Genetic disruption of mammalian endoplasmic reticulum-associated protein degradation: Human phenotypes and animal and cellular disease models

Endoplasmic reticulum-associated protein degradation (ERAD) is a stringent quality control mechanism through which misfolded, unassembled and some native proteins are targeted for degradation to maintain appropriate cellular and organelle homeostasis. Several in vitro and in vivo ERAD-related studies have provided mechanistic insights into ERAD pathway activation and its consequent events; however, a majority of these have investigated the effect of ERAD substrates and their consequent diseases affecting the degradation process. In this review, we present all reported human single-gene disorders caused by genetic variation in genes that encode ERAD components rather than their substrates. Additionally, after extensive literature survey, we present various genetically manipulated higher cellular and mammalian animal models that lack specific components involved in various stages of the ERAD pathway.     

Dopamine and the Dopamine Oxidation Product 5,6-Dihydroxylindole Promote Distinct On-Pathway and Off-Pathway Aggregation of α-Synuclein in a pH-Dependent Manner

The deposition of α-synuclein (α-syn) aggregates in dopaminergic neurons is a key feature of Parkinson's disease. While dopamine (DA) can modulate α-syn aggregation, it is unclear which other factors can regulate the actions of DA on α-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of α-syn by DA. We show that α-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of α-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of α-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of α-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with β-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with α-syn to generate the α-syn aggregates implicated in Parkinson's disease.     

Copper and Zinc Mediated Oligomerisation of Aβ Peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.     

Selinexor Overcomes Hypoxia-Induced Drug Resistance in Multiple Myeloma

Increased levels of the nuclear export protein, exportin 1 (XPO1), were demonstrated in multiple myeloma (MM) patients. Targeting XPO1 with selinexor (the selective inhibitor of nuclear export; SINE compound KPT-330) demonstrates broad antitumor activity also in patient cells resistant to bortezomib; hence, it is a promising target in MM patients. Hypoxia is known to mediate tumor progression and drug resistance (including bortezomib resistance) in MM cells. In this study, we tested the effects of selinexor alone or in combination with bortezomib in normoxia and hypoxia on MM cell survival and apoptosis in vitro and in vivo. In vitro, selinexor alone decreased survival and increased apoptosis, resensitizing MM cells to bortezomib. In vivo, we examined the effects of selinexor alone on tumor initiation and tumor progression, as well as selinexor in combination with bortezomib, on tumor growth in a bortezomib-resistant MM xenograft mouse model. Selinexor, used as a single agent, delayed tumor initiation and tumor progression, prolonging mice survival. In bortezomib-resistant xenografts, selinexor overcame drug resistance, significantly decreasing tumor burden and extending mice survival when combined with bortezomib.     

Novel Tumor Suppressor Function of Glucocorticoid-Induced TNF Receptor GITR in Multiple Myeloma

Glucocorticoid-induced TNF receptor (GITR) plays a crucial role in modulating immune response and inflammation, however the role of GITR in human cancers is poorly understood. In this study, we demonstrated that GITR is inactivated during tumor progression in Multiple Myeloma (MM) through promoter CpG island methylation, mediating gene silencing in primary MM plasma cells and MM cell lines. Restoration of GITR expression in GITR deficient MM cells led to inhibition of MM proliferation in vitro and in vivo and induction of apoptosis. These findings were supported by the presence of induction of p21 and PUMA, two direct downstream targets of p53, together with modulation of NF-κB in GITR-overexpressing MM cells. Moreover, the unbalanced expression of GITR in clonal plasma cells correlated with MM disease progression, poor prognosis and survival. These findings provide novel insights into the pivotal role of GITR in MM pathogenesis and disease progression.     

Effect of trace elements on the seminal oxidative status and correlation to sperm motility in infertile Saudi males

The impact of trace elements, especially zinc, selenium, copper, and magnesium, on male fertility has gained great interest and significance. Increased oxidative stress and altered trace element levels are probable etiological factors underlying male reproductive dysfunction and infertility. The present study focused on the evaluation of seminal oxidative markers, such as reactive oxygen species (ROS), malondialdehyde (MDA), and total antioxidant capacity (TAC), and trace element levels in the normozoospermic fertile control group (n = 40) and asthenozoospermic infertile group (n = 30). Semen from infertile men exhibited significantly higher ROS and MDA levels accompanied with significant decline in TAC and trace element (zinc and magnesium) levels. Furthermore, a significant correlation was observed between trace elements and oxidative markers with sperm motility. The current study revealed increased lipid peroxidation and oxidant-reductant imbalance that leads to deterioration of semen quality and male infertility. Thus, oxidative stress and trace elements can be considered important biomarkers of male infertility. Measurement of seminal oxidative stress with conventional seminological parameters must be integrated in fertility assessment from early stages to ensure healthy semen characteristics and fertility in men.     

