Mechanism Of Protein Degradation Research Articles

Cell-Autonomous and Non-cell-Autonomous Pathogenic Mechanisms in Huntington's Disease: Insights from In Vitro and In Vivo Models.

Huntington's disease (HD) is an autosomal dominant disorder caused by an expansion in the trinucleotide CAG repeat in exon-1 in the huntingtin gene, located on chromosome 4. When the number of trinucleotide CAG exceeds 40 repeats, disease invariably is manifested, characterized by motor, cognitive, and psychiatric symptoms. The huntingtin (Htt) protein and its mutant form (mutant huntingtin, mHtt) are ubiquitously expressed but although multiple brain regions are affected, the most vulnerable brain region is the striatum. Striatal medium-sized spiny neurons (MSNs) preferentially degenerate, followed by the cortical pyramidal neurons located in layers V and VI. Proposed HD pathogenic mechanisms include, but are not restricted to, excitotoxicity, neurotrophic support deficits, collapse of the protein degradation mechanisms, mitochondrial dysfunction, transcriptional alterations, and disorders of myelin. Studies performed in cell type-specific and regionally selective HD mouse models implicate both MSN cell-autonomous properties and cell-cell interactions, particularly corticostriatal but also with non-neuronal cell types. Here, we review the intrinsic properties of MSNs that contribute to their selective vulnerability and in addition, we discuss how astrocytes, microglia, and oligodendrocytes, together with aberrant corticostriatal connectivity, contribute to HD pathophysiology. In addition, mHtt causes cell-autonomous dysfunction in cell types other than MSNs. These findings have implications in terms of therapeutic strategies aimed at preventing neuronal dysfunction and degeneration.

The ubiquitin-proteasome system in plant responses to environments.

The ubiquitin-proteasome system (UPS) is a rapid regulatory mechanism for selective protein degradation in plants and plays crucial roles in growth and development. There is increasing evidence that the UPS is also an integral part of plant adaptation to environmental stress, such as drought, salinity, cold, nutrient deprivation and pathogens. This review focuses on recent studies illustrating the important functions of the UPS components E2s, E3s and subunits of the proteasome and describes the regulation of proteasome activity during plant responses to environment stimuli. The future research hotspots and the potential for utilization of the UPS to improve plant tolerance to stress are discussed.

Targeted Control of Chloroplast Quality to Improve Plant Acclimation: From Protein Import to Degradation.

The chloroplast is an important energy-producing organelle acting as an environmental sensor for the plant cell. The normal turnover of the entire damaged chloroplast and its specific components is required for efficient photosynthesis and other metabolic reactions under stress conditions. Nuclear-encoded proteins must be imported into the chloroplast through different membrane transport complexes, and the orderly protein import plays an important role in plant adaptive regulation. Under adverse environmental conditions, the damaged chloroplast or its specific components need to be degraded efficiently to ensure normal cell function. In this review, we discuss the molecular mechanism of protein import and degradation in the chloroplast. Specifically, quality control of chloroplast from protein import to degradation and associated regulatory pathways are discussed to better understand how plants adapt to environmental stress by fine-tuning chloroplast homeostasis, which will benefit breeding approaches to improve crop yield.

Deubiquitylatinase inhibitor b-AP15 induces c-Myc-Noxa-mediated apoptosis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors in east Asia. However, the molecular mechanism underlying its progression remains unclear. The ubiquitin-proteasome system (UPS) is a central mechanism for protein degradation and turnover. Accumulating evidence showed that more and more deubiquitinases could serve as attractive anti-cancer target. The expression of USP14 and UCH37 in esophagus squamous cell carcinoma tissues were examined by immunohistochemistry and western blot assays. Effect of b-AP15, a USP14 and UCH37 inhibitor, on ESCC cell growth was evaluated by cell viability assay. After cell lines being treated with b-AP15, cell cycle, apoptosis and the expression of related proteins were further explored to investigate the anti-ESCC mechanism of b-AP15. Results showed that deubiquitinating enzymes (DUBs) USP14 and UCH37 expressed at higher levels in ESCC tissues than in adjacent tissues. b-AP15 could inhibit cell proliferation and induce G2/M cell cycle arrest and apoptosis in ESCC cells. Mechanistically, b-AP15 treatment triggered Noxa-dependent apoptosis, which was regulated by c-Myc. Silencing Noxa and c-Myc could reduce b-AP15-induced apoptosis in ESCC cells. Our results revealed a novel mechanism of anti-tumor activity of b-AP15 in ESCC, and b-AP15 could be used as a potential therapeutic agent in ESCC.

