
==== Front
ACS Omega
ACS Omega
ao
acsodf
ACS Omega
2470-1343
American Chemical Society

10.1021/acsomega.4c01776
Article
Arabidopsis Dph4 is an Hsp70 Cochaperone with Iron-Binding Properties
https://orcid.org/0000-0003-4928-8458
Verma Amit Kumar †‡#
https://orcid.org/0000-0001-8353-8057
Sharma Priya §#
https://orcid.org/0000-0002-5444-3910
Islam Zeyaul *†∥
Biswal Anup Kumar †
Tak Yogesh ⊥
https://orcid.org/0000-0002-4821-9736
Sahi Chandan *†
† Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh 462066, India
‡ Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas 75390-9096, United States
§ Department of Botany, Faculty of Science, University of Delhi, Delhi 110007, India
∥ Diabetes Research Center (DRC), Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 34110 Doha, Qatar
⊥ Center for Alzheimer’s and Neurodegenerative Diseases, Peter O’Donnell Jr. Brian Institute, UT Southwestern Medical Center, Dallas, Texas 75390-9096, United States
* Email: zislam@hbku.edu.qa.
* Email: sahi@iiserb.ac.in.
28 08 2024
10 09 2024
9 36 3765037661
23 02 2024
30 07 2024
06 07 2024
© 2024 The Authors. Published by American Chemical Society
2024
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

J-domain proteins (JDPs) are obligate cochaperones of Hsp70s with a wide range of functions in protein homeostasis. Although the J-domain is required for the stimulation of Hsp70s ATPase activity, the functional specificity of JDPs is governed by domains or regions other than the J-domain. Jjj3/Dph4, a class III JDP, is required for diphthamide (DPH) biosynthesis in eukaryotes, including yeast and mammals. Dph4 has a conserved N-terminal J-domain and an uncharacterized C-terminal domain containing a signature CSL zinc finger motif. Previously, we showed that the Dph4 ortholog in Arabidopsis thaliana (atDjC13/AtJjj3/AtDph4) could restore DPH biosynthesis in yeast jjj3Δ mutant in a J-domain-dependent manner. Here, we characterize the C-terminal CSL motif of AtDph4 using yeast genetic and biochemical approaches. The CSL motif of AtDph4 is essential for DPH biosynthesis, and like human Dph4, AtDph4 showed distinct iron-binding activity, which is not present in its yeast counterpart. ScDph4 and AtDph4 proteins exhibit distinct iron-binding capabilities, as evidenced by UV–vis spectrophotometry, SEM-EDS (energy-dispersive spectroscopy function on the scanning electron microscope) and electron paramagnetic resonance (EPR) spectra analyses. Collectively, our data suggests that beyond their role as an Hsp70 cochaperone, Dph4 homologues in complex eukaryotes may have iron-binding abilities, indicating a potential role in iron-sulfur cluster assembly and iron homeostasis.

Department of Biotechnology, Ministry of Science and Technology, India 10.13039/501100001407 102/IFD/SAN/2131/20132014 Indian Institute of Science Education and Research Bhopal 10.13039/501100010713 NA Ministry of Education, India 10.13039/501100004541 NA Council of Scientific and Industrial Research, India 10.13039/501100001412 NA Department of Science and Technology, Ministry of Science and Technology, India 10.13039/501100001409 EMR/2015/001213 Department of Biotechnology, Ministry of Science and Technology, India 10.13039/501100001407 DBT/PR6885/GBD/27/488/2012 document-id-old-9ao4c01776
document-id-new-14ao4c01776
ccc-price
==== Body
pmcIntroduction

Proteins undergo a variety of post-translational modifications (PTMs) that affect their functions, localization, solubility, assembly, folding, interactions with nucleic acids, lipids, and other proteins, as well as their degradation.1−3 Among the several known PTMs that occur on multiple amino acids in different proteins, one exceptional modification is diphthamide (DPH), which is restricted to a single amino acid of a single protein in the entire proteome.4−6 DPH is a highly conserved and unique post-translational modification found in domain IV of eukaryotic Elongation Factor 2 (eEF2).5,6 Based on ribosomal frame-shift assays from the structural and functional yeast mutants, DPH is proposed to be essential for the maintenance of translational fidelity and the rate of translational elongation.7−11 Paradoxically, DPH can be ADP-ribosylated by bacterial toxins including Diphtheria toxin (DT) from Corynebacterium diphtheriae, Exotoxin A (ETA) from Pseudomonas aeruginosa, and Cholix toxin (ChxA) from Vibrio cholerae, resulting in a nonfunctional eEF2 that halts translation.12−15

DPH biosynthesis is a four-step process involving multiple proteins (Dph1-7, Kti11; Figure 1). In the first step, a 3-amino-3-carboxypropyl (ACP) radical is transferred from S-Adenosyl methionine (SAM) to the C2 position of the critical histidine residue in eEF2, leading to the formation of an ACP-modified eEF2 intermediate, catalyzed by Dph1-4 enzymes (Figure 1).16,17 Dph1-3 forms a multimeric protein complex, where Dph1 and 2 catalyze ACP transfer and Dph3 serves to channel electrons to the iron-sulfur clusters of Dph1 and Dph2 proteins, maintaining their redox state.16,17

Figure 1 Diphthamide biosynthesis pathway in eukaryotes. Diphthamide biosynthesis requires at least eight proteins (Dph1-7 and Kti13). The biosynthesis starts with the conversion of unmodified histidine to an ACP-intermediate that requires Dph1-4 proteins and the Kti13 protein. The intermediate then undergoes tetra methylation by Dph5 in the second step of the pathway, generating methylated diphthine. Dph7 then demethylates methylated diphthine, leading to the formation of diphthine, which is a substrate for the final enzyme of the pathway. Dph6 catalyzes the ATP-driven amidation of diphthine using ammonia as a source of the amide group, yielding diphthamide.

