Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca²⁺/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events.     

Oxidative Stress, NF-κB and the Ubiquitin Proteasomal Pathway in the Pathology of Calpainopathy

The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-κB signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-κB/IKK β signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-κB signaling could be considered for treatment of LGMD 2A.     

Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase: Signal transduction and synaptic transmission

The metabotropic glutamate receptors (mGluRs) fine-tune the efficacy of synaptic transmission. This unique feature makes mGluRs potential targets for the treatment of various CNS disorders. There is ample evidence to show that the ubiquitin proteasome system mediates changes in synaptic strength leading to multiple forms of synaptic plasticity. The present study describes a novel interaction between post-synaptic adaptors, long Homer-3 proteins, and one of the 26S proteasome regulatory subunits, the S8 ATPase, that influences the degradation of the metabotropic glutamate receptor 1α (mGluR1α). We have shown that the two human long Homer-3 proteins specifically interact with human proteasomal S8 ATPase. We identified that mGluR1α and long Homer-3s immunoprecipitate with the 26S proteasome both in vitro and in vivo. We further found that the mGluR1α receptor can be ubiquitinated and degraded by the 26S proteasome and that Homer-3A facilitates this process. Furthermore, the siRNA mediated silencing of Homer-3 led to increased levels of total and plasma membrane-associated mGluR1α receptors. These results suggest that long Homer-3 proteins control the degradation of mGluR1α receptors by shuttling ubiquitinated mGluR-1α receptors to the 26S proteasome via the S8 ATPase which may modulate synaptic transmission.     

Is the parasitization capacity of Trichogramma cordubensis influenced by the age of the females?

We investigated the parasitization capacity of Trichogramma cordubensis Vargas & Cabello (Hymenoptera: Trichogrammatidae) females aged 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h, using Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) eggs as hosts. Wasps were held without hosts during the period of ageing, therefore being increasingly time-limited with respect to parasitization as they got older. The total number of parasitized hosts decreased as the age of the parasitoid increased. However, the proportion of lifetime parasitism carried out on the first day increased with wasp age, up to 120-h old females. These results show that the parasitization capacity of ageing T. cordubensis females changes as they become time-limited. The consequences of such changes for biological control programs are discussed.     

The CaVβ Subunit Protects the I-II Loop of the Voltage-gated Calcium Channel CaV2.2 from Proteasomal Degradation but Not Oligoubiquitination

Ca_Vβ subunits interact with the voltage-gated calcium channel Ca_V2.2 on a site in the intracellular loop between domains I and II (the I-II loop). This interaction influences the biophysical properties of the channel and leads to an increase in its trafficking to the plasma membrane. We have shown previously that a mutant Ca_V2.2 channel that is unable to bind Ca_Vβ subunits (Ca_V2.2 W391A) was rapidly degraded (Waithe, D., Ferron, L., Page, K. M., Chaggar, K., and Dolphin, A. C. (2011) J. Biol. Chem. 286, 9598–9611). Here we show that, in the absence of Ca_Vβ subunits, a construct consisting of the I-II loop of Ca_V2.2 was directly ubiquitinated and degraded by the proteasome system. Ubiquitination could be prevented by mutation of all 12 lysine residues in the I-II loop to arginines. Including a palmitoylation motif at the N terminus of Ca_V2.2 I-II loop was insufficient to target it to the plasma membrane in the absence of Ca_Vβ subunits even when proteasomal degradation was inhibited with MG132 or ubiquitination was prevented by the lysine-to-arginine mutations. In the presence of Ca_Vβ subunit, the palmitoylated Ca_V2.2 I-II loop was protected from degradation, although oligoubiquitination could still occur, and was efficiently trafficked to the plasma membrane. We propose that targeting to the plasma membrane requires a conformational change in the I-II loop that is induced by binding of the Ca_Vβ subunit.     

Differential regulation of the calpain–calpastatin complex by the L-domain of calpastatin

Here we demonstrate that the presence of the L-domain in calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca^2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which calpastatin levels are increased. Our findings show that the L-domain of calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.     

Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin.     

Phosphorylation of theα subunit of the sodium channel by protein kinase C

The alpha subunit of the purified voltage-sensitive sodium channel from rat brain is rapidly phosphorylated to the extent of 3-4 mol phosphate/mol by purified protein kinase C. The alpha subunit of the native sodium channel in synaptosomal membranes is also phosphorylated by added protein kinase C as assessed by specific immunoprecipitation and polyacrylamide gel electrophoresis of labeled membranes. Our results suggest coordinate regulation of sodium channel phosphorylation state by cAMP-dependent and calcium/phospholipid-dependent protein kinases.     

