Soluble Intracellular Protein Research Articles

A Set of Active Promoters with Different Activity Profiles for Superexpressing Rhodococcus Strain.

Rhodococcus bacteria are a promising platform for biodegradation, biocatalysis, and biosynthesis, but the use of rhodococci is hampered by the insufficient number of both platform strains for expression and promoters that are functional and thoroughly studied in these strains. To expand the list of such strains and promoters, we studied the expression capability of the Rhodococcus rhodochrous M33 strain, and the functioning of a set of recombinant promoters in it. We showed that the strain supports superexpression of the target enzyme (nitrile hydratase) using alternative inexpensive feedings-acetate and urea-without growth factor supplementation, thus being a suitable expression platform. The promoter set included Ptuf (elongation factor Tu) and Psod (superoxide dismutase) from Corynebacterium glutamicum ATCC13032, Pcpi (isocitrate lyase) from Rhodococcus erythropolis PR4, and Pnh (nitrile hydratase) from R. rhodochrous M8. Activity levels, regulation possibilities, and growth-phase-dependent activity profiles of these promoters were studied in derivatives of the M33 strain. The activities of the promoters were significantly different (Pcpi < Psod ≪ Ptuf < Pnh), covering 103-fold range, and the most active Pnh and Ptuf produced up to a 30-50% portion of target protein in soluble intracellular proteins. On the basis of the mRNA quantification and amount of target protein, the production level of Pnh was positioned close to the theoretical upper limit of expression in a bacterial cell. A selection method for the laboratory evolution of such active promoters directly in Rhodococcus was also proposed. Concerning regulation, Ptuf could not be regulated (2-fold change), while others were tunable (6-fold for Psod, 79-fold for Pnh, and 44-fold for Pcpi). The promoters possessed four different activity profiles, including three with peak of activity at different growth phases and one with constant activity throughout the growth phases. Ptuf and Pcpi did not change their activity profile under different growth conditions, whereas the Psod and Pnh profiles changed depending on the growth media. The results allow flexible construction of Rhodococcus strains using the studied promoters, and demonstrate a valuable approach for complex characterization of promoters intended for biotechnological strain construction.

Protein O-mannosylation affects protein secretion, cell wall integrity and morphogenesis in Trichoderma reesei

Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1and ΔTrpmt4were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved inO-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved inO-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

Salt Tolerance Mechanism and Species Identification of the Plant Rhizosphere Bacterium JYZ-SD2.

A salt-tolerant microbe strain JYZ-SD2 was investigated to develop biological soil amendments to stimulate salix growth and acclimation in costal salt-affected soils. The salt tolerance mechanism of strain JYZ-SD2 was investigated by detecting the salt-tolerant growth characteristics, biofilm formation, ion distribution, secondary metabolites, and zymogram profiling. The strain was identified by physiological and biochemical characteristics (Biolog), 16S rDNA sequencing, and cry1/7/9 gene expressing. With increasing of NaCl concentration, strain JYZ-SD2 adapted to the increased osmotic pressure by prolonging the retardation period, slowing down the growth rate of the logarithmic phase, increasing spo0A gene expression, increasing biofilm formation, reducing Na+ uptake, and changing the expression of metabolites and intracellular soluble proteins. The results showed that strain JYZ-SD2 could be assigned to Bacillus cereus.

A High-Throughput Dose-Response Cellular Thermal Shift Assay for Rapid Screening of Drug Target Engagement in Living Cells, Exemplified Using SMYD3 and IDO1

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation–based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

Molecular characterization and transcriptional analysis of a crustacean heat shock protein 10 gene in shrimp Litopenaeus vannamei

Heat shock proteins (HSPs) are the most abundant and ubiquitous soluble intracellular proteins which conserved phylogenetically in all living organisms from archaebacteria to humans. Recent research achievements indicated that HSP10s might not only be involved in the responses to environmental stresses, but also play a pivotal role in the host defenses mechanism. In the present study, a cDNA of 715 bp for the Pacific white shrimp Litopenaeus vannamei HSP10 (designated as LvHSP10) was cloned via rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of LvHSP10 contained an open reading frame (ORF) of 309 bp, which encoded a protein of 102 amino acids. The protein sequence of LvHSP10 shared over 80 % similarity with previously identified HSP10s. There were a CPN10 domain and a chaperonins HSP10/CPN10 signature in the protein sequence of LvHSP10. The mRNA transcripts of LvHSP10 were constitutively expressed in all the tested tissues, including eyestalk, gill, gonad, heart, hemocytes, hepatopancreas, intestine, muscle, nerve and stomach, with the highest expression level in hepatopancreas. The mRNA expression profiles of LvHSP10 in hepatopancreas could be significantly induced by the stimulation of Vibrio parahaemolyticus, white spot syndrome virus (WSSV), and low and high pH challenge. These results provided useful information of the potential roles of LvHSP10 in the defense mechanism of shrimp against various biological stimulations and multiple environmental stresses.