Dph4 (DPH biosynthesis 4) or Jjj3 is a J-domain protein (JDP) that plays an essential but still elusive role in the first step of DPH biosynthesis in budding yeast (Figure 1).18,19 Although nonessential in budding yeast, Dph4 is essential for normal growth and development in more complex eukaryotes. Homozygous mouse mutants display growth retardation, developmental delay, embryonic lethality, preaxial polydactyly, and neurodegeneration.20 Dph4 is also implicated in apoptosis and signaling.21,22jjj3/dph4 knockout yeast cells are sensitive to a range of drugs and compounds, including rapamycin, caffeine, hygromycin B, cycloheximide, and wortmannin.23−25 JDPs are highly diverse cochaperones of Hsp70s that promote protein folding, assembly and disassembly of protein structures, transport of proteins within the cell, and degradation of misfolded, unstable, and aggregated proteins.26−28 All JDPs have a characteristic J-domain containing a conserved HPD (Histidine-Proline-Aspartate) tripeptide motif in a loop between helices II and III that is essential for the stimulation of the otherwise poor ATPase activity of Hsp70s.26,28,29 While the J-domain is essential for all Hsp70 cochaperone functions of JDPs, functional specificity of JDPs emerges from domains/regions outside the J-domain.26,27,30

JJJ3/Dph4 possesses an N-terminal J-domain and a CSL-type zinc finger motif at the C-terminal that consists of four conserved cysteine residues (Cys-X-Cys···Cys-X2-Cys), which tetrahedrally can coordinate both Zinc (Zn) and Iron (Fe). The last cysteine residue is followed by serine (S) and a leucine (L) residue, hence the name “zf-CSL’’ from the CSL tripeptide.19,31,32 The domain comprises a three-stranded β-sandwich with one sheet consisting of two parallel strands (β1 and β6) and one antiparallel strand (β5). The second sheet consists of three strands (β2, β3, and β4) running antiparallel to each other. A short α-helix separates the two sheets.31 The J-domain and a CSL domain are connected with a flexible linker region that maintains the structural integrity of the two domains and allows them to perform independent functions.31 Both mammalian (HsDph4) and Arabidopsis thaliana (AtDph4) orthologs can substitute for ScDph4, for DPH biosynthesis in budding yeast suggesting conservation of function.31,33 Furthermore, for their function in DPH biosynthesis, ScDph4 and its mammalian and plant counterparts require both their J-domain as well as the CSL zinc finger motif.31,33,34

Human Dph4 protein binds to both zinc and iron with its C-terminal CSL motif.31 However, it predominantly exists in an iron-bound state, as Fe-Dph4 has a better ability to stimulate the ATPase activity of Hsp70 compared with Zn-Dph4 under similar conditions.31 Fe-Dph4 has redox and electron carrier activity; it undergoes oligomerization, thus acting as a transient iron reservoir to maintain iron homeostasis.31 Interestingly, ScDph4 neither binds to iron nor donates electrons to the Fe–S cluster of the Dph1-2 heterodimer.32 Thus, Dph4′s role in the first step of the DPH pathway awaits further study. Previously, we reported the presence of the Dph4 ortholog, viz., AtDph4, in A. thaliana and showed that AtDph4 could restore DPH biosynthesis in the jjj3Δ mutant of budding yeast.33 In the study presented here, we show that the C-terminal CSL motif of atDjC13/AtDph4 is crucial for DPH biosynthesis. Biochemical studies reveal that, like its mammalian orthologs, AtDph4 also binds iron, a property also shared by Dph3, another protein having a crucial redox-active function in DPH biosynthesis.32 Using chimeric constructs, we show that AtDph4 is a highly specialized JDP that has two distinct activities. Through its J-domain, it works as an Hsp70 cochaperone, and its iron-binding CSL zinc finger motif has redox activity of unknown significance.

Materials and Methods

In-Silico Methods

Dph4 orthologs in various organisms were identified by BLASTP searches in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) and their specific databases, e.g., Saccharomyces Genome Database (https://www.yeastgenome.org/blast-sgd) for fungal species and the TAIR (https://www.arabidopsis.org/Blast/index.jsp)35−37 database for A. thaliana using ScDph4 as the query sequence. The top results with a significantly high score and low E-value were shortlisted and used to perform the “reverse BLAST” in SGD. Those sequences that resulted in the Dph4 protein were considered Dph4 orthologs.38 Multiple sequence alignment was done using MAFFT (https://mafft.cbrc.jp/alignment/server/),39 and alignment of the secondary structure was done using ESpript3 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).40 These orthologs were then examined for the presence of distinct domains and motifs using domain prediction at the NCBI Conserved Domain Database (CDD, Batch CD-search) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi).41 The IBS (Illustrator of Biological Sequences)42 tool was used to create the schematic representations. The tertiary structures were predicted with the α fold protein structure database (https://alphafold.ebi.ac.uk/)43 and visualized with PyMol (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC.).

Yeast Methods

The yeast genes (ScDph3 and ScDph4) were amplified by PCR from genomic DNA isolated from the diploid BY4741 strain. The A. thalianaDPH4 gene was amplified using specific primer pairs (Table S1) using Arabidopsis cDNA. Point mutations were generated using the site-directed mutagenesis technique.44 Dph4 and Dph3 chimera constructs were made using the PCR sewing method45 in the HIS-based marker plasmid pRS413.46 All constructs are listed in Table 1. For assessing DPH biosynthesis in yeast cells, their sensitivity to DT was monitored. Constructs were double transformed with a URA3-marked GAL-DT plasmid in dph4Δand dph3Δyeast strains using the LiAc/SS carrier DNA/PEG method.47 The functionality was tested by monitoring yeast growth on conditional expression of GAL-DT in a galactose-containing medium. The CDS of AtHsp70-1 (Hsc70-1) and AtHsp70-14 (NEF) were amplified from Arabidopsis cDNA (Table S1) and cloned in BamHI /XmaI sites of pSMT3 vector (Table 1) (Mossessova and Lima).72

