Team:SCAU-China/Design

MESEG

Design

Autophagy-Intelligent Selection System


Autophagy is a process in eukaryotes to resist biotic and abiotic stresses and recover and reuse intracellular materials, and thus plays an important role in the maintenance of cell homeostasis (Marshall and Vierstra, 2018). In the past decade, the mechanism of autophagy and the components involved in autophagic vesicle assembly have been extensively studied. According to the different ways of transporting cellular materials to the lysosome/vacuole, autophagy can be divided into macroautophagy, microautophagy and mega-autophagy, among which the macroautophagy is the most common one (Marshall and Vierstra, 2018). We will introduce the specific mechanism of macroautophagy (hereafter referred to as autophagy) in plants.

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


In plants, the cargo selection in autophagy is determined by various receptors. The receptor through its own ATG8 interacting motif (AIM, in animals referred to LC3 interacting region (LIR)), binds to the LDS (AIM/LIR docking site) on the ATG8-PE complex embedded in the autophagosome membrane. Therefore, the AIM portion functions as a binding site to the autophagosomes that later can be transported to the vacuole via autophagy. The core sequence (W/F/Y-XX-L/I/V) of AIM is very conservative, consisting of an aromatic amino acid (Trp/Phe/Tyr) followed by two random amino acids and an aliphatic amino acid (Leu/Ile/Val).

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


Therefore, we construct the fusion protein by combining the heavy metal binding protein metallothionein (MT), the AIM, and the fluorescent protein for visualization. In this construct MT-AIM-mCherry, MT can bind to the heavy metal ions entering the cell, while AIM can bind to ATG8-PE on the autophagosome membrane. Then the whole complex can be packed inside the autophagosome and later be transported to the vacuole. The autophagosome membrane fuses with the vacuole membrane and then releases the contents, so as to catch the heavy metal ions into the vacuole eventually. When expressing in C. reinhardtii, this fusion protein acts as an intelligent molecular magnet to enrich heavy metal ions through autophagy and store them in the vacuole, and thus reduce the toxicity of heavy metals to the algae. Our project will enhance the ability and service life of the algae for the bioremediation of heavy metal pollution.


We tested the molecular magnet construct first in yeasts. The homologous part of AIM in yeasts is the ARR domain with the CC domain as an auxiliary (Yamasaki and Noda, 2017). Therefore, we constructed a fusion protein MT-CC-mCherry-ARR instead. Please refer to Engineering and Project Model parts for details.

Video 1: Operation mode of the molecular magnet MT-AIM-mCherry fusion protein. The process shows how the heavy metal ions are attracted and caught, and then transported to the vacuole via autophagy.


We transformed the construct encoding the MT, AIM, and mCherry into C. reinhardtii using the pMO449 binary vector to ensure that the fusion protein can be highly expressed.

Figure 3: Schematic diagram of the plasmid to be transformed into C. reinhardtii.




Metallothionein-Molecular Magnet


Metallothionein (MT) is a metal binding protein with a low molecular weight of about 6500 Da and has about 30% cysteine residues (Smith and Nordberg, 2015). Two metal binding domains have been characterized in MT, α- and β- Cluster. Most organisms have MT genes, and both essential metals (such as zinc and copper) and unessential metals (such as cadmium and mercury) can easily induce MT synthesis (Smith and Nordberg, 2015). MT plays important roles in essential metal homeostasis, heavy metal detoxification, and cellular antioxidant defense (Hegelund et al., 2012). Transported by MT in organisms, MT is essential for the metabolism and kinetics of cadmium and copper. MT also functions in the metabolism of zinc and mercury (Wang et al., 2016).

In our project, we chose five MTs that chelate different metal ions, including the OsCAL1 and SpMTL (for cadmium ions), the HpCuMT (for copper ions), the HvMT4 (for zinc ions), and the SmtA (for copper, cadmium, and zinc ions).

Figure 4: The sources and names of different metallothioneins (MTs).


As a type of heavy metal hyperaccumulating plant, Sedum plumbizinccola hyperaccumulate Cd2+ under the help of SpMTL, an MT with high Cd2+-binding ability (Peng et al., 2017).

OsCAL1 is the MT involved in the Cd2+ transportation from root to stem in Rice (Oryza sativa), mainly expressed in root epidermis and xylem parenchyma cells. 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).

HpCuMT is the MT of Roman snail (Helix pomatia). Compared with other known copper binding proteins such as CUP1, HpCuMT has better binding ability to copper ions. A HpCuMT protein can bind to 12 copper ions while one CUP1 protein can bind to eight (Palacios, Ò., atrian et al., 2011; Palacios, O., Pagani et al., 2011). Moreover, HpCuMT binds to copper ions with high specificity and residence time (Berger et al., 1997).

HvMT4 from barley (Hordeum vulgare) binds to both copper and zinc, and has a higher affinity for zinc and a variety of binding forms, including Zn2MT, Zn3MT, and Zn4MT (Hegelund et al., 2012).

SmtA from Synechococcus elongatus, already tested in the previous iGEM projects (BBa_K1342003), binds to the cadmium, zinc, and copper ions (Jianguo Shi, 1992), and forms the complexes such as Cd4SmtA and Zn4SmtA (blindauer et al., 2001). Please refer to Improvement for details.


Our Fusion Protein-Intelligent Molecular Magnet


In order to improve the efficiency of algae bioremediation of heavy metals, we constructed a fusion protein consisting of the core protein domain in autophagy (AIM), the metallothionein (MT), and the red fluorescent protein (mCherry), and expressing it in C. reinhardtii. Because of the challenge of algae transformation, we first tested our design in yeasts, and later in algae. Please refer to the Engineering Success and Proof of Concept page for details.




References


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