Team:SCAU-China/design

MESEG

Design

Autophagy-Intelligent identification system:


Autophagy, as a way of biological stress resistance and intracellular material recovery and reuse, plays an important role in the maintenance of cell homeostasis. In the past decade, the mechanism of autophagy and the components involved in autophagic vesicle assembly have been studied in detail. According to the occurrence scale of autophagy, it can be divided into macroautophagy, microautophagy and mega-autophagy. In general, macroautophagy is the most common. Next, we will introduce the specific mechanism with macroautophagy.

Figure 1: Types of autophagy (Bu et al., 2020).


In plants, the cargo selection of macroautophagy is determined by various receptors. The receptor binds to LDS (LIR / AIM docking site) on ATG8-PE complex on autophagosome membrane through its own AIM structure (ATG8 interacting motif (AIM) is called LC3 interacting region in animals (LIR)). And finally locate the goods in the autophagosome. The AIM structure is very conservative in the biology world. The core sequence is W/F/Y-XX-L/I/V, an aromatic amino acid (Trp, Tyr or Phe) followed by two random amino acids and an aliphatic amino acid (Leu, Ile and Val). AIM acts as a hub to bind substances which will be degraded by autophagy pathway.

Figure 2: Cargo selection in macroautophagy (Marshall and vierstra, 2018).


So we prepared to construct the fusion protein of AIM- Metallothionein (MT)-fluorescent protein. In this way, metallothionein can bind the heavy metal ions entering the cell and bind to ATG8-PE on the autophagosome membrane by AIM, and then the autophagosome will be transported to the vacuole. The autophagosome membrane fuses with the vacuole membrane and then releases the contents, so as to finally pull the heavy metal ions into the vacuole. This will reduce the toxicity of heavy metals to cells, enrich heavy metal ions through autophagy, enhance the ability and service life of Chlamydomonas reinhardtii to deal with heavy metal pollution. We will verify it firstly in yeast. Since the autophagy system of yeast is slightly different from that of plants, in yeast, we use the ARR domain which homologous to AIM and the CC domain which will be an auxiliary worker to ARR domain (Yamasaki and Noda, 2017). So we will construct a fusion protein with the basic structure of MT-CC-mCherry-ARR. The design will be improved according to the experiment results.


Refer to the engineering and model page for details.

Video 1: Operation mode of fusion protein in autophagy pathway.


We will transfer the fusion protein composed of aim, MT and mCherry red fluorescent protein into Chlamydomonas reinhardtii by pMO449 binary vector to ensure that our fusion protein can be highly expressed.

Figure 3: Schematic diagram of the carrier to be transformed into Chlamydomonas




Metallothionein-molecular magnet:


Metallothionein (MT) is a low molecular weight metal binding protein with a molecular weight of about 6500 Da. It is composed of about 30% cysteine residues containing sulfhydryl groups. In terms of structure, two different metal binding domains have been characterized in MT, α- and β- Cluster. Most organisms have MT coding genes, and both essential metals (such as zinc and copper) and unessential metals (such as cadmium and mercury) can easily induce MT synthesis. MT is important in the metabolism and kinetics of cadmium and copper because these metals are transported by MT in organisms. Cadmium that does not bind to MT is toxic and can cause toxic damage to cells. MT also provides various important functions for zinc and mercury. In addition, MT protein plays a role in the metabolism of essential metals and the prevention of metal toxicity. In general, MT plays an important role in essential metal homeostasis, heavy metal detoxification and cellular antioxidant defense (Wang et al., 2016, Smith and Nordberg, 2015).


We selected five metallothionein for our project. They are OsCAL1, SpMTL that can chelate with cadmium ions, HpCuMT that can chelate with copper ions, HvMT4 that can chelate with zinc ions, and finally SMTA, which can chelate with copper cadmium and zinc ions at the same time.

Figure 4: Source and name of metallothionein


We have reasons to choose these proteins. As a kind of hyperaccumulation plant, Sedum plumbizinccola absorbs heavy metals from the natural metal containing soil in which they exist, and accumulates a large amount of metals in aboveground tissues without any phytotoxic effect, which can hyperaccumulate Cd2+ and Zn2+. Scientists found a SpMTL protein with high binding ability to cadmium in its body. Because SpMTL has higher affinity for cadmium, we will use it as a representative of cadmium absorption (Peng et al., 2017).


Rice (Oryza sativa) OsCAL1 is mainly expressed in root epidermis and xylem parenchyma cells, and involves in the specific transport of Cd2+ from rice root to stem. Cd2+ may be transported by coordinating with the three sulfhydryl groups in OsCAL1 to form a stable Cd:3(SH-) complex (Luo et al., 2018). Because OsCAL1 has high specificity for Cd2+, we will use it as a representative of cadmium absorption. HpCuMT comes from roman snail (helix pomatia). A HpCuMT protein can bind 12 copper ions. Compared with known Cu binding proteins such as CUP1 (one CUP1 protein can bind 8 copper ions), HpCuMT has better binding potential. Moreover, HpCuMT binds copper ions with high specificity and residence time (Palacios, Ò., atrian et al., 2011, Palacios, O., Pagani et al., 2011, Berger et al., 1997). So we will take it as a representative of copper absorption. HvMT4 from barely (Hordeum vulgare) has binding ability to both copper and zinc, but it has higher affinity for zinc and has a variety of binding forms, including Zn2MT, Zn3MT and Zn4MT (Hegelund et al., 2012). So we will take it as a representative of zinc absorption. SMTA from Synechococcus elongatus was applied as a metallothionein absorbing cadmium in previous iGEM projects (BBa_K1342003). We found that SMTA not only has the binding effect of cadmium. It also has a good binding effect of zinc and copper (Jianguo Shi, 1992). We will upgrade the previous SMTA. For details, please refer to the improvement page. Zn2+ can form Zn4SMTA complex with SMTA by nine cysteine residues. Similarly, Cd2+ can also form Cd4SMTA complex with SMTA (blindauer et al., 2001). The combination form of SMTA and copper is unclear. We finally regard it as the representative of absorbing cadmium, copper and zinc.




Fusion-intelligent molecular magnet


We fused AIM that isthe key molecule in the autophagy system and metallothionein with mCherry red fluorescent protein and transferred them into Chlamydomonas in order to improve the resistance of Chlamydomonas to heavy metals and enrich heavy metals by the autophagy system. Because Chlamydomonas transformation is more difficult and complex than yeast or bacteria, we will use yeast to make verification firstly and Chlamydomonas will be used for verification in parallel. Please refer to the engineering success and proof of concept page for detail.




References


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  2. Blindauer, C.A., Harrison, M.D., Parkinson, J.A., Robinson, A.K., Cavet, J.S., Robinson, N.J., and Sadler, P.J. (2001). A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity. P NATL ACAD SCI USA 98, 9593-9598.
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