Enhanced killing of Escherichia coli using a combination of polyhexamethylene biguanide hydrochloride and 1-bromo-3-chloro-5,5- dimethylimidazolidine-2,4-dione.

The bactericidal activities of polyhexamethylene biguanide hydrochloride (PHMB), 1-bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione (BCDMH) and the combination of the two (designated as PB) were compared using Escherichia coli as the test organism. PB exhibited strong bactericidal activity: 10 mg/L PHMB combined with 8 mg/L BCDMH resulted in approximately 5.74 log10 reduction (LR), whereas 320 mg/L PHMB or 20 mg/L BCDMH was about 5.53 and 6.56 LR, respectively. Analyses using scanning electron microscopy, flow cytometry and atomic absorption spectroscopy indicated that PB, PHMB and BCDMH disrupted cell membranes and changed membrane structure and permeability, resulting in the leakage of intracellular soluble proteins and ions. PB exerted stronger effects on potassium and magnesium leakage, membrane potential and permeability than BCDMH did. PB caused less protein leakage than PHMB did. These results suggest that at a relatively low concentration, PB exhibited good bactericidal activity and physiological effect on E. coli.

Inhibitor activity of different medicinal plants extracts from Thuja orientalis, Nigella sativa L., Azadirachta indica and Bougainvillea spectabilis against Zucchini yellow mosaic virus (ZYMV) infecting Citrullus lanatus

ABSTRACTZucchini yellow mosaic virus (ZYMV) has a wide distribution in Mecca regions and is considered to be a serious threat on watermelon production. The aim of this study was to evaluate the antiviral activity of five medicinal plant extracts, against ZYMV-infecting watermelon plants. The experiment consisted of four application of soaking seeds, in vitro and in vivo experiments. The results of greenhouse experiments with individually treatments by decoction and infusion of Nigella sativa (NS) seeds in soaking and in-vitro experiment, bougainvillea and neem in pre-inoculation and thuja extract following nigella decoction in post inoculation were efficient to reduce and\\or inhibit symptoms expression infecting watermelon seedling, whereas, decoction exhibited higher antiviral activity against ZYMV than infusion. The decoction and infusion of NS seed increased the total photosynthesis pigments, total soluble intracellular protein and total phenol contents. In conclusion, the results of the present investigation propose that the bougainvillea and neem extracts may perhaps play a central function in the mechanism of acquired systemic resistance and may produce pathogenesis-related proteins that play a significant role in watermelon seedling resistance against ZYMV.

Early Perspective: Microplate Applications of the Cellular Thermal Shift Assay (CETSA)

The cellular thermal shift assay (CETSA) was introduced in 2013 as a means to assess drug binding in complex environments such as cell lysates, live cells, and even tissues. The assay principle relies on the well-proven biophysical concept of ligand-induced thermal stabilization of proteins, which in CETSA applications is measured as a persistent presence of soluble protein at elevated temperatures. Given its recent development, we have just started to learn about the benefits and pitfalls of the method as it is applied to a growing number of protein target classes, the majority of which are intracellular soluble proteins. One of the early technology developments concerned the transfer of the original assay procedure from PCR tubes and Western blot detection of soluble protein to a homogeneous assay in high-density microplates. A move to high-throughput formats is essential for a more systematic application in drug discovery settings, as well as in academic efforts for validating chemical probes through studies of structure–activity relationships. This perspective aims at providing an overview of knowledge gained in microplate formatting of CETSA and makes an attempt at forecasting future applications.

Like prions: the propagation of aggregated tau and α-synuclein in neurodegeneration.

The abnormal aggregation of a small number of known proteins underlies the most common human neurodegenerative diseases. In tauopathies and synucleinopathies, the normally soluble intracellular proteins tau and α-synuclein become insoluble and filamentous. In recent years, non-cell autonomous mechanisms of aggregate formation have come to the fore, suggesting that nucleation-dependent aggregation may occur in a localized fashion in human tauopathies and synucleinopathies, followed by seed-dependent propagation. There is a long prodromal phase between the formation of protein aggregates and the appearance of the first clinical symptoms, which manifest only after extensive propagation, opening novel therapeutic avenues.

Proteomic Investigation of Rhizoctonia solani AG 4 Identifies Secretome and Mycelial Proteins with Roles in Plant Cell Wall Degradation and Virulence.