Mitochondrial degradation of amyloidogenic proteins — A new perspective for neurodegenerative diseases

This perspective article outlines mechanisms of mitochondrial import and protein degradation and how these have been linked to alpha-synuclein and Amyloid beta (Aβ) homeostasis. Our aim is to underpin and stimulate the debate on the recent conception of mitochondria as protein degrading organelles, which suggests that mitochondria are more directly involved in neurodegenerative diseases than previously assumed.

Establishment of a system for screening autophagic flux regulators using a modified fluorescent reporter and CRISPR/Cas9

Autophagy is a mechanism of bulk protein degradation that plays an important role in regulating homeostasis in many organisms. Among several methods for evaluating its activity, a fluorescent reporter GFP-LC3-RFP-LC3ΔG, in which GFP-LC3 is cleaved by ATG4 following autophagic induction and degraded in lysosome, has been used for monitoring autophagic flux, which is the amount of lysosomal protein degradation. In this study, we modified this reporter by exchanging GFP for pHluorin, which is more sensitive to low pH, and RFP to mCherry, to construct pHluorin-LC3-mCherry reporter. Following starvation or mTOR inhibition, the increase of autophagic flux was detected by a decrease of the fluorescent ratio of pHluorin to mCherry; our reporter was also more sensitive to autophagy-inducing stimuli than the previous one. To establish monitoring cells for mouse genome-wide screening of regulators of autophagic flux based on CRISPR/Cas9 system, after evaluating knockout efficiency of clones of Cas9-expressing MEFs, we co-expressed our reporter and confirmed that autophagic flux was impaired in gRNA-mediated knockout of canonical autophagy genes. Finally, we performed genome-wide gRNA screening for genes inhibiting starvation-mediated autophagic flux and identified previously reported genes such as Atgs. Thus, we have successfully established a system for screening of genes regulating autophagic flux with our pHluorin-LC3-mCherry reporter in mice.

2166-P: Sel1L-Hrd1 ERAD Is a Key Regulator of Beta-Cell Function and Identity

βcell survival and dedifferentiation are two hotly-debated mechanisms underlying βcell loss in type 2 diabetes; however, the underlying molecular mechanism remains largely unclear. Here we compare two principal protein quality control mechanisms, ER-associated protein degradation (ERAD) and autophagy and find that ERAD plays an important role in βcell dedifferentiation. Mice with βcell-specific Sel1L-Hrd1 deletion exhibit early-onset hyperglycemia and glucose intolerance as early as ~5 weeks of age. Surprisingly, in contrast to other tissue specific models of Sel1L-Hrd1 ERAD deficiency in which prohormones accumulate within the ER, Sel1L-Hrd1 ERAD deficiency impairs βcell function by attenuating insulin expression without an overt defect in proinsulin maturation and also triggers marked βcell dedifferentiation. There is, however, no overt activation of unfolded protein response (UPR) or proinsulin aggregation. In striking contrast to ERAD deficiency, loss of macroautophagy, a major mechanism for bulk protein degradation and organelle turnover, in βcells causes massive cell loss and hyperglycemia, but not βcell dedifferentiation. Thus, our data reveal a novel role of Sel1L-Hrd1 ERAD in the maintenance of βcell identify and demonstrate that impairment of ERAD contributes to the progression of type 2 diabetes in a manner distinct from that of autophagy. Disclosure N. Shrestha: None. X. Li: None. Y. Ji: None. R.B. Reinert: None. P. Arvan: None. L. Qi: None. Funding National Institutes of Health

Parkin Facilitates Proteasome Inhibitor-Induced Apoptosis via Suppression of NF-κB Activity in Hepatocellular Carcinoma

