#pone-0025831-g001" target="_blank">Figure 1</a>. Risperidone (1 mg/kg, i.p.) was administered to some of the EGF-treated rats for 14 days at the adult stage. (A) The location of dialysis probe was examined and is shown in rat brain atlas. Six rats were excluded for incorrect probe placement. The digit represents the distance from the bregma. (B) PPI levels were monitored in control, EGF-treated and EGF+risperidone-treated animals following microdialysis. (C) Pulse-alone startle responses and PPI levels were monitored in control, EGF-treated and EGF+risperidone-treated animals following microdialysis. (D) Basal concentrations of dopamine in dialysates were monitored for 150 min, dopamine release was evoked by perfusion of 80 mM KCl over 60 min (solid bar), and then monitored over 150 min. Data represent dopamine concentrations in 30-min fractions (nM, mean ± SEM, N = 11–13 rats per group). There was a significant interaction between time and dopamine release [F(12,198) = 2.72, P = 0.002]. *P<0.05, ***P<0.001, compared with controls and ++P<0.01, compared with EGF-treated rats by Fisher's LSD.</p