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- Mishra, Laxmi S., 1983-
(författare)
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FtsH metalloproteases and their pseudo-proteases in the chloroplast envelope of Arabidopsis thaliana
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- By cleaving peptide bonds, proteases either activate or degrade proteins and maintain protein quality control in response to various developmental stimuli and environmental factors. My work has focused on elucidating the role of the filamentation temperature sensitive protein H (FtsH) proteases. FtsHs belong to a membrane-embedded class of proteases found in eubacteria, animals and plants, which are located in the organelles of endosymbiosis (mitochondria and chloroplasts). They possess an AAA+ (ATPase associated with various cellular activities) and a peptidase M41 domain containing the HEXXH consensus sequence in the Zn2+ metalloprotease domain. FtsH proteases are known to form ring-like homo- or hetero-hexameric complexes. Arabidopsis thaliana, the model plant used in this study, contains seventeen AtFtsH proteases, of which twelve are presumably proteolytically active and five presumably proteolytic inactive members, known as AtFtsHi (i for inactive). In AtFtsHi members, the HEXXH motif is either deleted (AtFtsHi3) or mutated (AtFtsHi1, 2, 4, 5). Twelve AtFtsHs (AtFtsH 1, 2, 5–9, 11, 12 and AtFtsHi 1-5) are targeted to the chloroplast, whereas the remaining three (AtFtsH 3, 4 and 10) are mitochondrial. In Paper I, we demonstrate that AtFtsH12 interacts with AtFtsHi1, 2, 4, 5 to form a heteromeric complex. Abundance of these AtFtsH12-AtFtsHi complexes alters the accumulation of TIC (translocon on the inner chloroplast membrane) complexes. Transgenic mi12 (miRNA) knockdown plants that express lower amounts of AtFtsH12 displayed a pale-seedling and an aberrant chloroplast phenotype. mi12 plants displayed lowered total chlorophyll (Chla+Chlb) amount compared to wild type (WT), complementation lines and native AtFtsH12 promoter overexpressor (ox12) lines. Our biochemical studies identified drastic modifications in the total proteome of mi12 seedlings. N-terminome analyses of mi12 seedlings showed undisturbed plastidic protein maturation. In Paper II, we have shown that single mutants depleted in AtFTSHI1, 2, 4 or 5 are embryo-lethal, suggesting the pseudo-proteases to have an indispensable role in seed germination. This study further identified “weak” Atftshi1, Atftshi4, Atftshi3-1(kd) and Atftshi3-2 homozygous mutants, which develop into plants with altered photosynthetic efficiency. Field experiments were performed to determine the Darwinian fitness of these homozygous as well as heterozygous AtFtsHi mutants. The results suggested AtFtsHi enzymes to be critical during early developmental stages. A complete Atftshi3 knockdown mutant (Atftshi3-1(kd)) was identified (described in Paper III), which is not embryo-lethal and tolerates drought better than WT plants. Atftshi3-1(kd) leaves were smaller with fewer and smaller stomatal aperture. Above ground, Atftshi3-1(kd) leaves displayed lowered stomatal conductance and increased WUEi (intrinsic water-use efficiency), while below ground, the root-associated bacterial community showed a typical drought stress response. Upregulated transcripts of the ABA-responsive genes in leaves of Atftshi3-1(kd) compared to WT indicate the drought tolerance to be controlled independently of ABA. To conclude, AtFtsHi pseudo-proteases affect various stages of plant development and abiotic stress management, especially drought.
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| 2. |
- Orädd, Fredrik, 1994-
(författare)
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Determining the effects of regulatory parameters on the structural dynamics of P-type ATPase membrane transporters
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are macromolecular machines with roles in all cellular activities and structures. The functional properties of each protein is the result of its combination of 3D-structure and inherent dynamics, and a wealth of structural and dynamic mechanisms have evolved to regulate protein activity. P-type ATPases are membrane transport proteins that hydrolyze ATP to move cations across membranes. These proteins are involved in important biological functions such as Ca2+ signaling and Cu+ homeostasis, making proper regulation critical. Adenylate kinase (AdK) is a small, soluble protein that plays a role in energy homeostasis by interconverting ATP, AMP, and ADP, which are bound by two substrate binding domains. In this thesis, the effect of regulatory parameters on the structural dynamics of Cu+-ATPases and the sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA) was investigated, together with the reaction dynamics of AdK.In Paper III, the human Cu+-ATPase ATP7B was simulated with (holo) and without (apo) Cu+ bound to the regulatory metal binding domains (MBDs, with MBD-1 closest to the core protein). In the holo state, the MBD chain was more dynamic and extended, and MBD-2 approached the membrane Cu+ entry site. In Paper IV, the stability of the interaction between MBD-2 and the Cu+-entry site was evaluated using MD simulations, showing that the interaction was stable in the cytosol-open E1 state, but not in the lumen-facing E2P state. An interaction site between MBD-3 and the cytoplasmic domains was also found, where MBD-3 might inhibit activity by interfering with functional motions. Finally, in Paper II, Cu+ entry into the membrane high-affinity Cu+-binding site was simulated, showing that a proposed initial binding site was transient and that the Cu+ ion could move deeper into the membrane domain. In Paper I, we used time-resolved X-ray solution scattering (TR-XSS) to show a simultaneous closing of the substrate binding domains in AdK, which included a partial unfolding and refolding event in the ATP-binding domain. Paper VI demonstrated that a novel time-resolved setup based on detector readout at the MAX IV beamline CoSAXS could trigger and detect AdK structural dynamics.In Paper V, TR-XSS experiments showed that the rate-limiting step in skeletal-muscle SERCA1a was an E1-to-E2P intermediate at both low and high Ca2+ concentrations. An inhibitory effect at high Ca2+ concentration was explained by a fraction of SERCA molecules stalling in the ATP-binding/phosphorylation step. In Paper VII, TR-XSS experiments showed that the housekeeping isoform SERCA2b, which is slower but has higher Ca2+ affinity than the other SERCA isoforms, shared the same rate-limiting step as the SERCA1a isoform, but with a longer rise-time. Deletion of the SERCA2b luminal extension (LE) shifted the rate-limiting step to ATP-binding/phosphorylation, possibly because of LE-stabilization of the ATP-bound structure. These papers demonstrated the capability of TR-XSS to detect changes in rate-limiting steps and to investigate how protein structural dynamics respond to mutations and inhibitory conditions.
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