Identification Of Acetylated Proteins In Silkworm Body And Its Effects On Silk Protein Synthesis

Our ancestors trained the wild silkworm into domesticated silkworm through artificial domestication and selection, and the yield of silk has been improved by about 10 times. In 1930’s, the yield of silk by silkworm increased by nearly 20% due to the use of the first hybrid variety of silkworm. Since then, the yield of silk by silkworm has reached the peak, and is difficult to exceed. So far, there is no revolutionary breakthrough for the improvement of silk yield. Nowadays, to achieve new breakthrough, we must look for the relevant factors about silk protein synthesis in the molecular level, and obtain the theoretical basis of silk protein synthesis and regulation. At present, the research on gene regulation of silk protein synthesis is getting more and more attention. In this study, we firstly studied the related regulation and effect of acetylation modification on silk protein synthesis and provided a theoretical basis for the improvement of silk yield and quality.Lysine acetylation could change the protein function, therefore, we initially identified the acetylome in the silkworm at a large scale using lysine-acetylation(Kac) peptide enrichment coupled with nano-HPLC/MS/MS method. Overall, a total of 342 acetylated proteins with 667 Kac sites were firstly identified with the average degree of acetylation was 1.9. There were twenty Kac proteins with five or more Kac sites and there were at least seven highly intensively acetylated proteins, which contained eleven or more Kac sites, most of which were nutrient-storage proteins, such as storage proteins, vitellogenin and apolipophorin as well as sericin2 protein. Sequence motifs analysis around Kac sites revealed five significantly enriched Kac motifs, including KH, KY, KF, K*F and F*K, which account for 55.7% of all Kac sites. The Kac sites were also significantly enriched on coils structure. Functional analysis showed the acetylated proteins were primarily involved in the pathways about substance and energy metabolism as well as the regulation for biosynthesis and storage of amino acids. In conclusion, the substance and energy metabolism can be strictly regulated by lysine acetylation PTM, which may also regulate the biosynthesis of protein and amino acids.The larval stage is the only feeding stage of the silkworm and in this stage, the silkworm must reserve enough nutrients for silk protein synthesis and subsequent development. The above results showed that a large number of Kac sites are present on all four types of nutrient-storage proteins such as lipid-storage apolipophorin, 30 K and vitellogenin as well as amino acid-storage Storage proteins(SPs), implying lysine acetylation may represent a common regulatory mechanism of nutrient storage, transport, and metabolism in the silkworm. The protein expression profiling and acetylation profiling from different developmental stages further confirmed that the protein and acetylation levels of SPs showed a high abundance in early and middle stages of 5th instar larva, however, a low abundance in late stage of 5th instar larva and spinning stage. As the SP proteins are responsible for the storage of amino acids, the plenty of SP proteins and their stored amino acids synthesized in early and middle stages of 5th instar larva may provide the amino acids source for the synthesis of large amounts of silk protein in late stage of 5th instar larva. The acetylation level of SP proteins changed along with the change of protein level. Therefore, lysine acetylation may be involved in the control of release and utilization of amino acids stored by SP proteins, and thus indirectly regulate the synthesis of silk protein by controlling the supply of amino acids. Combined with the acetylation profiling of other nutrient storage proteins in larva, we confirmed that lysine acetylation may represent a new regulatory mechanism of nutrient storage and utilization in the silkworm. The preliminary study on molecular mechanism showed that lysine acetylation could influence the protein level of SP2 through competing with ubiquitination and suppressing the ubiquitin-mediated proteasomal degradation, thereby stabilizing the protein. Taken together, we suggested that lysine acetylation could regulate the stabilization and hydrolysis of nutrient storage proteins by crosstalk with lysine ubiquitination, thus control the storage and release of nutrients stored by them. Therefore, acetylation modification is involved in the regulation of silk protein synthesis as new mechanism.Sericins are a type of glue proteins that coat and cement silk fibers, including Ser1, Ser2 and Ser3. The nano-HPLC/MS/MS revealed that there are about twenty Kac sites on Ser2 protein. The protein and acetylation profiling in different developmental stages further discovered a high acetylation level in other sericins, such as Ser1 and Ser3. According to the morphological changes from powder to solid-like form of acetylated starch, we predicted that the highly acetylation of sericins is helpful to enhance the adhesion of sericins on silk fiber surface. This may be due to the formation of hydrogen bonds between the carbonyl oxygen atom of acetyl group in sericin and the hydrophilic groups from side chains of amino acids on fiber surface, where a large mount of hydrophilic amino acids were distributed, such as tyrosine, lysine, threonine, glutamic acid, aspartic acid, cysteine and serine. According to the transformation from insolubilization to solubilization after deacetylation in chitin when treated with hot water at an alkaline condition, we predicted that the degumming process of silk is actually a deacetylation process of sericins, providing a new theoretical basis for the improvement of degumming technology of silk.Finally, we studied the effect of acetylation on BmN cells and silk synthesis. Firstly, the acetylation level of the cells was up-regulated and down-regulated by TSA and C646, respectively. The result showed that up-regulation of acetylation could improve the activity of anti-apoptosis. Then we further treated 5th larva with TSA and C646, respectively, and prepared the acetylated silk. The mechanics test showed that the decrease of acetylation level can lead to the decrease of breaking strength and elongation at break of silk, and the elongation at break of silk is increased when the acetylation level was increased. However, the effect of acetylation on the mechanical properties of silk is somewhat low. IR spectrum analysis showed acetylation had little effect on the structure of silk fiber. The crystal structure of acetylated and control silk was also stable and has no change by XRD analysis, suggesting that acetylation could only change the properties of sericin proteins but not silk fibroin. The SEM observation showed that there remained some attached sericins on the acetylated silk, however, the surface of control or deacetylated silk was clean, suggesting acetylation of sericins is helpful to enhance the adhesion of sericins on silk fiber surface.In conclusion, we firstly found that acetylation could regulate the silk protein synthesis through regulating the supply of amino acids. The high acetylation was also found in sericins firstly, and further confirmed that acetylation is helpful to enhance the adhesion of sericins on silk fiber surface. The above results showed a new regulatory mechanism of silk protein synthesis and could provide a new theoretical basis for the increase of silk yield and improvement of degumming, dyeing, printing and finishing of silk.