Table 1 List of Plasmid Constructs Used in the Study

plasmid	gene	base vector	source	
pCS944	ScDph4	pRS413-TEF	this study	
pCS68	ScDph4	pRS414-TEF	this study	
pCS1073	CaDph4	pRS414-TEF	this study	
pCS1074	KlDph4	pRS414-TEF	this study	
pCS977	AtDph4	pRS413-TEF	this study	
pCS1101	AtDph4	pRS414-TEF	this study	
pCS1076	HsDph4	pRS414-TEF	this study	
pCS1103	AtDph4H39Q	pRS414-TEF	this study	
pCS1102	AtDph4C158Y	pRS414-TEF	this study	
pCS945	ScDph4_P114A	pRS413-TEF	this study	
pCS946	ScDph4_del: P114	pRS413-TEF	this study	
pCS947	ScDph4_C152A	pRS413-TEF	this study	
pCS941	ScDph4_C113A	pRS414-TEF	this study	
pCS1104	AtDph4_add:113P	pRS414-TEF	this study	
pCS1106	AtDph4_C114A	pRS414-TEF	this study	
pCS1107	AtDph4_C155A	pRS414-TEF	this study	
pCS13	AtDph4-C	pRS413-TEF	this study	
pCS1096	ScDph3	pRS413-TEF	this study	
pCS1099	ScDph4-J+ ScDph3	pRS413-TEF	this study	
pCS1100	ScDph4-J+ AtDph4-C	pRS413-TEF	this study	
pCS77	Gal-DT	pYES2	Thakur et al.31	
pCS172	empty vector	pRS314	Sahi and Craig34	
pCS210	ScDph4	pET28a	this study	
pCS213	AtDph4	pET28a	this study	
pCS1299	AtHsp70-1	pSMT3	this study	
pCS1535	AtHsp70-14	pSMT3	this study	

Western Analysis

Western analysis was performed as described in ref (33). Briefly, an equal number of cells in the exponential phase were used to isolate the total protein. The cells were treated with 0.1 M NaOH before being resuspended in SDS sample buffer (62.5 mM Tris HCl, pH 6.8, 5% glycerol, 2% SDS, 2% β-mercaptoethanol, and 0.01% bromophenol blue). To detect protein, anti-Dph4 or anti-HA (Sigma-Aldrich) rabbit antibodies were used. Image Quant software (Molecular Dynamics, Sunnyvale, CA) was used for quantification.

Protein Expression and Purification

All Dph4 proteins were overexpressed inEscherichia coli Rosetta cells at an OD600 of 0.6–0.8, with IPTG (0.25 mM) and an O/N incubation at 16 °C and 200 rpm. His-tagged proteins were purified using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin. For purification, the cells were resuspended in lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% Glycerol, 1 mM DTT) and incubated on ice for 30 min with the addition of lysozyme, 1 mM PMSF, and a protease inhibitor cocktail (Sigma-Aldrich). Cells were then lysed by sonication (45% amplitude, 10s O/N, 20s OFF, 10 min). The lysate was then clarified by centrifugation at 15,000 rpm at 4 °C for 30 min. The supernatant was incubated for an hour with the Ni-NTA resin (Qiagen, Cat. No. 30210) at 4 °C. After passing the flow-through, the beads were washed with the lysis buffer thrice, and finally, His-tagged protein was eluted in 1 mL fractions using an imidazole gradient (50 mM Tris pH 8.0. 150 mM NaCl, 10% Glycerol, 1 mM DTT) with imidazole salt ranging from 250 to 600 mM. Protein-elution fractions were concentrated using MF-Millipore membrane filters with a pore size of 10 kDa. The aliquots were flash-frozen in liquid nitrogen and stored at −80 °C. Polyclonal anti-AtDph4 antibodies were raised against full-length 6xHis-AtDph4 protein in CD-1 mice in the animal facility of IISER Bhopal.

AtHsp70-1-pSMT3 and AtHsp70-14-pSMT3 constructs were overexpressed in Rosetta cells. Protein expression was induced at OD600 0.8 using 0.5 mM IPTG at 16 °C for 20 h. The cells were harvested by centrifugation at 6,000 g for 10 min. Cells were resuspended in lysis buffer (50 mM Tris pH7.5, 100 mM KCl, 0.5% triton X-100, 1 mM PMSF, 0.5 mM DTT, 20 mM imidazole, and 1 mg/mL lysozyme) and incubated at ice temperature for 30 min. The cells were sonicated for complete lysis using a probe sonicator. The lysate was centrifuged at 16,000 g for 30 min. The supernatant was incubated with Ni-NTA agarose beads for 3 h at 4 °C. Flow-through was collected, and Ni-NTA beads were washed in wash buffer 1 (50 mM Tris pH7.5, 100 mM KCl, 0.5% triton X-100, 1 mM PMSF, 0.5 mM DTT, and 20 mM imidazole) and wash buffer 2 (50 mM Tris pH7.5, 100 mM KCl, 0.5% triton X-100, 1 mM PMSF, 0.5 mM DTT, and 30 mM imidazole). For AtHsp70-1, the bound protein was incubated with 2 units of alkaline phosphatase (per mg of bound protein) for 10 min at room temperature and then washed, additionally, with wash buffer 2. Bound protein was eluted in an elution buffer (50 mM Tris (pH 7.5), 100 mM KCl, and 250 mM imidazole). The eluted protein was dialyzed against a dialysis buffer (50 mM Tris pH 7.5, 100 mM KCl, 500 μM DTT, 1 mM MgCl2) in the presence of SENP1 (Ulp protease) for 12 h. The cleaved tag was bound to the Ni-NTA agarose beads for 2 h at 4 °C, and the protein without tag was collected as flow-through.

ATPase Assay

About 0.5 μM purified Hsp70 (AtHsp70-1) was incubated with 8 μM Hsp40 (AtDph4 or AtDPHH39Q) and NEF (AtHsp70-14) in ATPase assay buffer (50 mM Tris, pH 7,5; 100 mM KCl, 500 μM DTT) with 1 mM ATP. Also, 50 μL of the reaction mixture was transferred to a 96-well plate in triplicate, which was incubated at 25 °C. To each well, 150 μL of malachite green reagent mix (0.081% (w/v) malachite green in 6N H2SO4, 5.7% ammonium heptamolybdate in 6 N HCl, and 0.7% (w/v) poly(vinyl alcohol) in water; all in 2:1:3 ratio) was added and incubated for 10 min at room temperature before stopping the reaction with 3.7% trisodium citrate. The absorbance was recorded at 620 nm in a plate reader (Eon microplate spectrophotometer, BioTek instruments). ATP hydrolysis was quantified according to the phosphate standard curve prepared by using dipotassium phosphate.

