γ‐Secretase Inhibition Induces Muscle Hypertrophy in a Notch‐Independent Mechanism

A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ‐secretase. Notch‐1 is one of the substrates of γ‐secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch‐1, a number of proteins have been identified to undergo proteolysis by γ‐secretase. To date, the specific role of γ‐secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ‐secretase inhibitor DAPT (N‐N[‐(3,5‐Difluorophenacetyl‐l ‐alanyl)]‐S‐328 phenylglycine‐t‐butyl‐ester) induces muscle hypertrophy. Knockdown of Notch‐1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ‐secretase‐dependent effects on muscle hypertrophy in chick myogenic cells are Notch‐1‐independent. We also investigate the effects of γ‐secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label‐free proteomic approach. Data overview of interaction network obtained from STRING show that after γ‐secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ‐secretase in skeletal muscle hypertrophy.     

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.     

The stress response neuropeptide CRF increases amyloid‐β production by regulating γ‐secretase activity

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid‐β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ‐secretase internalization. Co‐immunoprecipitation studies establish that γ‐secretase associates with CRFR1; this is mediated by β‐arrestin binding motifs. Additionally, CRFR1 and γ‐secretase co‐localize in lipid raft fractions, with increased γ‐secretase accumulation upon CRF treatment. CRF treatment also increases γ‐secretase activity in vitro, revealing a second, receptor‐independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ‐secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ‐secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ‐secretase.     

Suppression,disaggregation, and modulation of γ‐Synuclein fibrillation pathway by green tea polyphenol EGCG

Oligomerization of γ‐Synuclein is known to have implications for both neurodegeneration and cancer. Although it is known to co‐exist with the fibrillar deposits of α‐Synuclein (Lewy bodies), a hallmark in Parkinson's disease (PD), the effect of potential therapeutic modulators on the fibrillation pathway of γ‐Syn remains unexplored. By a combined use of various biophysical tools and cytotoxicity assays we demonstrate that the flavonoid epigallocatechin‐3‐gallate (EGCG) significantly suppresses γ‐Syn fibrillation by affecting its nucleation and binds with the unstructured, nucleus forming oligomers of γ‐Syn to modulate the pathway to form α‐helical containing higher‐order oligomers (~158 kDa and ~ 670 kDa) that are SDS‐resistant and conformationally restrained in nature. Seeding studies reveal that these oligomers although “on‐pathway” in nature, are kinetically retarded and rate‐limiting species that slows down fibril elongation. We observe that EGCG also disaggregates the protofibrils and mature γ‐Syn fibrils into similar SDS‐resistant oligomers. Steady‐state and time‐resolved fluorescence spectroscopy and isothermal titration calorimetry (ITC) reveal a weak non‐covalent interaction between EGCG and γ‐Syn with the dissociation constant in the mM range (K_d ~ 2–10 mM). Interestingly, while EGCG‐generated oligomers completely rescue the breast cancer (MCF‐7) cells from γ‐Syn toxicity, it reduces the viability of neuroblastoma (SH‐SY5Y) cells. However, the disaggregated oligomers of γ‐Syn are more toxic than the disaggregated fibrils for MCF‐7cells. These findings throw light on EGCG‐mediated modulation of γ‐Syn fibrillation and suggest that investigation on the effects of such modulators on γ‐Syn fibrillation is critical in identifying effective therapeutic strategies using small molecule modulators of synucleopathies.     

Role of γ‐glutamyltranspeptidase in the pathogenesis of Helicobacter pylori infection