Rhizoctonia solani AG 4 is a soilborne necrotrophic fungal plant pathogen that causes economically important diseases on agronomic crops worldwide. This study used a proteomics approach to characterize both intracellular proteins and the secretome of R.solani AG 4 isolate Rs23A under several growth conditions, the secretome being highly important in pathogenesis. From over 500 total secretome and soluble intracellular protein spots from 2-D gels, 457 protein spots were analyzed and 318 proteins positively matched with fungal proteins of known function by comparison with available R.solani genome databases specific for anastomosis groups 1-IA, 1-IB, and 3. These proteins were categorized to possible cellular locations and functional groups and for some proteins their putative roles in plant cell wall degradation and virulence. The majority of the secreted proteins were grouped to extracellular regions and contain hydrolase activity.

Inhibitory activity of different medicinal extracts from Thuja leaves, ginger roots, Harmal seeds and turmeric rhizomes against Fig leaf mottle-associated virus 1 (FLMaV-1) infecting figs in Mecca region.

Fig leaf mottle-associated virus-1 (FLMaV-1) is a closterovirus newly identified in fig trees, in the Mecca region, suffering from mosaic disease symptoms and apparently is compromising the fig plantation in the country. In the present study, we demonstrated the efficiency of two in vivo experiments including pre and post treatments using Thuja leaf, ginger roots, Harmal seeds and turmeric rhizome extracts on symptoms expression of rooted cuttings infected with FLMaV-1- and their impact on virus multiplication. Results showed that individual treatments with ginger roots and turmeric rhizomes in pre-grafting experiments and Thuja extract following Harmal seeds in post grafting experiments were efficient against symptom development. In addition, results showed that the total photosynthesis pigments; total soluble intracellular proteins and total phenol contents were higher in infected treated cuttings compared with healthy ones, thus it was taken as evidence on a mutual interaction between these extracts and virus multiplication.

The flavonoid, rutin, ameliorates neurodegeneration in the retina of diabetic rats (CAM1P.164)

Abstract Diabetic retinopathy remains the leading cause of blindness worldwide. Dietary flavonoid, rutin may protect the diabetes induced retinal damage. The purpose of this study was to examine the ameliorative effect of rutin on diabetic rat retina through neurotrophic, inflammatory and immunological parameters. Diabetes was induced in male Wistar rats by streptozotocin (65mg/kg). Rutin (100mg/kg) was orally administered to normal and diabetic rats after two weeks of diabetes induction and continued for five weeks. After treatments, retinas were isolated and analyzed for potential neuroinflammatory and immunological markers. Our results indicate that the reduced level of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the diabetic retina were significantly improved by rutin. Increased levels of soluble intracellular adhesion molecule (sICAM-1) and monocyte chemotactic protein (MCP-1) in the diabetic retinas were significantly attenuated to normal level by rutin. Reduced level of GSH in diabetic retina was increased and the increased level of TBARS in the diabetic retina was significantly decreased by rutin administration. The increased level of caspase-3, and decreased level of Bcl-2 in diabetic retina were significantly ameliorated to normal levels by rutin. These results suggest the effectiveness of rutin in protection of diabetic induced damage to the retina by maintaining neurotrophic support, and antioxidants levels.

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.

Heterologous expression of a recombinant lactobacillal β-galactosidase in Lactobacillus plantarum: effect of different parameters on the sakacin P-based expression system.

BackgroundTwo overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase.ResultsVarious factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35–40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase.ConclusionsThe over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.

Development of an enzyme-linked-receptor assay based on Syrian hamster β2-adrenergic receptor for detection of β-agonists

β-Adrenergic agonists (β-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of β-agonists, a β2-adrenergic receptor (β2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant β2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of β-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized β2-AR proteins by β-agonists. The IC50 and limit of detection values for ractopamine were 30.38μgL−1 and 5.20μgL−1, respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of β-agonists in animal feeds.

Total soluble protein profiles of Beauveria bassiana and their relationship with virulence against Helicoverpa armigera

Intracellular total soluble proteins of Beauveria bassiana are believed to play an important role in virulence against insect hosts. Thirty B. bassiana isolates collected from different geographical regions and host ranges were characterised by total soluble proteins present in cells, using the SDS–PAGE technique to differentiate the isolates based on virulence and host insect origin. In vitro analysis of total soluble protein profiles of 30 isolates was studied to understand the relationship of isolates with their host of origin and virulence against Helicoverpa armigera. There was a positive relationship between virulence and host origin. All the non-virulent isolates are grouped together. Similarly, highly virulent isolates against H. armigera were grouped together. The relationship between total soluble proteins and pathogenicity was positively correlated. Thirty isolates shared only 22% similarity in their protein profiles.