Biochemical Methods

UV–visible spectroscopy for ScDph4 and AtDph4 proteins was carried out on the Cary 5000 UV–vis NIR spectrophotometer (Agilent Technologies), and EPR studies were performed on Biospin (Bruker EPR), Model A200, at 9.42 GHz in the quartz tube under standard conditions as described previously.31 Energy-dispersive X-ray Spectroscopy (EDS) was done for estimating the elemental composition of proteins by Scanning Electron Microscopy (Zeiss microscope), Ultraplus.48,49,73

Results

Omnipresent Nature of Dph4 Proteins

Dph4 is involved in the first step of the DPH biosynthetic pathway in Saccharomyces cerevisiae.19 To better understand the conservation and functionality of the Dph4 protein, we identified orthologs of this protein in different organisms including Candida albicans, Kluyveromyces lactis, Homo sapiens, andA. thaliana. Dph4 protein orthologues were found in most but not all of the organisms (Table S2). The multiple sequence alignment of identified orthologs indicates high sequence similarity among them (Figure 2A). The identified orthologues possess the characteristic J-domain at the N-terminus along with the conserved HPD motif and a C-terminal CSL zinc finger motif containing four conserved cysteine residues (Figure 2B). As expected, the predicted structural organization of ScDph4, CaDph4, KlDph4, and AtDph4 were similar to the reported structure of Human Dph4 protein31 (Figure 2C).

Figure 2 Multiple sequence alignment (MSA) and structural comparison of Dph4 proteins. (A) Five representative Dph4 protein sequences from S. cerevisiaeScDph4, C. albicansCaDph4, K. lactisKlDph4, H. sapiensHsDph4, and A. thalianaAtDph4 were aligned using MAFFT, and the alignment of the secondary structure was done using ESpript3. Identical residues are highlighted in red, and similar residues are shown in boxes. (B) Domain organization of Dph4 protein. The domains were predicted using the NCBI conserved domain database (CDD), and the structure was made using the IBS tool (Illustrator of biological sequences) (C) Structure of Dph4 protein. The structure comprises a N-terminal J-domain characterized by four α-helices and a C-terminal zF-CSL motif consisting of a three-stranded β-sandwich with one sheet comprising two parallel strands and one antiparallel strand. The second sheet in the β-sandwich is composed of strands running antiparallel to each other. The two β-sheets are separated by a short stretch α-helix. The N-terminal and C-terminal are connected through a linker region (α-helix). The Dph4 protein structure is predicted using α fold 2 tool.

Dph4 Function is Highly Conserved across the Species

To test if the function of Dph4 is evolutionarily conserved across the organisms, including plants, we performed a yeast complementation experiment where the dph4 yeast knockout strain was tested for sensitivity to DT in the presence of Dph4 orthologues from various organisms. Full-length ORFs of these orthologues were cloned in a yeast expression vector under the TEF1 promoter for moderate overexpression. These constructs carrying different Dph4 orthologues were transformed into a dph4Δ strain harboring the catalytic A-chain subunit of DT (dph4Δ+DT) under the conditional regulation of the galactose inducible promoter. As expected, the yeast strain lacking Dph4 was resistant to DT on the galactose-containing medium. On the contrary, dph4Δ, when complemented with yeast ScDph4 (wild-type) and, with all of the orthologs, were DT sensitive and dead on the same media (Figure 3A), indicating that heterologous expression of different Dph4 orthologs can substitute for ScDph4 and can reconstitute the DPH biosynthesis pathway in yeast.

Figure 3 Dph4 protein is functionally conserved across organisms. (A) Five μL of 10-fold serial dilutions of dph4Δ cells harboring GAL-DT (dph4ΔGAL-DT) along with an empty pRS413 plasmid empty plasmid (−) or JDP expressing constructs containing Dph4 orthologs from S. cerevisiae (ScDph4), C. albicans (CaDph4), K. lactis (KlDph4), H. sapiens (HsDph4), A. thaliana (AtDph4) and driven under TEF promoter were spotted on Histidine drop-out plates and incubated at 23 °C for 3 days. (B) C-term zf-CSL motif-dependent function of AtDph4. Five μL of 10-fold serial dilutions of dph4Δ cells harboring GAL-DT (dph4ΔGAL-DT) along with an empty pRS413 plasmid empty plasmid (−) or JDP expressing constructs; pRS413-TEF-ScDph4 (ScDph4), pRS413-TEF-AtDph4(AtDph4), pRS413-TEF-AtDph4H39Q (AtDph4H39Q), and pRS413-TEF-atDjC13C158Y (AtDph4 C158Y) were spotted on His drop-out plates and incubated at 30 °C for 3 days.

While DPH biosynthesis genes have been studied in S. cerevisiae, Schizosaccharomyces pombe, Mus musculus, andH. sapiens,10,18−21,50 relatively little is known about them in plants. Only recently, in Arabidopsis, the Dph1 protein was identified and reported to be involved in the first step of DPH biosynthesis, which is known to regulate protein synthesis and plant growth.51 In addition to the highly conserved J-domain, Dph4 orthologs also have a conserved zinc finger CSL motif (zf-CSL) at their C-terminus, which is shown to be important for its function in DPH biosynthesis.31,52 We investigated whether the zf-CSL of Arabidopsis Dph4 is required for its function. A “C158A″ mutation was created in the C-terminal zf-CSL motif. A plasmid carrying this mutation was transformed into dph4Δ+DT. This mutation rendered the protein inactive, and as a result, the cells carrying these mutated genes survived on galactose medium in the presence of DT (Figure 3B). This was similar to the mutation in the J-domain (AtDph4H39Q), which was shown previously to be inactive for carrying out DPH biosynthesis in budding yeast.33

J-domain proteins are known to enhance the ATPase activity of Hsp70s, promoting effective interactions with client proteins and thereby facilitating Hsp70-mediated protein folding and supporting various cellular processes. To determine whether AtDph4 retains this capability, Hsp70 ATPase assays were performed. The ability of wild-type (AtDph4) and J-domain mutant (AtDph4H39Q) proteins to stimulate the basal ATPase activity of cytosolic AtHsp70-1 (AT5G02500), in the presence of the cytosolic nucleotide exchange factor AtHsp70-14 (AT1G79930), was monitored. AtDph4 stimulated the ATPase activity of AtHsp70-1 by ∼1.5 fold, while AtDph4H39Q could not (Figure S3). This suggests that AtDph4 may function as a Hsp70 cochaperone in carrying out DPH biosynthesis in Arabidopsis.