γ‐Glutamyltranspeptidase and asparaginase have been shown to play important roles in Helicobacter pylori colonization and cell death induced by H. pylori infection. In this study, the association of γ‐glutamyltranspeptidase and asparaginase was elucidated by comparing activities of both deamidases in H. pylori strains from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer. γ‐Glutamyltranspeptidase activities in H. pylori strains from patients with gastric cancer were significantly higher than in those from patients with chronic gastritis or gastric ulcers. There was a wide range of asparaginase activities in H. pylori strains from patients with gastric cancer and these were not significantly than those from patients with other diseases. To identify the contributions of γ‐glutamyltranspeptidase and asparaginase to gastric cell inflammation, human gastric epithelial cells (AGS line) were infected with H. pylori wild‐type and knockout strains and inflammatory responses evaluated by induction of interleukin‐8 (IL‐8). IL‐8 response was significantly decreased by knockout of the γ‐glutamyltranspeptidase‐encoding gene but not by knockout of the asparaginase‐encoding gene. Additionally, IL‐8 induction by infection with the H. pylori wild‐type strain was significantly decreased by adding glutamine during infection. These findings indicate that IL‐8 induction caused by γ‐glutamyltranspeptidase activity in H. pylori is mainly attributable to depletion of glutamine. These data suggest that γ‐glutamyltranspeptidase plays a significant role in the chronic inflammation caused by H. pylori infection.     

Solution properties of γ‐crystallins: Compact structure and low frictional ratio are conserved properties of diverse γ‐crystallins

γ‐crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ‐crystallins have high contents of sulfur‐containing methionine and cysteine, but this reaches extremes in fish γM‐crystallins with up to 15% Met. In addition, fish γM‐crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ‐crystallins and in the related β‐crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ‐crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β‐sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full‐length and truncated versions of human αB‐crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ‐crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ‐crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.     

Opposite expression of securin and γ‐H2AX regulates baicalein‐induced cancer cell death

Securin and γ‐H2AX have been shown to regulate cell survival and genomic stability. However, it is still unknown how the expression and regulation of these proteins is altered following treatment with baicalein, a natural flavonoid extracted from the Scutellaria baicalensis root. In the present study, we investigate the possible roles of securin and γ‐H2AX in baicalein‐induced cancer cell death. Baicalein reduced cell viability in a variety of human cancer cell lines, including bladder, cervical, colon, and lung cancer cells. Interestingly, baicalein treatment (40–80 µM for 24 h) markedly inhibited securin expression, while the levels of γ‐H2AX were elevated. Abnormal spindle formation and chromosomal segregation were induced by baicalein. Furthermore, wild type HCT116 cancer cells had a higher incidence of cytotoxicity and apoptosis than securin‐null HCT116 cells following treatment with baicalein. In contrast, baicalein increased the levels of γ‐H2AX to a similar extent in both cell types. Transfection with H2AX siRNA further increased baicalein‐induced cell death. Additionally, blockade of the AKT pathway by treatment with wortmannin or AKT shRNA lowered the levels of γ‐H2AX and enhanced cytotoxicity in baicalein‐treated cells. Taken together, our findings suggest that the opposing effects of baicalein on securin and γ‐H2AX levels may be involved in the regulation of cell viability and genomic stability by this compound. J. Cell. Biochem. 111: 274–283, 2010. © 2010 Wiley‐Liss, Inc.     

Aph‐1 is required to regulate Presenilin‐mediated γ‐secretase activity and cell survival in Drosophila wing development

Aph‐1 is a multipass transmembrane protein and an essential component of the Presenilin (Psn)‐mediated γ‐secretase complex. During protease assembly, Aph‐1 stabilizes the newly synthesized Psn holoprotein to facilitate generation of the active form of Psn, which is a Psn‐NTF/Psn‐CTF heterodimer produced through a Presenilinase‐initiated endoproteolytic cleavage of the Psn holoprotein. Although it is clear that loss of Aph‐1 activity leads to failure of Psn heterodimer formation, little is understood about whether Aph‐1 plays a role in regulating γ‐secretase activity in addition to assisting Psn maturation. Using various modified Psn forms that do not require endoproteolysis or have a large deletion of the cytosolic loop, we show that in Drosophila Aph‐1 is still required for γ‐secretase activity independent of its role in promoting Psn endoproteolysis. In addition, our results indicate that Aph‐1 is required to promote cell survival in the wing imaginal disc; aph‐1 mutant cells are lost either through cell death or because of a defect in cell proliferation. This function of Aph‐1 is independent of its role in regulating γ‐secretase activity, but possibly involves downregulating the activity of uncleaved Psn holoprotein. genesis 47:169–174, 2009. © 2009 Wiley‐Liss, Inc.     