Teaching cases: A 1-week-old newborn with hypercalcemia and palpable nodules: subcutaneous fat necrosis

Abstract: We present a 1-week-old newborn with subcutaneous fat necrosis complicated by hypercalcemia. She received conservative treatment of adequate hydration and restricted supplementary vitamin D.The case: A 1-week-old term newborn girl was brought to her physician with a 2-day history of subcutaneous masses. The girl had been born by vacuum-assisted vaginal delivery with a birth weight of 3.5 kg. She did not require resuscitation but was observed for 24 hours in a special care nursery because of tachypnea. The patient was discharged home after 48 hours, and her course over the next 5 days was unremarkable.On physical examination, the newborn was afebrile and in no obvious distress. She had multiple firm, mobile, mildly tender subcutaneous nodules with overlying erythema (Figure 1). The largest mass on palpation was located in the left deltoid area and measured 2 × 2.5 cm (Figure 1). Two smaller lesions were located in the left posterior axillary area (Figure 1) and a fourth lesion was in the right posterior auricular region. The remainder of the physical examination and the results of a complete blood count were normal. The newborn''s total serum calcium level was elevated (2.94 [normal 1.96–2.66] mmol/L) as was her ionized calcium level (1.36 [normal 1.14–1.29] mmol/L. A biopsy was not performed because the infant was well and the results of clinical investigations were consistent with subcutaneous fat necrosis.Open in a separate windowFigure 1: A 5-day-old infant with lesions of subcutaneous fat necrosis. The largest mass (black arrow) measured 2 × 2.5 cm. Two smaller lesions (white arrows) were located in the left posterior axillary area.Subcutaneous fat necrosis of the newborn is a relatively uncommon condition that occurs in the first several weeks after birth. The incidence is unknown; however, it is more frequently reported after perinatal distress than after uncomplicated deliveries, and maternal risk factors include gestational diabetes and preeclampsia.¹ Skin lesions are characterized by indurated nodules that range from flesh-coloured to blue and by plaques on the face, trunk and buttocks as well as on the arms and legs near the trunk. Figure 2 is a representative microscopic image of this condition.² The differential diagnosis is bacterial cellulitis, erysipelas and sclerema neonatorum.³Open in a separate windowFigure 2: Left: A typical photomicrograph from a different patient showing lobular panniculitis with sparing of the dermis and epidermis (original magnification × 20). Right: A high-power view shows that the inflammatory infiltrate is mixed and composed of histiocytes, lymphocytes, neutrophils and eosinophils. Cleft-like spaces (arrow) suggestive of dissolved crystals can be seen at the periphery of some of the fat cysts (original magnification × 400). Reproduced with permission from Macmillan Publishers Ltd: Journal of Perinatology (Diamantis et al.²) © 2006.Although subcutaneous fat necrosis of the newborn is often benign and self-limited, the most important concern is hypercalcemia, which can lead to neurologic or cardiac problems, nephrocalcinosis and nephrolithiasis. Clinical signs of newborn hypercalcemia include irritability, poor feeding and vomiting. Skin lesions typically resolve over a period of weeks to several months; however, hypercalcemia can persist longer and requires serial monitoring. The treatment of hypercalcemia ranges from conservative measures such as hydration and restriction of vitamin D and calcium to more aggressive interventions such as furosemide, glucocorticoid or bisphosphonate therapy in severe cases.⁴In our patient, mild hypercalcemia was accompanied by mild elevations in the ratio of calcium to creatinine in the urine and a normal 1,25-dihydroxyvitamin D level. Because our patient was otherwise well, we opted for conservative management. In 2 months, her calcium level had normalized and the lesions completely regressed.Fahed Aljaser MD Michael Weinstein MD Division of Pediatric Medicine Hospital for Sick Children University of Toronto Toronto, Ont.     

Methionine regulates copper/hydrogen peroxide oxidation products of Abeta.

Metal-catalysed oxidation (MCO) may play a causative role in the pathogenesis of Alzheimer's disease (AD). Amyloid beta peptide (Abeta), the major biomarker of AD, in the presence of copper ions reduces Cu(2+) to Cu(+) and catalyses the formation of H(2)O(2) that subsequently induces radicals through Fenton chemistry. Abeta is also subject to attack by free radicals, where the presence of Cu(2+) in conjunction with H(2)O(2) catalyses oxygenation, primarily at the methionine sulfur atom. This work investigates MCO of Abeta, to gain further insight into the role of oxidative stress in AD. By combining a fluorescence assay with gel electrophoresis to monitor MCO reactions of Abeta (1-28) in the presence and absence of methionine it was determined that methionine can both protect some residues against MCO and promote the oxidation of Tyr(10) specifically. Electrospray ionization mass spectrometric analysis of methionine MCO products indicated the formation of methionine sulfoxide, methionine sulfone and related hydroxylated products. Similar products could be formed from the oxidation of Met(35) of Abeta and may relate to changes in properties of the peptide following MCO.     

Neurotoxic,redox-competent Alzheimer's beta-amyloid is released from lipid membrane by methionine oxidation

The amyloid beta peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Abeta peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Abeta) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Abeta. However, unlike the unoxidized peptide, Met(O)Abeta is unable to penetrate lipid membranes to form ion channel-like structures, and beta-sheet formation is inhibited, phenomena that are central to some theories for Abeta toxicity. Our results show that, like the unoxidized peptide, Met(O)Abeta will coordinate Cu2+ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Abeta production contributes to the elevation of soluble Abeta seen in the brain in Alzheimer's disease.