AtDph4 has Fe-Binding Activity

The CSL-class zinc-binding family of proteins is known to possess metal-binding capacity due to the presence of negatively charged residues in sequence, including four conserved cysteine residues, with the last cysteine followed by a serine and a leucine residue, forming a metal-binding knuckle.52 The NMR solution structure of the human Dph4 ortholog has been solved and has been shown to have electron carrier activity, which was attributed to its iron-binding property.31 This prompted us to investigate whether AtDph4 also possessed similar metal-binding properties and compare it with ScDph4. ScDph4 and AtDph4 were expressed and purified from E. coli using His-tag affinity chromatography. A striking difference in color between these two proteins was observed after elution. At equal concentrations (Figure 4A), AtDph4 was brown in color, while ScDph4 was colorless under the same conditions (Figure 4B). We corroborate this observation with an earlier report by Dong et al., where they compared the brown-colored ScDph3, another zf-CSL motif-containing protein, involved in the DPH biosynthesis pathway, to the colorless ScDph4 protein.32

Figure 4 AtDph4 has Fe-binding ability. (A–B) ScDph4 and AtDph4 protein expression profile. (A) Coomassie blue-stained SDS-PAGE for protein purification showing profiles for ScDph4 and AtDph4. Lanes 1–7 indicate loading of different sample fractions at various steps, viz. 1 whole cell extract, 2 supernatant, 3 pellet, 4 bead-bound protein, 5 bead after elution, 6 flow-through, 7 eluted proteins; M marker. (B) Preparations of ScDph4 protein (Left) and Arabidopsis AtDph4 (Right) purified from E. coli Rosetta strain. (C) UV–visible absorption spectra of ScDph4 (red) and AtDph4 (blue) are plotted against wavelength. The peaks at 350 and 490 nm are marked by the arrow symbol. (D) SEM-EDS analysis of ScDph4 and AtDph4. Backscattered electron images in the SEM display compositional contrast that results from different atomic number elements and their distribution. (E) EPR spectra of ScDph4 and AtDph4. Comparison of EPR spectra of ScDph4 with AtDph4 protein preparation recorded at −195.79 °C. The observed resonance of the AtDph4 protein is marked for its g values.

Metal-binding proteins, including Dph3, TFIIS, and rubredoxin, have been shown to exhibit unique UV–vis spectral profiles with peaks at 350 and 490 nm and a shoulder peak at 570 nm,32,53 typical characteristic of iron-binding proteins. So, we used UV–visible spectrophotometry to thoroughly characterize the metal-binding property of the AtDph4 protein. The UV–vis spectrum for AtDph4 also showed a similar UV–vis profile, with peaks at 350, 490, and 570 nm suggesting AtDph4 might have some metal affinity. On the other hand, ScDph4, which was colorless, did not show any of these peaks (Figure 4C). While such UV–vis spectrophotometry indicates iron binding, it does not directly specify the qualitative iron-binding properties of the proteins under investigation.54 Methods such as electron paramagnetic resonance (EPR) spectroscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) are the approaches to determine the elemental composition of metal-containing compounds or proteins.40−42 In principle, SEM exhibits the compositional contrast that results from different atomic number elements and their distribution, while EDS analysis involves the generation of an X-ray spectrum from the entire scan area of the SEM. To show that AtDph4 actually binds iron, we subjected the ScDph4 and AtDph4 proteins to EDS function on the SEM and observed the presence of iron. As indicated in the SEM-EDS spectra (Figure 4D), traces of iron were detected in the AtDph4 protein, but not in ScDph4, further confirming that AtDph4 has an affinity for iron.

In biological systems, iron can be present in either a ferrous (Fe2+) or ferric (Fe3+) state, depending upon the transition of electrons in the d-subshell orbits. We utilized this property of iron to detect its presence in protein preparations by employing EPR spectroscopy. The detection of iron by EPR spectroscopy depends in principle on the presence of an odd number of electrons, resulting in at least one unpaired electron. This creates an energy difference between the two possible spin states of an electron when an external magnetic field is applied.55 The EPR spectrum of AtDph4 showed resonance at a g value of 4.3, confirming the ferric state, but the same observation was not evident for ScDph4. This further validates that AtDph4 indeed binds to iron (Figure 4E). Put together, we conclude that although functionally similar in the budding yeast model, the Arabidopsis Dph4 protein has a metal-binding ability in contrast to its yeast counterpart.

Sequence Diversity in the C-Terminal Region Defines the Iron-Binding Property of Dph4 Orthologs

Our UV–visible spectrophotometry and SEM-EDS spectral analysis showed a difference between AtDph4 and ScDph4. To understand the underlying reasons for this difference in iron-binding properties, we undertook a rigorous sequence analysis and homology modeling of Dph4 homologues. Sequence comparison of the Dph4 proteins from different organisms revealed the presence of proline (P) between the first two cysteine residues that form the first loop of the metal-binding knuckle (Figure 5A) in some yeast strains, viz., CPRC in ScDph4, compared to CRC in AtDph4, and the HsDph4 sequence. This suggests that the presence of two amino acids in budding yeast as compared to one in human and Arabidopsis proteins could have an impact on its loop length and thereby on iron binding. This was substantiated by a structural overlap of this part of the proteins, indicating that an extra proline residue between Cys113 and Cys116 causes an increase in the overall length of the loop of Dph4 proteins in yeast species, which might influence their metal-binding abilities (Figure 5B). Since Dph3 and Dph4 share the CSL zinc finger motif, we then compared the sequence of AtDph4 with ScDph3, which is reported to possess iron-binding ability.32 Interestingly, the CSL motif of AtDph4 was found to be 42.31% similar to the CSL motif of ScDph3, but only 24.24% similar to ScDph4′s CSL motif. According to the NCBI BlastP pairwise alignment, there is no significant similarity between AtDph4 and ScDph4 in the CSL motif. Furthermore, Dph3 proteins have a single proline residue in between the two cysteines, strengthening our loop length hypothesis that sequences with a single amino acid in between the two cysteines, and thus reduced loop length, may coordinate bind iron better than those with more than one amino acid. This is consistent with our data that AtDph4, like HsDph4 and ScDph3, binds to iron and is thus colored, while ScDph4 is colorless.

