scholarly journals Heme oxygenase-2 is post-translationally regulated by heme occupancy in the catalytic site

2020 ◽  
Vol 295 (50) ◽  
pp. 17227-17240 ◽  
Author(s):  
Liu Liu ◽  
Arti B. Dumbrepatil ◽  
Angela S. Fleischhacker ◽  
E. Neil G. Marsh ◽  
Stephen W. Ragsdale
Keyword(s):  
Heme Oxygenase ◽  
Protein Level ◽  
Translational Regulation ◽  
Degradation Rate ◽  
Cellular Protein ◽  
Catalytic Site ◽  
Heme Binding ◽  
Direct Role ◽  
Low Protein ◽  
Pathophysiological Role

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. HO1 expression is transcriptionally regulated; however, HO2 expression is constitutive. How the cellular levels and activity of HO2 are regulated remains unclear. Here, we elucidate the mechanism of post-translational regulation of cellular HO2 levels by heme. We find that, under heme-deficient conditions, HO2 is destabilized and targeted for degradation, suggesting that heme plays a direct role in HO2 regulation. HO2 has three heme binding sites: one at its catalytic site and the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, an HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared with the WT HO2. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2's role in maintaining heme homeostasis, paving the way for future investigation into HO2's pathophysiological role in heme deficiency response.

scholarly journals Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

2020 ◽  
Author(s):  
Liu Liu ◽  
Arti B. Dumbrepatil ◽  
Angela S. Fleischhacker ◽  
E. Neil G. Marsh ◽  
Stephen W. Ragsdale
Keyword(s):  
Heme Oxygenase ◽  
Protein Level ◽  
Translational Regulation ◽  
Transcriptional Control ◽  
Degradation Rate ◽  
Cellular Protein ◽  
Catalytic Site ◽  
Heme Binding ◽  
Low Protein ◽  
Pathophysiological Role

ABSTRACTHeme oxygenase-2 (HO2) and −1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2’s role in maintaining heme homeostasis, paving the way for future investigation into HO2’s pathophysiological role in heme deficiency response.

scholarly journals Timescale of degradation-driven protein level fluctuation in the yeast Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Bahareh Mahrou ◽  
Azady Pirhanov ◽  
Moluk Hadi Alijanvand ◽  
Yong Ku Cho ◽  
Yong-Jun Shin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Protein Level ◽  
Transcriptional Control ◽  
Degradation Rate ◽  
Modulation Frequency ◽  
Cellular Protein ◽  
Yeast Saccharomyces Cerevisiae ◽  
Protein Levels ◽  
Protein Degradation Rate

Generating robust, predictable perturbations in cellular protein levels will advance our understanding of protein function and enable control of physiological outcomes in biotechnology applications. Previous studies have shown that controlling RNA transcription achieves perturbations in protein levels over a timescale of several hours. Here, we demonstrate the potential for harnessing the protein degradation machinery to achieve robust, rapid control of a specific protein level in the yeast Saccharomyces cerevisiae. Using a light-driven protein degradation machinery and red fluorescent proteins as reporters, we show that under constant transcriptional induction, repeated triangular fluctuations in protein levels can be generated by controlling the protein degradation rate. Consistent with previous results using transcriptional control, we observed a continuous decrease in the magnitude of fluctuations as the modulation frequency increased, indicating low-pass filtering of input perturbation. However, compared to hour-scale fluctuations observed using transcriptional control, modulating the protein degradation rate enabled five to ten minute-scale fluctuations. Our study demonstrates the potential for repeated control of protein levels by controlling protein degradation rate, at timescales much shorter than that achieved by transcriptional control.

Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux

2019 ◽  
Vol 166 (4) ◽  
pp. 309-315 ◽  
Author(s):  
Taro Watanabe ◽  
Noriyuki Kioka ◽  
Kazumitsu Ueda ◽  
Michinori Matsuo
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Translational Regulation ◽  
Cholesterol Efflux ◽  
Degradation Rate ◽  
Active Form ◽  
Density Lipoprotein ◽  
Protein Levels ◽  
Kinase C ◽  
Pkc Activation

Abstract ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/βI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

The performance of calves given concentrate diets treated with formaldehyde

1973 ◽  
Vol 24 (4) ◽  
pp. 613 ◽  
Author(s):  
GJ Faichney ◽  
HL Davies
Keyword(s):  
Food Consumption ◽  
Protein Level ◽  
Rumen Fluid ◽  
Amino Nitrogen ◽  
Peanut Meal ◽  
Plasma Urea ◽  
Protein Levels ◽  
Low Protein ◽  
Formaldehyde Treatment ◽  
Protein Diets