Background: The ubiquitin-proteasome system (UPS) is a tight homeostatic control mechanism of intracellular protein degradation and turnover involved in many human diseases. Proteasome inhibitors were initially developed as anticancer agents with potentially beneficial in the suppression of tumor growth. However, clinical trials of patients with solid tumors fail to demonstrate the same efficacy of these proteasome inhibitors. Methods: The expression of Parkin was examined by quantitative PCR and western blotting analyses and immunohistochemical assay. The effect of Parkin on tumor suppression in hepatocellular carcinoma (HCC) cells in vitro was measured using cell viability assay and cell death assay, soft agar clonogenic assay in vitro and a Xenografted tumor model in vivo. The underlying mechanisms in which Parkin -mediated NF-κB activity inhibition were determined via luciferase reporter assays, immunofluorescence staining, nuclear protein extraction assay, immunoprecipitation, and western blot analysis. Findings: Lower Parkin expression correlates with poor survival in patients with HCC. Ectopic Parkin expression enhances proteasome inhibitor-induced apoptosis and tumor suppression in HCC cells in vitro and in vivo. In contrast, knockdown of Parkin expression promotes apoptosis resistance and tumor growth. Mechanically, Parkin promotes TNF receptor-associated factor (TRAF) 2 and TRAF6 degradation and thus facilitates nuclear factor-kappa B (NF-κB) inhibition, which finally results in apoptosis. Interpretation: These findings reveal a direct molecular link between Parkin and protein degradation in the control of NF-κB pathway and may provide a novel UPS-dependent strategy for the treatment of HCC by induction of apoptosis. Funding: This work was supported by the Natural Science Foundation of China (grant numbers 81672874, 81502194), the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (GDUPS), the Distinguished Young Scholar of Guangdong Province (grant number 2015A030306033), the Natural Science Foundation of Guangdong Province (grant number 2015A030313468), the Young Scholar of Science and Technology of Guangdong Province (grant number 2016TQ03R801), the Innovative Academic Team of Guangzhou Education System (grant number 1201610014), the Natural Science Foundation research team of Guangdong Province (grant number 2018B030312001), the Research Team of Department of Education of Guangdong Province (grant number 2017KCXTD027),the Guangzhou key medical discipline construction project fun, Guangdong traditional Chinese medicine bureau project (grant number 20161178), and Guangzhou traditional Chinese medicine and traditional Chinese and western medicine science and technology project (grant numbers 2016A011020, 20182A011025). Declaration of Interest: The authors have no professional, personal or financial conflicts of interests relating to this publication. Ethical Approval: For the clinical materials used in our study, prior patient consent and approval from the Institutional Research Ethics Committee of Taizhou hospital were obtained.

IKKβ slows Huntington’s disease progression in R6/1 mice

Neuroinflammation is an important contributor to neuronal pathology and death in neurodegenerative diseases and neuronal injury. Therapeutic interventions blocking the activity of the inflammatory kinase IKKβ, a key regulator of neuroinflammatory pathways, is protective in several animal models of neurodegenerative disease and neuronal injury. In Huntington's disease (HD), however, significant questions exist as to the impact of blocking or diminishing the activity of IKKβ on HD pathology given its potential role in Huntingtin (HTT) degradation. In cell culture, IKKβ phosphorylates HTT serine (S) 13 and activates HTT degradation, a process that becomes impaired with polyQ expansion. To investigate the in vivo relationship of IKKβ to HTT S13 phosphorylation and HD progression, we crossed conditional tamoxifen-inducible IKKβ knockout mice with R6/1 HD mice. Behavioral assays in these mice showed a significant worsening of HD pathological phenotypes. The increased behavioral pathology correlated with reduced levels of endogenous mouse full-length phospho-S13 HTT, supporting the importance of IKKβ in the phosphorylation of HTT S13 in vivo. Notably, many striatal autophagy genes were up-regulated in HD vs. control mice; however, IKKβ knockout partially reduced this up-regulation in HD, increased striatal neurodegeneration, and enhanced an activated microglial response. We propose that IKKβ is protective in striatal neurons early in HD progression via phosphorylation of HTT S13. As IKKβ is also required for up-regulation of some autophagy genes and HTT is a scaffold for selective autophagy, IKKβ may influence autophagy through multiple mechanisms to maintain healthy striatal function, thereby reducing neuronal degeneration to slow HD onset.

Protein synthesis inhibition induces proteasome assembly and function

Protein synthesis and degradation balance have a crucial role in maintenance of cellular homeostasis and function. The ubiquitin-proteasome system is one of the major cellular proteolytic machineries responsible for the removal of normal, abnormal, denatured or in general damaged proteins. Proteasome is a multisubunit enzyme that consists of the 20S core and the 19S regulatory complexes giving rise to multiple active forms. In the present study we investigated the crosstalk between protein synthesis and proteasome-mediated protein degradation. Pharmacological protein synthesis inhibition led to increased proteasome function and assembly of 30S/26S proteasome complexes, in human primary embryonic fibroblasts. The enhancement in proteasome function counted for the degradation of ubiquitinated, misfolded and oxidized proteins. Additionally, it was found that heat shock proteins 70 and 90 are probably involved in the elevated proteasome assembly. Our results provide an insight on how the mechanisms of protein synthesis, protein degradation and heat shock protein chaperones machinery interact under various cellular conditions.

