Alpha-synuclein (αSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons αSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following αSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic αSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against αSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.Parkinson's disease (PD) is the second most common neurodegenerative disorder (1,2). Accruing evidence points to a causative role for the presynaptic protein alpha-synuclein (αSyn) in PD pathogenesis. αSyn is a major constituent of Lewy Bodies-cellular inclusions that are the hallmark pathological feature of PD and other neurodegenerative disorders collectively
The amyloid ␤ (A␤) peptide that accumulates in Alzheimer's disease brain is derived from the proteolytic processing of the amyloid precursor protein by ␤-and ␥-secretase activities. The ␤-secretase enzyme ␤-site amyloid precursor protein-cleaving enzyme (BACE) generates the N terminus of A␤ by cleavage at either Asp 1 (␤-site) or Glu 11 (␤-site), ultimately leading to the production of full-length A␤1-40/42 or truncated A␤11-40/42. The functional significance of this variable cleavage site specificity as well as the relative pathological impact of full-length versus N-terminally truncated A␤ remains largely unknown. In our analysis of BACE reactivity in cell culture, we found that the preference of the protease for either ␤-or ␤-cleavage was strongly dependent on intracellular localization. Within the endoplasmic reticulum, ␤-site proteolysis predominated, whereas in the trans-Golgi network, ␤-cleavage was favored. Furthermore, the contrasting cleavage site specificities of BACE were not simply due to differences in organelle pH or the oligosaccharide composition of the glycoproteins involved. Examination of post-mortem brain specimens revealed significant levels of A␤11-40/42 within insoluble amyloid pools. Taken together, these data support an important role for ␤-cleavage in the process of cerebral amyloid deposition and localize the processing event to the trans-Golgi network.Senile plaques, lesions composed largely of aggregated amyloid ␤ (A␤) 1 protein, are a pathologic hallmark of Alzheimer's disease (AD) (1, 2). A␤ is derived from proteolytic processing of the type 1 membrane glycoprotein APP (3, 4), and its deposition most likely represents a crucial causative event in AD pathogenesis (5). The membrane-anchored aspartyl protease BACE acts on APP first at its ␤-cleavage site (6 -10), generating a membrane-bound C-terminal stub (C99) whose subsequent proteolysis by a second enzyme, ␥-secretase, yields A␤. In an alternative cellular pathway precluding A␤ production, APP is initially cleaved by ␣-secretase activity, ultimately leading to the release of a shorter peptide known as p3 (Fig. 1A) (11).Full-length A␤ encompasses a well-defined 40-or 42-amino acid residue stretch within the APP backbone (A␤1-40 and A␤1-42). However, in cerebral amyloid deposits, numerous N-terminally truncated variants of A␤40 and A␤42 (NtA␤), frequently harboring additional structural modifications, have been isolated (2,12,13). Whereas the functional significance of this N-terminal heterogeneity remains unclear, a variety of NtA␤ species aggregate more quickly in vitro than their fulllength counterparts (14). Whereas most types of NtA␤ are assumed to arise from the proteolysis of full-length peptides after their release from cells in the central nervous system, two such variants, A␤11-40 and A␤11-42, are generated directly from APP by BACE proteolysis at an alternative site, termed ␤Ј, between Tyr 10 and Glu 11 of A␤ (8,15,16). This event initially produces a shorter C-terminal stub (C89), which then acts as a substrate for ␥-se...
Introducing mutations within the amyloid precursor protein (APP) that affect β- and γ-secretase cleavages results in amyloid plaque formation in vivo. However, the relationship between β-amyloid deposition and the subcellular site of Aβ production is unknown. To determine the effect of increasing β-secretase (BACE) activity on Aβ deposition, we generated transgenic mice overexpressing human BACE. Although modest overexpression enhanced amyloid deposition, high BACE overexpression inhibited amyloid formation despite increased β-cleavage of APP. However, high BACE expression shifted the subcellular location of APP cleavage to the neuronal perikarya early in the secretory pathway. These results suggest that the production, clearance, and aggregation of Aβ peptides are highly dependent on the specific neuronal subcellular domain wherein Aβ is generated and highlight the importance of perikaryal versus axonal APP proteolysis in the development of Aβ amyloid pathology in Alzheimer's disease.