Induction of Heat Shock Protein 70 Ameliorates Ultraviolet-Induced Photokeratitis in Mice

Acute ultraviolet (UV) B exposure causes photokeratitis and induces apoptosis in corneal cells. Geranylgeranylacetone (GGA) is an acyclic polyisoprenoid that induces expression of heat shock protein (HSP)70, a soluble intracellular chaperone protein expressed in various tissues, protecting cells against stress conditions. We examined whether induction of HSP70 has therapeutic effects on UV-photokeratitis in mice. C57 BL/6 mice were divided into four groups, GGA-treated (500 mg/kg/mouse) and UVB-exposed (400 mJ/cm2), GGA-untreated UVB-exposed (400 mJ/cm2), GGA-treated (500 mg/kg/mouse) but not exposed and naive controls. Eyeballs were collected 24 h after irradiation, and corneas were stained with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). HSP70, reactive oxygen species (ROS) production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and protein kinase B (Akt) expression were also evaluated. Irradiated corneal epithelium was significantly thicker in the eyes of mice treated with GGA compared with those given the vehicle alone (p < 0.01). Significantly fewer TUNEL-positive cells were observed in the eyes of GGA-treated mice than controls after irradiation (p < 0.01). Corneal HSP70 levels were significantly elevated in corneas of mice treated with GGA (p < 0.05). ROS signal was not affected by GGA. NF-κB activation was reduced but phospho-(Ser/Ther) Akt substrate expression was increased in corneas after irradiation when treated with GGA. GGA-treatment induced HSP70 expression and ameliorated UV-induced corneal damage through the reduced NF-κB activation and possibly increased Akt phosphorilation.

Nucleocytoplasmic Coagulation: An Injury-Induced Aggregation Event that Disulfide Crosslinks Proteins and Facilitates Their Removal by Plasmin

Cellular injury causes a myriad of processes that affect proteostasis. We describe nucleocytoplasmic coagulation (NCC), an intracellular disulfide-dependent protein crosslinking event occurring upon late-stage cell death that orchestrates the proteolytic removal of misfolded proteins. Invitro and invivo models of neuronal injury show that NCC involves conversion of soluble intracellular proteins, including tubulin, into insoluble oligomers. These oligomers, also seenin human brain tissue following neurotrauma, actasa cofactor and substrate for the plasminogen-activating system. In plasminogen(-/-) mice, levels of misfolded β-tubulin were elevated and its clearance delayed following neurotrauma, demonstratinga requirement for plasminogen in the removal of NCC constituents. While additional invivo studies will further dissect this phenomenon, our study clearly shows that NCC, a process analogous to the formation of thrombi, generates an aggregated protein scaffold that limits release of cellular components and recruits clearance mechanisms to the site of injury.

Production of Extracellular Biopolymers and Identification of Intracellular Proteins and Rhizobium tropici

The objective of this study was to identify species of rhizobia (from the IPA 403 and IPA 49 isolates), to assess the physico-chemical characteristics of the biopolymers produced by these rhizobia and to determine the soluble intracellular proteins that are present in these rhizobia. The polysaccharides containing acetyl and pyruvic acid groups that were produced by different strains that had been cultivated in yeast extract mannitol (YEM) medium for 132, 144, and 168 h were evaluated for yield, viscosity, and concentration. Based on the analysis of their partial 16S rDNA sequences, both isolates were identified as Rhizobium tropici. The polymers produced in liquid YEM medium were recovered, dried and weighed to determine culture yield. Soluble intracellular proteins were identified through the techniques of 2D-PAGE and mass spectrometry for cultures that were cultivated for 168 h. The largest biopolymer yield and the highest viscosity and concentration of acetyl and pyruvic acids were obtained from the IPA 403 isolate after 168 h of culture. The proteins that were identified for the CIAT 899 isolate included elongation factor TU, a chaperone; GroE/GroEs and a putative glycosyltransferase, all of which catalyze the production of polysaccharides. For the IPA 403 strain, dinitrogenase and nitrogenase iron proteins were found. In the IPA 49 strain, glyceraldehyde-3-phosphate dehydrogenase was found along with two other proteins, the beta subunit of an electron-transferring flavoprotein and a dehydrogenase.

Heterozygosity for the proteasomal Psmc1 ATPase is insufficient to cause neuropathology in mouse brain, but causes cell cycle defects in mouse embryonic fibroblasts

The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurones. We previously reported that the 26S proteasome was essential for neuronal homeostasis and survival in mouse brains following conditional genetic homozygous knockout of a key subunit of the multi-meric 26S proteasome (19S ATPase Psmc1). Here, we investigated the effects of Psmc1 heterozygosity in the mouse brain and primary mouse embryonic fibroblasts. Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1+/−) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1+/− MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1+/− MEFs.