Five groups of Friesian bull calves were given concentrate diets containing 70 % barley in which low (12 %), medium (15 %), and high (19%) protein levels were obtained by varying the amount of peanut meal included. The effects of protein level and of formaldehyde treatment of the complete diet at the low and medium protein levels were studied in terms of liveweight gain, voluntary food consumption, digestibility of the diet, ammonia nitrogen in rumen fluid, and urea and a-amino nitrogen in blood plasma. Observations were begun when the calves reached 70 kg liveweight and continued until they reached 130 kg liveweight. The calves given the low protein diets grew more slowly than those given the higher protein diets. The calves given the high protein diet grew no better than those given the medium protein diets. Formaldehyde treatment was associated with an increase in the rate of liveweight gain of 9% (P = 0.11) at the low protein level but had practically no effect at the medium protein level. The treatment did not adversely affect voluntary food consumption but was associated with decreases in the digestibility of nitrogen and in rumen ammonia levels and small increases in plasma urea levels.

Effects of variations in dietary protein levels on hair growth and pelt quality in mink (Mustela vison)

2000 ◽  
Vol 80 (4) ◽  
pp. 633-642 ◽  
Author(s):  
Palle V. Rasmussen ◽  
Christian F. Børsting
Keyword(s):  
Protein Content ◽  
Dietary Protein ◽  
Protein Level ◽  
Hair Growth ◽  
Fibre Length ◽  
Hair Follicles ◽  
Protein Levels ◽  
Low Protein ◽  
Pregnancy And Lactation ◽  
Hair Fibre

The effect of different and shifting dietary protein levels on hair growth and the resulting pelt quality in mink was studied. Two groups of pastel female mink were fed either 59% (high protein, HP) or 40% (low protein, LP) of metabolisable energy (ME) from protein during pregnancy and lactation. Shortly after weaning, kits from females fed the LP diet were put on a new LP diet (21% protein of ME). Kits from females fed HP were randomly distributed to four experimental groups fed a new HP diet (34% protein of ME) and three of these groups were shifted to diets with 21% protein at different times during June until September. Skin biopsies were taken at 4, 6, 23 and, 29 wk of age. Histological techniques and computer-assisted light microscopy were used to determine the ratio of activity (ROA) of underfur and guard hairs, respectively, defined as the number of growing hairs as a percentage of the total number of hairs. The hair fibre length and thickness were determined by morphometric methods and correlated with fur properties of dried pelts judged by sensory methods. It was documented that 40% of ME from protein during pregnancy and lactation was sufficient for mink kits to express their genetic capacity to produce hair follicles. In males, a reduced protein level from the age of 15 wk or 22 wk until pelting disturbed moulting, indicated by a low ROA of underfur hairs at 23 wk, and consequently reduced the growth and development of the winter coat. A constantly low protein level from conception until the age of 29 wk did not disturb moulting, but led to a reduction of primeness and especially of the underfur length and fibre thickness of the winter coat. A low protein level from the age of 9 wk only reduced the thickness of the underfur fibres. Hair growth, final fur volume, and general quality of the winter coat of males were influenced negatively and to the same degree in all groups fed the LP diet in part of the growth period. The number of underfur hairs per area (hair density) of the winter coat was not influenced by the dietary treatment meaning that the protein content of 21% of ME in the LP diet was high enough for the mink to express its genetic capacity to develop hair follicles. However, this low protein content led to a reduction of hair fibre length and hair fibre thickness of the underfur. Overall, this study demonstrated that hair growth and hair properties in pelts are very dependent on the dietary protein supply in the period from 22 wk of age until pelting, irrespective of the supply in the preceding periods. Key words: Fur properties, hair fibres, nutrition, pelage, protein requirement

scholarly journals The Arabidopsis locus AT3G03890 encodes a dimeric β-barrel protein implicated in heme degradation

2020 ◽  
Vol 477 (24) ◽  
pp. 4785-4796
Author(s):  
Jia Wang ◽  
Qi Guo ◽  
Xiaoyi Li ◽  
Xiao Wang ◽  
Lin Liu
Keyword(s):  
Crystal Structure ◽  
Recombinant Protein ◽  
Water Molecule ◽  
Heme Oxygenase ◽  
Protein Complex ◽  
Biosynthetic Pathway ◽  
Initial Step ◽  
Heme Iron ◽  
Heme Binding ◽  
New Protein

Plant tetrapyrroles, including heme and bilins, are synthesized in plastids. Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to the linear tetrapyrrole biliverdin as the initial step in bilin biosynthesis. Besides the canonical α-helical HO that is conserved from prokaryotes to human, a subfamily of non-canonical dimeric β-barrel HO has been found in bacteria. In this work, we discovered that the Arabidopsis locus AT3G03890 encodes a dimeric β-barrel protein that is structurally related to the putative non-canonical HO and is located in chloroplasts. The recombinant protein was able to bind and degrade heme in a manner different from known HO proteins. Crystal structure of the heme–protein complex reveals that the heme-binding site is in the interdimer interface and the heme iron is co-ordinated by a fixed water molecule. Our results identify a new protein that may function additionally in the tetrapyrrole biosynthetic pathway.