Functional characterization of p.Pro409His variant in HNF1A, a hypomorphic mutation involved in pancreatic β-cell dysfunction.

HNF1A is a gene coding for the transcription factor HNF1-α, mutated in some forms of MODY and type 2 diabetes mellitus characterized by a strong genetic component. The penetrance of HNF1A variants differs considerably; thus, to assess the genetic risk of diabetes in carrier subjects of a HNF1A mutant allele, a functional characterization of mutant forms is of paramount importance. The HNF1A gene was sequenced in two patients with partly discordant diabetic phenotype, carrying the p.Pro409His variant. To evaluate the pathogenicity of the variant, we measured the transactivation power of the corresponding P408H HNF1-α mutant mouse form on HNF1-α target promoters. We found a lower but detectable activity of transactivation of the mutant form compared with the wild-type form and we excluded mechanisms of protein degradation or nuclear mislocalization. The HNF1A mutation p.Pro409His can be considered a mild variant that confers a moderate risk of type 2 diabetes mellitus in heterozygous carriers.

F-box protein-32 down-regulates small-conductance calcium-activated potassium channel 2 in diabetic mouse atria

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation, but the underlying ionic mechanism for this association remains unclear. We recently reported that expression of the small-conductance calcium-activated potassium channel 2 (SK2, encoded by KCCN2) in atria from diabetic mice is significantly down-regulated, resulting in reduced SK currents in atrial myocytes from these mice. We also reported that the level of SK2 mRNA expression is not reduced in DM atria but that the ubiquitin-proteasome system (UPS), a major mechanism of intracellular protein degradation, is activated in vascular smooth muscle cells in DM. This suggests a possible role of the UPS in reduced SK currents. To test this possibility, we examined the role of the UPS in atrial SK2 down-regulation in DM. We found that a muscle-specific E3 ligase, F-box protein 32 (FBXO-32, also called atrogin-1), was significantly up-regulated in diabetic mouse atria. Enhanced FBXO-32 expression in atrial cells significantly reduced SK2 protein expression, and siRNA-mediated FBXO-32 knockdown increased SK2 protein expression. Furthermore, co-transfection of SK2 with FBXO-32 complementary DNA in HEK293 cells significantly reduced SK2 expression, whereas co-transfection with atrogin-1ΔF complementary DNA (a nonfunctional FBXO-32 variant in which the F-box domain is deleted) did not have any effects on SK2. These results indicate that FBXO-32 contributes to SK2 down-regulation and that the F-box domain is essential for FBXO-32 function. In conclusion, DM-induced SK2 channel down-regulation appears to be due to an FBXO-32-dependent increase in UPS-mediated SK2 protein degradation.

Mechanisms of Niemann-Pick type C1 Like 1 protein degradation in intestinal epithelial cells.

Intestinal Niemann-Pick C1 Like 1 (NPC1L1) protein plays a key role in cholesterol absorption. A decrease in NPC1L1 expression has been implicated in lowering plasma cholesterol and mitigating the risk for coronary heart disease. Little is known about the mechanisms responsible for NPC1L1 protein degradation that upon activation may lead to a reduction in NPC1L1 protein levels in intestinal epithelial cells (IECs). In current studies, the human intestinal Caco-2 and HuTu-80 cell lines expressing NPC1L1-hemagglutinin fusion protein were used to investigate the mechanisms of NPC1L1 protein degradation. Incubation with the proteasome inhibitors MG-132 and lactacystin (10 μM, 24 h) significantly increased NPC1L1 protein levels in IECs. Also, the inhibition of the lysosomal pathway with bafilomycin A1 (80 nM, 24 h) resulted in a significant increase in NPC1L1 protein levels. Immunoprecipitation studies showed that NPC1L1 protein is both a poly- and monoubiquinated polypeptide and that the inhibition of the proteasomal pathway remarkably increased the level of the polyubiquinated NPC1L1. The surface expression of NPC1L1 was increased by the inhibition of both proteasomal and lysosomal pathways. Furthermore, the pharmacological inhibition of mitogen-activated protein kinase pathway (PD-98059, 15 μM, 24 h) and siRNA silencing of ERK1/2 resulted in a significant decrease in NPC1L1 protein levels in IECs. In conclusion, our results showed that basal level of intestinal cholesterol transporter NPC1L1 protein is modulated by both ubiquitin proteasome- and lysosome-dependent degradation as well as by ERK1/2-dependent pathway. The modulation of these pathways may provide novel clues for therapeutic intervention to inhibit cholesterol absorption and lower plasma cholesterol.