Regeneration of the progesterone 11α-hydroxylase of Rhizopus nigricans by the use of periodate

Summary The cell-free progesterone 11-hydroxylase enzyme of Rhizopus nigricans can be directly regenerated by periodate oxidation. This permits action of the enzyme over a period of hours with an activity similar to that in the presence of an NADPH generating system.     

CD3ζ-Chain Expression of Human T Lymphocytes Is Regulated by TNF via Src-like Adaptor Protein-Dependent Proteasomal Degradation

Decreased expression of the TCR ζ-chain has been reported in several autoimmune, inflammatory, and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. Although ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is less clear. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data have reported that SLAP regulates immunoreceptor signaling. We have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation. TNF treatment of human T lymphocytes (15-40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose-dependent manner (p < 0.05) and decreases activation-induced IL-2 expression (p < 0.01). Although blocking of the lysosomal compartment fails to restore TNF-induced CD3 ζ-chain downregulation, inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalization of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p < 0.05 and p < 0.01, respectively), whereas TNF-induced ζ-chain downregulation was inhibited by gene silencing of SLAP with small interfering RNA. SLAP levels of the CD4(+) T lymphocytes isolated from patients with rheumatoid arthritis were more than 2-fold higher than that of the healthy donors' (p < 0.05); moreover, TNF treatment did not alter the SLAP expression of the CD4(+) cells of anti-TNF therapy-treated patients. Our present data suggest that TNF modulates T cell activation during inflammatory processes by regulating the amount of CD3 ζ-chain expression via a SLAP-dependent mechanism. These data provide evidence for SLAP-dependent regulation of CD3 ζ-chain in the fine control of TCR signaling.     

Acceleration of the depolymerization of amyloid β fibrils by ultrasonication

Amyloid fibrils, rigid and filamentous aggregates associated with various diseases, are often difficult to depolymerize into monomers. Ultrasonication is a strong agitation that accelerates nucleation above the critical concentration of amyloid fibrillation. We examined the effects of ultrasonication on the fibrils of amyloid β(1–40) as well as on monomers. Ultrasonic pulses accelerated spontaneous fibrillation when the peptide concentration was above 1 μM. On the other hand, ultrasonic pulses accelerated the depolymerization of fibrils into monomers at 1 μM. These results indicate that, although amyloid fibrillation is a reversible process determined by thermodynamic stability, kinetically trapped supersaturation and physical difficulty of dissolving rigid fibrils prevent the smooth phase transitions. We propose that, in addition to accelerating the nucleation of fibrillation and fragmentation of fibrils above the critical concentration, ultrasonication is useful for dissolving fibrils below the critical concentration.     

Effect of progesterone pretreatment on the uptake of estradiol-17β by the uterine epithelium of the rat

Summary Progesterone pretreatment of ovariectomised rats resulted in a moderate (44%) lowering of the level of nuclear estradiol receptors found in the uterine epithelium 2 h after a single injection of this estrogen.     

Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

Ovarian cancer, one of inflammation-associated cancers, is the fifth leading cause of cancer deaths among women. Inflammation in the tumor microenvironment is associated with peritoneal tumor dissemination and massive ascites, which contribute to high mortality in ovarian cancer. Tumor suppressor p53 is frequently deleted or mutated in aggressive and high-grade ovarian cancer, probably aggravating cancer progression and increasing mortality. We therefore investigated the influence of p53 on proinflammatory chemokines in ovarian cancer cells. A PCR array of the chemokine network revealed that ovarian cancer cells with low or mutated p53 expression expressed high levels of proinflammatory chemokines such as CXCL1, 2, 3 and 8. Transient transfection of p53 into p53-null ovarian cancer cells downregulated proinflammatory chemokines induced by tumor necrosis factor-α (TNF), a proinflammatory cytokine abundantly expressed in ovarian cancer. Furthermore, p53 restoration or stabilization blocked TNF-induced NF-κB promoter activity and reduced TNF-activated IκB. Restoration of p53 increased ubiquitination of IκB, resulting from concurrently reduced proteasome activity followed by stability of IκB. A ubiquitination PCR array on restoration of p53 did not reveal any significant change in expression except for Mdm2, indicating that the balance between p53 and Mdm2 is more important in regulating NF-κB signaling rather than the direct effect of p53 on ubiquitin-related genes or IκB kinases. In addition, nutlin-3, a specific inducer of p53 stabilization, inhibited proinflammatory chemokines by reducing TNF-activated IκB through p53 stabilization. Taken together, these results suggest that p53 inhibits proinflammatory chemokines in ovarian cancer cells by reducing proteasomal degradation of IκB. Thus, frequent loss or mutation of p53 may promote tumor progression by enhancing inflammation in the tumor microenvironment.