scholarly journals Effects of Dietary Protein Levels on Bamei Pig Intestinal Colony Compositional Traits

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Dong Wang ◽  
Guoshun Chen ◽  
Lili Song ◽  
Mingjie Chai ◽  
Yongfeng Wang ◽  
...  
Keyword(s):  
Dietary Protein ◽  
Protein Level ◽  
Crude Protein ◽  
Microbial Flora ◽  
Protein Levels ◽  
Low Protein ◽  
Dietary Crude Protein ◽  
Protein Diets ◽  
Crude Protein Level ◽  
Microbial Diversities

Diets containing different crude protein levels (16%, 14%, and 12%) were created to feed Bamei pigs in order to study the effect of these compositions on intestinal colonies. Therefore, 27 healthy Bamei pigs of similar weight ( 20.99   kg ± 0.16   kg ) were selected and randomly divided into three groups for microbial diversity analysis. The results of this study show that microbial diversities and abundances in Bamei pig jejunum and caecum samples after feeding with different dietary protein levels were significantly different. Dietary crude protein level exerted no significant effect on the Shannon index for cecum microbes in these pigs, while Simpson, ACE, and Chao1 indices for group I were all significantly higher than those of either the control group or group II ( P < 0.05 ). Indeed, data show that microbial diversities and abundances in the 14% protein level group were higher than those in either the 16% or 12% groups. Dominant bacteria present in jejunum and cecum samples given low-protein diets were members of the phyla Firmicutes and Bacteroidetes. Data show that as dietary crude protein level decreases, representatives of the microbial flora genus Lactobacillus in jejunum and cecum samples gradually increases. Values for the KEGG functional prediction of microbial flora at different dietary protein levels also show that genes of jejunum and cecum microorganisms were mainly enriched in the “metabolism” pathway and indicate that low protein diets increase intestinal metabolic activity. Therefore, we recommend that Bamei pig dietary protein levels are reduced 2% from their existing level of 16% crude protein. We also suggest that essential synthetic amino acids (AA) are added to optimize this ideal protein model as this will increase intestinal flora diversity in these pigs and enhance health. These changes will have a positive effect in promoting the healthy growth of Bamei pigs.

The associative effect of level of energy and protein intake in the dairy cow

1970 ◽  
Vol 37 (3) ◽  
pp. 481-491 ◽  
Author(s):  
F. J. Gordon ◽  
T. J. Forbes
Keyword(s):  
Energy Intake ◽  
Milk Yield ◽  
Protein Level ◽  
Protein Intake ◽  
Latin Square ◽  
Metabolizable Energy ◽  
Energy Output ◽  
Additional Energy ◽  
Protein Nitrogen ◽  
Low Protein

SummaryEight lactating cows were used in a Latin square experiment, to study the associative effects of level of energy and protein intake on milk yield and composition. Four diets were used, supplying 80 and 120% of estimated energy requirements and 80 and 120% of estimated protein requirements. The level of energy intake significantly affected milk yield, milk energy output, percentage butterfat, ash and non-protein nitrogen. The level of protein intake only significantly affected milk energy output and the non-protein nitrogen content of the milk. Although only the interaction of the effects of energy and protein intake on the milk content of solidsnot-fat (SNF) and ash was significant, it was evident that the effect of each of these factors on milk yield or composition was related to the level of the other in the diet.Input-output relationships within each protein level were used to compute the response in milk energy output and bodyweight change to a change in energy intake. These showed a greater partitioning of additional energy toward milk energy output with the high than with the low protein level. Multiple regression analysis within each level of protein intake was used to partition energy intake between that used for maintenance, milk energy output and liveweight change. The results showed efficiencies of utilization of metabolizable energy for milk output of 63 and 50% on the high- and low-protein diets, respectively.Nitrogen balance data are presented.

The pre-disposition to mottling of certain wheats

1968 ◽  
Vol 8 (30) ◽  
pp. 85 ◽  
Author(s):  
HJ Moss
Keyword(s):  
Protein Content ◽  
Protein Level ◽  
Starch Granules ◽  
Gluten Strength ◽  
Predisposing Factor ◽  
Factors Associated ◽  
Protein Levels ◽  
Low Protein ◽  
Flour Protein

Mottling in wheat is the condition whereby starchy and vitreous sections are found together in a grain or a sample. The occurrence of mottling in nine varieties was studied in relation to flour protein content, proportion of large starch granules, and gluten strength. When protein levels fell below 10 per cent the varieties differed in mottling behaviour. Some varieties lost vitreousness but became opaque rather than mottled; others became mottled. It was concluded that although low protein level is a predisposing factor, mottling is aggravated by factors associated with high dough stability and a high proportion of large starch granules. The interaction of these factors is used to explain the differing susceptibilities of varieties to mottling.

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