Abstract P1-05-05: eEF1A2 facilitates PTEN-GSK3β mediated Aurora-A protein degradation during S-G2 phase inactivated in PTEN-deficient breast cancer

Abstract The AURKA gene, encoding Aurora kinase-A (Aurora-A), is frequently amplified and overexpressed across multiple cancer types correlating with poor prognosis. Although the AURKA gene is frequently amplified in human cancers, underlying mechanism(s) for Aurora-A protein stability through different phases of cell cycle are not well elucidated. Inhibiting the kinase activity and promoting protein degradation are two well-validated conceptual strategies for targeting protein kinases in cancers. Here, we demonstrate that Eukaryotic Elongation Factor 1 Alpha 2 (eEF1A2) facilitates PTEN-GSK3β mediated Aurora-A protein degradation through the SCF complex (SKP1-Cul1-FBXW7) during the S/G2 phase of proliferating cells. In contrast, this mechanism is inactivated in cancer cells accompanying PTEN-GSK3β pathway deficiency. Mechanistically, eEF1A2 interacts with Aurora-A, GSK3β, FBXW7 and Cul1-E3 ligase, as the SCF complex, to facilitate Aurora-A polyubiquitination for 26S proteasomal degradation. eEF1A2 promotes PTEN phosphorylation at T366 and stability, inactivates AKT and activates GSK3β which in turn phosphorylates Aurora-A at S283, S284 and S342. The phosphorylation of Aurora-A at S342 is detected during S/G2 phase of cell mitosis in parallel with eEF1A2-SCF complex formation with active form of GSK3β and neddylated Cul1. Conversely, genetic ablation of EEF1A2 and PTEN, activation of AKT, inhibition of GSK3β, expression of Aurora-A phosphodeficient-mutant attenuates the Aurora-A protein degradation which is corroborated in Aurora-A overexpressing mouse mammary carcinomas and human breast carcinomas. This study identifies a novel mechanism of Aurora-A protein degradation mediated eEF1A2-PTEN-GSK3β pathway and provides a framework for the discovery of Aurora-A therapeutic targets in breast cancer that harbors deficiency of PTEN tumor suppressor pathway. Citation Format: Treekitkarnmongkol W, Solis LM, Kai K, Thompson AM, Tian W, Wistuba II, Sasai K, Jltsumori Y, Sahin AA, Hawke DH, Lee JM, Qin L, Bawa-Khalfe T, Rad R, Wong KK, Abbott CM, Katayama H, Sen S. eEF1A2 facilitates PTEN-GSK3β mediated Aurora-A protein degradation during S-G2 phase inactivated in PTEN-deficient breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-05.

On the Mechanism of Hyperthermia-Induced BRCA2 Protein Degradation.

The DNA damage response (DDR) is a designation for a number of pathways that protects our DNA from various damaging agents. In normal cells, the DDR is extremely important for maintaining genome integrity, but in cancer cells these mechanisms counteract therapy-induced DNA damage. Inhibition of the DDR could therefore be used to increase the efficacy of anti-cancer treatments. Hyperthermia is an example of such a treatment—it inhibits a sub-pathway of the DDR, called homologous recombination (HR). It does so by inducing proteasomal degradation of BRCA2 —one of the key HR factors. Understanding the precise mechanism that mediates this degradation is important for our understanding of how hyperthermia affects therapy and how homologous recombination and BRCA2 itself function. In addition, mechanistic insight into the process of hyperthermia-induced BRCA2 degradation can yield new therapeutic strategies to enhance the effects of local hyperthermia or to inhibit HR. Here, we investigate the mechanisms driving hyperthermia-induced BRCA2 degradation. We find that BRCA2 degradation is evolutionarily conserved, that BRCA2 stability is dependent on HSP90, that ubiquitin might not be involved in directly targeting BRCA2 for protein degradation via the proteasome, and that BRCA2 degradation might be modulated by oxidative stress and radical scavengers.