Figure 5 Sequence and structural diversity in the Dph4 CSL motif. (A) Multiple sequence alignment of Arabidopsis yeast and human Dph4 orthologs, highlighting the differences in the iron-binding residues. (B) Secondary structure alignment of Dph4 orthologs to highlight the variation in loop length. (C) Five μL of 10-fold serial dilutions of dph4Δ cells harboring GAL-DT (dph4 GAL-DT) along with an empty pRS413 plasmid empty plasmid (−) or Dph4 protein expressing constructs; pRS413-TEF-ScDph4 (ScDph4), pRS413-TEF-ScDph4_P114A (ScDph4_P114A), pRS413-TEF-ScDph4_del P114 (ScDph4_del P114), pRS413-TEF-AtDph4_C152A (AtDph4_C152A), pRS413-TEF-AtDph4_C113A (AtDph4_C113A), pRS413-TEF-AtDph4 (AtDph4), pRS413-TEF-AtDph4_C114A (AtDph4_C114A), and pRS413-TEF-AtDph4_C155A (AtDph4_C155A) were spotted on His drop-out plates and incubated at 30 °C for 3 days.

We set out to determine if the proline residue is specifically required for the ScDph4 functionality or if it is just contributing to the overall loop length, which is crucial for the ScDph4 function. We first created ScDph4 constructs where the proline residue was either replaced with an alanine or deleted and tested their functionality in the dph4Δ+DT strain. Our results show that the P114A substitution did not affect the functionality of ScDph4, as dph4Δ+DT cells harboring P114A mutations carrying ScDph4 plasmids were unable to grow on galactose-containing plates (Figure 5C). In contrast, proline deletion completely abrogated ScDph4 function, as the cells survived on the galactose-containing medium. Next, we tested the importance of loop length in the Arabidopsis Dph4 protein. For this, we created an AtDph4 construct where a proline was inserted between the cysteine residues (P114), forming the metal-binding loop, and tested its functionality in the dph4Δ+DT strain. Interestingly, the presence of this extra proline renders AtDph4 inactive in budding yeast as it failed to complement the dph4Δ+DT strain (Figure 5D).

Next, we investigated the functional relevance of the conserved cysteines in the zf-CSL motif of ScDph4 and AtDph4. For this, cysteine 113 and 152 from ScDph4 and cysteine 114 and 155 from AtDph4 were chosen and replaced with alanine using site-directed mutagenesis. These mutation-carrying constructs were tested for their ability to complement the Dph4 deficiency in the dph4Δ+DT strain. As expected, these alanine substitutions evade both the ScDph4 and AtDph4 functions as they failed to rescue the dph4Δ+DT strain on galactose-containing medium (Figure 5D), signifying the highly conserved nature of cysteine residues in the formation of a metal-binding loop proving the essentiality of cysteine residues in both ScDph4 and AtDph4 function.

To test the expression of AtDph4 mutants, all of the constructs were transformed into the dph4Δ strain. The total protein lysate of the yeast transformants was analyzed by Western using the AtDph4 antibodies. While the wild-type (AtDph4) and J-domain mutant (AtDph4H39Q) proteins were expressed at comparable levels, most other mutants were expressed at lower levels in budding yeast (Figure S1).

To analyze if these proteins were properly folded and if their levels were sufficiently high to perform their role in DPH biosynthesis, we took an indirect approach. All of the constructs expressing AtDph4, AtDph4H39Q, AtDph4C158Y, AtDph4_add:113P, AtDph4_C114A, and AtDph4_C155A were transformed into the ydj1Δ strain. We hypothesized that, if properly folded, having a functional J-domain, all of the proteins, except AtDph4H39Q, should interact with Hsp70 and rescue the temperature sensitivity of yeast cells lacking Ydj1. Ydj1 is the most abundant J-domain protein in the yeast cytosol. Its deficiency makes yeast cells sick, which can be efficiently rescued by overexpression of J-domains containing proteins.34 All of the AtDph4 variants, except the J-domain mutation, AtDph4H39Q, were able to rescue the “slow-growth” phenotype of the Ydj1 deletion strain, signifying their ability to stimulate the weak ATPase activity of cytosolic Hsp70 (Figure S2). These results indicate two things: First, these variants are expressed at sufficiently high levels. Second, these proteins are stable and properly folded in vivo and their inability to rescue DPH biosynthesis defect of dph4Δ is due to compromised function in yeast cells.

ScDph3 and AtDph4 are Functionally Distinct

AtDph4 shares the conserved zf-CSL motif with the Dph3 protein, which has iron-binding activity and works as a redox-active protein during DPH biosynthesis.32 Since AtDph4 shares common structural elements, sequential features (Figure 6A,B), and also iron-binding ability, we examined if AtDph4 could replace Dph3 in dph3Δ cells and restore DPH biosynthesis. For this, we transformed the full-length AtDph4 construct in dph3Δ+DT cells. Our results showed that AtDph4 could not complement the DPH biosynthesis defects of Δdph3 (Figure 6C). Since the similarity between Dph3 and AtDph4 is only in the zf-CSL motif, we reasoned that possibly the presence of a J-domain tethers AtDph4 to Hsp70, thereby compromising its ability to substitute for Dph3 in budding yeast. So, we generated an AtDph4-C construct encompassing only residues 77-174 of AtDph4 lacking the N-terminal J-domain. Like full-length AtDph4, expression of C-terminus too could not restore DPH biosynthesis as dph3Δ+DT cells expressing the AtDph4-C mutant grew like dph3Δ [Gal-DT] cells with an empty plasmid (Figure 6C). We further tested this idea using an alternative approach. Using the PCR sewing method, we inserted the J-domain fragment of ScDph4 (residues 1-73) to the N-terminus of Dph3 to make a chimeric construct [ScDph4-J+ScDph3], assuming this protein would functionally behave like AtDph4. We made another chimera construct [ScDph4-J+AtDph4-C] where the same J-domain fragment was fused with AtDph4-C (77-174) as a control. The functionality of these chimeric proteins was tested in both dph3Δ+DT and dph4+DT cells. The [ScDph4-J+ScDph3] chimeric protein rescued dph3Δ+DT cells but failed to do so in dph4Δ+DT cells; on the other hand, [ScDph4-J+AtDph4-C] rescued the dph4Δ+DT cells but failed to complement dph3Δ+DT cells. Our results show that even though these two proteins display remarkable structural and biochemical similarity, AtDph4 and ScDph3 are functionally distinct and have very specific and nonredundant requirements in the DPH biosynthesis process.