MUC1 is a substrate for γ‐secretase

Understanding the underlying mechanisms by which a normal cell avoids the oncogenic potential of MUC1 signaling requires further definition of the pathways by which the MUC1 cytoplasmic tail is processed in both normal and tumor‐derived cells. In the present study we describe the processing pathway initiated by TACE/ADAM17 cleavage of MUC1. Utilizing the human uterine epithelial cell line, HES, derived from normal endometrium, we show that endogenous full length MUC1 undergoes regulated intramembranous proteolysis mediated by presenillin‐dependent γ‐secretase. Cytokine‐stimulated HES cells exposed to γ‐secretase inhibitors accumulated a membrane‐associated 15 kDa fragment of the MUC1 C‐terminal subunit (CTF15). Inhibitors of TACE/ADAM17‐mediated shedding inhibited accumulation of MUC1‐CTF15 and MUC1 ectodomain release to a similar extent consistent with MUC1‐CTF15 being a product of TACE/ADAM17 action. Reduction of catalytically active γ‐secretase complex by nicastrin siRNA treatment also resulted in CTF15 accumulation. Furthermore, mature nicastrin, the substrate receptor for γ‐secretase, co‐immunoprecipitated with CTF15 in the presence of γ‐secretase inhibitors indicating the formation of CTF15: nicastrin complexes. MUC1‐CTF15 accumulation in response to γ‐secretase inhibition was demonstrated in both normal and tumor‐derived cells from humans and mice indicating that this processing pathway exists in many cell contexts. We did not detect products of MUC1 cleavage by γ‐secretase in the presence of various proteasomal inhibitors indicating that subsequent degradation is either non‐proteasomal or extremely efficient. We suggest that this efficient pathway attenuates potential signaling mediated by cytoplasmic tail fragments. J. Cell. Biochem. 108: 802–815, 2009. © 2009 Wiley‐Liss, Inc.     

γ‐Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ‐Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ‐Tubulin Ring Complexes (γ‐TuRCs). While the subunits that constitute γ‐Tubulin Small Complexes (γ‐TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ‐TuRC‐specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ‐TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ‐TuRCs on astral MTs. γ‐TuRCs locate along the length of astral MTs, and depletion of γ‐TuRC‐specific proteins increases MT dynamics and causes the plus‐end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down‐regulation rescues spindle orientation defects induced by γ‐TuRC depletion. Therefore, we propose a role for γ‐TuRCs in regulating spindle positioning by controlling the stability of astral MTs.     

Fission yeast Pcp1 links polo kinase‐mediated mitotic entry to γ‐tubulin‐dependent spindle formation

The centrosomal pericentrin‐related proteins play pivotal roles in various aspects of cell division; however their underlying mechanisms remain largely elusive. Here we show that fission‐yeast pericentrin‐like Pcp1 regulates multiple functions of the spindle pole body (SPB) through recruiting two critical factors, the γ‐tubulin complex (γ‐TuC) and polo kinase (Plo1). We isolated two pcp1 mutants (pcp1‐15 and pcp1‐18) that display similar abnormal spindles, but with remarkably different molecular defects. Both mutants exhibit defective monopolar spindle microtubules that emanate from the mother SPB. However, while pcp1‐15 fails to localise the γ‐TuC to the mitotic SPB, pcp1‐18 is specifically defective in recruiting Plo1. Consistently Pcp1 forms a complex with both γ‐TuC and Plo1 in the cell. pcp1‐18 is further defective in the mitotic‐specific reorganisation of the nuclear envelope (NE), leading to impairment of SPB insertion into the NE. Moreover pcp1‐18, but not pcp1‐15, is rescued by overproducing nuclear pore components or advancing mitotic onset. The central role for Pcp1 in orchestrating these processes provides mechanistic insight into how the centrosome regulates multiple cellular pathways.     

Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Poly‐γ‐glutamate (γ‐PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the γ‐PGA synthesis ywsC‐ywtAB genes in its chromosome, cannot produce γ‐PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in γ‐PGA‐producing B. subtilis mutant strains. Mutant B. subtilis PGA6‐2 stably produces high levels of γ‐PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6‐2 is not only a γ‐PGA producer, but it is also a candidate for the genetic and metabolic engineering of γ‐PGA production. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.     