El dominio STK de la proteína de resistencia a la bacteriosis vascular de yuca RXAM1 interactúa con una E3 Ubiquitin Ligasa

La yuca (Manihot esculenta) representa el pilar de la seguridad alimentaria para cerca de mil millones de personas, principalmente en las zonas tropicales. Uno de los factores limitantes de la producción de yuca es la bacteriosis vascular causada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Recientemente se identificó el gen RXam1 el cual confiere resistencia parcial de yuca a cepas de Xam. RXam1 codifica una proteína con un dominio LRR (Leucine Rich Repeats) extracelular y un dominio STK (Serina Treonina Kinasa) citoplasmático; estas proteínas son conocidas como RLKs (Receptor Like Kinases). En este estudio se realizó el tamizaje de una librería de ADNc de yuca mediante doble híbrido de levadura para identificar las posibles proteínas que interactúan con el dominio STK de RXam1. El tamizaje de 3x108 clones permitió identificar y confirmar cinco clones de ellos los cuales corresponden al mismo gen, el cual codifica para una proteína que presenta un dominio central de dedos de zinc CHY, seguido por un dominio C-terminal “RING finger” y un “Zinc ribbon” el cual fue denominado CRFE3-1 (Cassava RING Finger E3 ligase). La interacción entre STK y CRFE3-1 fue altamente especifica ya que se demostró también por doble híbrido que STK no interactúa con una E3 ligasa de Arabidopsis, altamente similar a CRFE3-1, así como tampoco CRFE3-1 interactúa con el dominio STK de un RLK de lechuga similar a RXam1. La identificación de CRFE3-1 sugiere que mecanismos de degradación proteica son importantes para regular la actividad de RXam1.

Autophagy and Ubiquitin-Proteasome System.

Millions of protein molecules are synthesized per minute in each cell, and simultaneously, millions of protein molecules are degraded. Mutated and misfolded newly synthesized proteins are rapidly degraded to prevent the toxicity caused by the accumulation of these protein fragments. There are two main mechanisms of intracellular protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP). There is a certain relationship between these two mechanisms, and there are some molecules that initiate compensatory effects to prevent disease progression.

Inhibition of Skp1-Cullin-F-box complexes during bovine oocyte maturation and preimplantation development leads to delayed development of embryos†

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

Trehalose Limits Fragment Antibody Aggregation and Influences Charge Variant Formation in Spray-Dried Formulations at Elevated Temperatures.

The preparation of PLGA rods for sustained release applications via a hot-melt extrusion process employs heat and mechanical shear. Understanding protein stability and degradation mechanisms at high temperature in the solid state is therefore important for the preparation of protein-loaded PLGA rods. The stability of a model protein, labeled Fab2, has been investigated in solid-state formulations containing trehalose at elevated temperatures. Spray-dried formulations containing varying levels of trehalose were exposed to temperatures ranging from 90 to 120 °C. Measurement of aggregation and chemical degradation rates suggests that trehalose limits Fab2 degradation in a concentration-dependent manner, but the effect tends to saturate when the mass ratio of trehalose to protein is around 1 in the solid formulation. The Fab2 secondary structure and spray-dried particle morphology were studied using circular dichroism and scanning electron microscopy techniques, respectively. On the basis of temperature and trehalose-dependent aggregation kinetics as well as changes in spray-dried particle morphology, a mechanism is proposed for the trehalose stabilization of proteins in solid state at elevated temperatures. The results reported here suggest that when fragment antibodies in the solid state are formulated with trehalose as excipient, a high temperature process such as hot-melt extrusion can be successfully accomplished with minimal degradation.

A new method for enamel amino acid racemization dating: A closed system approach

Abstract Analysis of the predictable breakdown of proteins and amino acids in ancient biominerals enables age estimation over the Quaternary. We postulate that enamel is a suitable biomineral for the long-term survival of endogenous amino acids. Analysis of multiple amino acids for geochronological studies is typically achieved using a RP-HPLC method. However, the low concentrations of amino acids coupled with high concentrations of inorganic species make accurate determination of amino concentrations challenging. We have developed a method for the routine preparation of multiple enamel samples using biphasic separation. Furthermore, we have shown that amino acids that exhibit effectively closed system behaviour can be isolated from enamel through an exposure time of 72 h to bleach. Elevated temperature experiments investigating the processes of intra-crystalline protein degradation (IcPD) do not appear to match the patterns from fossil samples, reinforcing the need for a comprehensive understanding of the underlying mechanisms of protein degradation. This novel preparative method isolates intra-crystalline amino acids suitable for the development of mammalian geochronologies based on enamel protein degradation. The lower rates of racemization in enamel (cf. Bithynia opercula) suggest that the enamel AAR may be able to be used as a relative dating technique over time scales >2.8 Ma. Enamel AAR has the potential to directly date mammalian remains past the limit of all other current direct dating methods, providing an invaluable tool for geochronological studies.