Figure 6 AtDph4 and ScDph3 are structurally similar, though distinct functionally. (A) Dph3 and Dph4 proteins share the zf-CSL motif. The domain organization was predicted using NCBI CDD, and the structural representation was done using the IBS tool. (B) Sequence alignment of the C-terminal zf-CSL motif of Dph4 with the zf-CSL motif of ScDph3 and HsDph3 proteins; the alignment was visualized in Espript3. Similar residues are shown in boxes, and identical residues are highlighted in red. (C) Five μL of 10-fold serial dilutions of wild-type cells harboring GAL-DT along with an empty pRS413 plasmid (WT-GAL-DT) and dph3Δ cells either with an empty plasmid (−) or different gene expressing constructs pRS413-TEF-AtDph4 (AtDph4), pRS413-TEF-Dph3 (Dph3), pRS413-TEF-AtDph4-C terminal fragment (AtDph4-C), pRS413-TEF-chimera construct of J-domain of Dph4 fused with Dph3 (ScDph4-J+Dph3), and pRS413-TEF- chimera construct of J-domain of ScDph4 fused with AtDph4-C terminal (ScDph4-J+AtDph4-C) were spotted on His DO plates containing glucose and galactose and incubated at 30 °C for 3 days.

Discussion

DPH is an archaebacterial and eukaryotic post-translational modification that has been conserved during evolution. Its pathobiological relevance is well established, but its biological significance is still elusive. DPH’s presence in domain IV of eEF2 suggests its role in the maintenance of translational fidelity.16,17 DPH has also been implicated in specific translational regulation under stress; it regulates selenoprotein translation and internal ribosomal entry site (IRES) mediated translation DPH biosynthesis machinery is also highly conserved.56,57 All Dph1-7 genes have been found conserved in yeast, mice, and humans.16,17 So far, only two proteins involved in the first step of DPH biosynthesis have been identified in plants, AtDph1 and AtDph4.33,51 A recent publication established the existence of DPH in plants and showed that AtDph1 (AT5G62030) is essential for translational fidelity; just like the yeast mutants, dph1 mutants are sensitive to translational inhibitors and have defects in the TOR pathway, thus displaying abnormalities in growth.33,51 In a previous study, we discovered the Arabidopsis ortholog for the Dph4 protein, AtDph4 (atDjC13/AT4G10130), and showed that AtDph4 can replace Jjj3/ScDph4 for DPH biosynthesis in budding yeast, suggesting that its function is conserved across species.33,51

Dph4 is a bipartite protein with an N-terminal J-domain and a C-terminal CSL zinc finger motif. Dph4′s J-domain is required for DPH biosynthesis and also for stimulation of Hsp70 protein ATPase activity.31,58 The J-domain of AtDph4 is known to be required for DPH biosynthesis.33 Other than being a Hsp70 cochaperone, human Dph4 has roles such as iron binding, redox, and electron carrier activity governed by its C-terminal motif. Human Dph4 binds to both zinc and iron via four conserved cysteine residues in the CSL motif.31,58 These residues are critical for the function of yeast and human Dph4 genes in DPH biosynthesis.31,34 Consistent with that, C158Y, C114A, and C155A substitutions in AtDph4 rendered the protein nonfunctional, resulting in no DPH synthesis and thus resistance to DT in the yeast cells lacking ScDph4. Moreover, purified AtDph4 could bind iron, as verified by UV–visible and SEM-EDS spectra. This suggests that like HsDph4 and Dph3, AtDph4 could act as an electron donor for Dph1 and Dph2 iron–sulfur clusters.31,58 Similar to HsDph4 and other Zn-CSL motif-containing proteins, AtDph4 might also coordinate with the zinc metal. However, further experiments are needed to confirm this possibility. Beyond DPH biosynthesis, AtDph4 may also have a moonlighting iron-binding/redox function. AtDph4 orthologs could be functioning like DJA6 and DJA5, which are Arabidopsis type-I J-proteins that bind iron via conserved cysteine residues in the cysteine-rich (CR) domain and enable iron incorporation into Fe–S clusters through their interactions with the sulfur utilization factor (SUF) apparatus via their J-domain.59 The absence of these two proteins results in severe abnormalities in the accumulation of chloroplast Fe–S proteins, photosynthetic dysfunction, and considerable intracellular iron overload.59 Another JDP, AtHSCB, plays an important role in the regulation of the Fe–S assembly pathway in plant mitochondria, thus maintaining iron homeostasis.60−62 Just like the chloroplast and mitochondrial JDPs, AtDph4, being localized to the cytoplasm, might be a component of CIA (cytosolic iron–sulfur protein assembly) machinery and regulate the iron homeostasis in the cytosol. A recent study that examined the impact of iron and sulfate deficiency on levels of DPH modification, discovered that the iron- and sulfate-deficient Arabidopsis mutants have slightly increased levels of unmodified eEF2 than wild-type controls.33,51 DPH modification states are thus dependent on iron and sulfur availability, as four proteins involved in the first step require iron for their function, while Dph1 and 2 contain ISCs, and Dph3 and Dph4 bind iron via CSL motifs. However, whether or not the iron-binding activity of AtDph4 is important in vivo needs to be further investigated.

Sequence analysis and homology modeling of the metal-binding loop in Dph4 homologues support the idea of an optimum loop length on iron binding. This is further corroborated by the fact that HsDph4, AtDph4 as well as Dph3 proteins, which have shorter metal-binding knuckles bind iron and ScDph4 does not. Interestingly, the P114A substitution did not affect the functionality of ScDph4, while its deletion completely abrogated the ScDph4 function. On the other hand, the presence of extra proline in AtDph4 had a deleterious effect on protein function. This indicates that structural changes in this region are not only responsible for unique metal-binding abilities in complex eukaryotes such as plants and humans but also have a greater functional relevance in different organisms.

Despite the remarkable biochemical similarity shared by AtDph4 and Dph3, our results from the chimeras do not unequivocally prove an evolutionary relationship between these two proteins. Although they are involved in the same cellular process (DPH biosynthesis), both AtDph4 and Dph3 are functionally distinct. We believe that Dph3 is the main protein for all of the redox activities and acts as a reductant in Fe–S cluster assembly during DPH biosynthesis. On the other hand, Dph4 orthologs in more complex eukaryotes, like humans and plants, work as an Hsp70 cochaperone, playing a role in assisting the formation of Fe–S cluster assembly and, at the same time, buffering the electron transfer activity of Dph3; however, this needs to be experimentally validated.