Effect of propeptide amino acid substitution in γ‐carboxylation,activity and expression of recombinant human coagulation factor IX

The production of recombinant vitamin K dependent (VKD) proteins for therapeutic purposes is an important challenge in the pharmaceutical industry. These proteins are primarily synthesized as precursor molecules and contain pre–propeptide sequences. The propeptide is connected to γ‐carboxylase enzyme through the γ‐carboxylase recognition site for the direct γ‐carboxylation of VKD proteins that has a significant impact on their biological activity. Propeptides have different attitudes toward γ‐carboxylase and certain amino acids in propeptide sequences are responsible for the differences in γ‐carboxylase affinity. By aiming to replace amino acids in hFIX propeptide domain based on the prothrombin propeptide, pMT‐hFIX‐M14 expression cassette, containing cDNA of hFIX with substituted ?14 residues (Asp to Ala) was made. After transfection of Drosophila S2 cells, expression of the active hFIX was analyzed by performing ELISA and coagulation test. A 1.4‐fold increase in the mutant recombinant hFIX expression level was observed in comparison with that of a native recombinant hFIX. The enhanced hFIX activity and specific activity of the hFIXD‐14A (2.2 and 1.6 times, respectively) were further confirmed by comparing coagulation activity levels of substituted and native hFIX. Enrichment for functional, fully γ‐carboxylated hFIX species via barium citrate adsorption demonstrated 2‐fold enhanced recovery in the S2‐expressing hFIXD‐14A relative to that expressed native hFIX. These results show that changing ?14 residues leads to a decrease in the binding affinity to substrate, increase in γ‐carboxylation and activity of recombinant hFIX. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:515–520, 2018     

Non‐apoptotic function of caspases in a cellular model of hydrogen peroxide‐associated colitis

Oxidative stress, caused by reactive oxygen species (ROS), is a major contributor to inflammatory bowel disease (IBD)‐associated neoplasia. We mimicked ROS exposure of the epithelium in IBD using non‐tumour human colonic epithelial cells (HCEC) and hydrogen peroxide (H₂O₂). A population of HCEC survived H₂O₂‐induced oxidative stress via JNK‐dependent cell cycle arrests. Caspases, p21^WAF1 and γ‐H2AX were identified as JNK‐regulated proteins. Up‐regulation of caspases was linked to cell survival and not, as expected, to apoptosis. Inhibition using the pan‐caspase inhibitor Z‐VAD‐FMK caused up‐regulation of γ‐H2AX, a DNA‐damage sensor, indicating its negative regulation via caspases. Cell cycle analysis revealed an accumulation of HCEC in the G₁‐phase as first response to oxidative stress and increased S‐phase population and then apoptosis as second response following caspase inhibition. Thus, caspases execute a non‐apoptotic function by promoting cells through G₁‐ and S‐phase by overriding the G₁/S‐ and intra‐S checkpoints despite DNA‐damage. This led to the accumulation of cells in the G₂/M‐phase and decreased apoptosis. Caspases mediate survival of oxidatively damaged HCEC via γ‐H2AX suppression, although its direct proteolytic inactivation was excluded. Conversely, we found that oxidative stress led to caspase‐dependent proteolytic degradation of the DNA‐damage checkpoint protein ATM that is upstream of γ‐H2AX. As a consequence, undetected DNA‐damage and increased proliferation were found in repeatedly H₂O₂‐exposed HCEC. Such features have been associated with neoplastic transformation and appear here to be mediated by a non‐apoptotic function of caspases. Overexpression of upstream p‐JNK in active ulcerative colitis also suggests a potential importance of this pathway in vivo.     

Knockout of pgdS and ggt genes improves γ‐PGA yield in B. subtilis

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ‐glutamic acid) (γ‐PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ‐PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ‐PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ‐PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ‐PGA productivity. Biotechnol. Bioeng. 2013; 110: 2006–2012. © 2013 Wiley Periodicals, Inc.