Senile plaques (SPs), one of two defining lesions of Alzheimer's disease (AD), are composed of a mixture of full-length Abeta1-40/42, and N- or C-terminally truncated Abeta peptides, including Abeta11-40/42. Sequential proteolysis of amyloid precursor protein (APP) by beta- and gamma-secretases produces Abeta1-40/42, but beta-site APP-cleaving enzyme 1 (BACE1), the major beta-secretase, also generates Abeta11-40/42, and BACE1 overexpression in cultured cells results primarily in secretion of Abeta11-40/42. The ratio of Abeta11-40/42 to Abeta1-40/42 depends on the ratio of BACE1 to APP, and Abeta11-40/42 can be generated from both full-length APP and its carboxy-terminal fragment (C99). Here, we investigated the role of Abeta11-40/42 in the pathogenesis of AD and Down's syndrome (DS) brains. We demonstrated significant amount of Abeta11-42 in DS brains by Western blots. While pyroAbeta11-42-modified Abeta species existed predominantly in mature SP cores in AD brain sections, both unmodified free Abeta11-40 and pyro-modified Abeta11-40 are detected in vascular amyloid deposits by immunohistochemistry. Using novel ELISAs for quantifying free Abeta11-40/42 and pyroAbeta11-40/42, we showed that insoluble Abeta11-42 predominated in extracts of AD and DS brains. This is the first systematic study of Abeta11-40/42 in neurodegenerative Abeta amyloidosis implicating Abeta11-40/42 in SP formation of AD and DS brains. The detection of Abeta11-42 in young DS brain suggests an early role for this N-terminally truncated Abeta peptide in the pathogenesis of SPs in AD and DS.
Amyloid beta peptides (A beta) are generated by the proteolytic processing of the amyloid beta precursor protein (APP). The newly identified beta-site APP-cleaving enzyme (BACE) cleaves APP at Asp1 as well as between Tyr10 and Glu11 of A beta, producing C-terminal fragments (CTFs) C99 and C89, respectively. Subsequent cleavage by gamma-secretase gives rise to A beta 1-40/42 and A beta 11-40/42. Although both full-length and A beta peptides truncated at residue 11 have been identified in amyloid plaques in the AD brain, the relative proportion of these two cleavage products produced by BACE and secreted into the medium by cultured cells is unknown. Using cell lines stably overexpressing BACE, we found that A beta 11-40 and A beta 11-42 are major A beta cleavage products generated by BACE. We further showed that BACE utilizes both full-length APP as well as C99 as substrates for the production of C89, and that A beta 11-40/42 can be generated by sequential cleavage of single APP molecules by BACE and gamma-secretase. Taken together, the abundance of A beta 11-40/42 produced by BACE suggests that their roles in AD pathogenesis may be underestimated.
Purpose: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC. Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma.Experimental Design: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second ratelimiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity.Results: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance.Conclusions: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted.
Prefabrication is considered as a modern method of construction, transferring part of on-site work into factories and generating a new decoupling point in supply chain. Management of prefabricated component (PC) suppliers plays a key role in the performance of prefabricated construction project (PCP). Evaluating and improving supplier management maturity (SMM) become critical issues for large contractors. Previous research on PCP supply chain mainly focused on technology, process and performance aspects, while paying little attention to supplier relationship management. This study proposes an assessment criteria system of supplier management from five dimensions, including procurement process, operation efficiency, relationship coordination and strategy alignment and corporate social responsibility. A maturity grid with five levels is designed to present continuous improvement of supplier management. 34 large PCP construction firms listed in contractor directory were investigated in China through semi-structured interview and questionnaire survey. Independent two-samples t-test and one-way analysis of variance were adopted to explore SMM of different groups according to business scope, procurement method and construction experience. The results indicate that the overall maturity level of supplier management is relatively low, implying that supply activity cannot achieve inter-organization integration. Management on relationship coordination and strategy alignment lag behind procurement management and operation efficiency. Single business contractors have higher SMM than diversified ones. Contractors integrated in engineering, procurement and construction are superior to design-bid-build contractors in supplier management, especially in relationship coordination and strategic alignment. Construction experience is conductive to supplier operation efficiency. Noticeably, there are no statistically significant differences in the performance of corporate social responsibility among different groups. These results will contribute to developing a benchmark framework for contractors to evaluate SMM and achieve continuous improvements in PCP.
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