At this point, we cannot rule out the possibility that AtDph4 may be involved in functions other than DPH biosynthesis, as well. ScDph4 is known to interact with protein kinases involved in different processes.63 Likewise, AtDph4 interacts with the mitogen-activated protein kinase 1 (AtMPK1),64 which is activated upon wounding, indicating a role of AtDph4 in defense response. A recent study reveals that AtDph4 plays a role in plant defense against Turnip mosaic potyvirus infection; consequently, AtDph4 loss of function mutants displayed significantly enhanced disease progression in comparison to WT plants.65HsDph4 is regulated via HSF2 transcriptional regulation under extracellular stress conditions.66 The methylation of DPH4 promoters influences diphthamide synthesis and toxin sensitivity in tumor cells, demonstrating that DPH gene expression impacts diphthamide synthesis.67 According to in-silico expression data at GENEINVESTIGATOR68 and eEFP browser (http://bar.utoronto.ca/efp2/Arabidopsis/Arabidopsis_eFPBrowser2.html).69AtDph4 is expressed in almost all stages of growth and development as well as under a variety of biotic and abiotic conditions. Interestingly, an ARE element-AAACCA, which is required for anaerobic induction, was also found in the AtDph4 promoter. Anaerobic conditions are required for ISC assembly.66,70 Thus, AtDph4 like HsDph4 may have a role in growth and development as well as the biotic or abiotic stress response.

The Dph4 homologues in complex eukaryotes like plants and humans exhibit metal-binding capabilities. Such a kind of novel gains of functions has been reported in other JDPs as well. For example, Cwc23, a type III JDP possesses an additional C-terminal RNA Recognition motif (RRM) in its Arabidopsis and human ortholog that imparts these proteins a unique RNA binding ability, which is absent in the S. cerevisiae ortholog, ScCwc23.71 Considering that fungi and animals are monophyletic and plants diverged much earlier, the most parsimonious interpretation is that the iron-binding property was possibly lost in the common ancestor of Saccharomycetaceae. Such events of neofunctionalization could be instrumental in generating evolutionary novelty with a greater adaptability to more complex environments.

Conclusions

In conclusion, our research work highlights the significance of the C-terminal domain of type III JDP, AtDph4 and reveals its novel iron-binding abilities. As genes involved in the DPH biosynthesis pathway are characterized in different systems, including plants, the importance of this unusual, yet conserved post-translational modification in growth and development is coming to light. It remains to be seen if the ability of AtDph4 to bind iron is important in either DPH biosynthesis or in a yet unknown cellular process requiring redox activity. Nevertheless, this study presents an example of how structural modifications in JDPs are responsible for imparting unique combinations of functions in complex eukaryotes, enabling adaptation to complex environments.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.4c01776.Table S1. List of primers used in this study. Table S2: List of Dph4 orthologs found in different species. Figure S1: Expressions of AtDph4 mutants. Figure S2: AtDph4 mutants rescue the ydj1Δstrain. Figure S3: AtDph4 stimulates ATPase activity of AtHsp70-1 (PDF)

Supplementary Material

ao4c01776_si_001.pdf

Accession Codes

Arabidopsis thaliana Dph4 (O82623), Homo sapiens Dph4 (Q6P3W2), Saccharomyces cerevisiae Dph4 (P47138), andSaccharomyces cerevisiae Dph3 (Q3E840).

Author Contributions

# A.K.V. and P.S. contributed equally. C.S., Z.I., and A.K.V. conceptualized and planned the experiment. A.K.V., Z.I., A.K.B., Y.T., and P.S. carried out the experiments. A.K.V., Z.I., and P.S. performed the in-silico analysis. A.K.V., P.S., and C.S. analyzed the results and wrote the manuscript.

A.K.V. thanks GATE fellowship, MHRD, India; P.S. and A.K.B. thank CSIR, New Delhi, for fellowship. This work was supported by funds from the Department of Biotechnology (DBT/PR6885/GBD/27/488/2012), the Department of Science and Technology (EMR/2015/001213), RGYI (SAN No.102/IFD/SAN/2131/2013-2014), and intramural funds from IISER Bhopal to C.S.

The authors declare no competing financial interest.

Acknowledgments

We thank Elizabeth Craig (UW-Madison, USA) for donating yeast strains and plasmids. We thank IISER Bhopal’s Central instrumentation facility for providing SEM, EPR, and UV–vis facilities.

Abbreviations

JDPs J-domain proteins

Hsp70 heat shock protein 70

DPH diphthamide

DT diphtheria toxin

Fe-Dph4 iron-bound Dph4

Zn-Dph4 zinc-bound Dph4

PTMs post-translational modifications

EF2 elongation factor 2
==== Refs
References

Walsh P. ; Bursać D. ; Law Y. C. ; Cyr D. ; Lithgow T. The J-protein Family: Modulating Protein Assembly, Disassembly and Translocation. EMBO Rep. 2004, 5 (6 ), 567–571. 10.1038/sj.embor.7400172.15170475
Seet B. T. ; Dikic I. ; Zhou M.-M. ; Pawson T. Reading Protein Modifications with Interaction Domains. Nat. Rev. Mol. Cell Biol. 2006, 7 (7 ), 473–483. 10.1038/nrm1960.16829979
Ramazi S. ; Zahiri J. Post-Translational Modifications in Proteins: Resources, Tools and Prediction Methods. Database 2021, 2021 . 10.1093/database/baab012.
Yang X.-J. Multisite Protein Modification and Intramolecular Signaling. Oncogene 2005, 24 (10 ), 1653–1662. 10.1038/sj.onc.1208173.15744326
Robinson E. A. ; Henriksen O. ; Maxwell E. S. Elongation Factor 2. J. Biol. Chem. 1974, 249 (16 ), 5088–5093. 10.1016/S0021-9258(19)42331-4.4368673
Van Ness B. G. ; Howard J. B. ; Bodley J. W. ADP-Ribosylation of Elongation Factor 2 by Diphtheria Toxin. Isolation and Properties of the Novel Ribosyl-Amino Acid and Its Hydrolysis Products. J. Biol. Chem. 1980, 255 (22 ), 10717–10720. 10.1016/S0021-9258(19)70366-4.7000782
Kimata Y. ; Kohno K. Elongation Factor 2 Mutants Deficient in Diphthamide Formation Show Temperature-Sensitive Cell Growth. J. Biol. Chem. 1994, 269 (18 ), 13497–13501. 10.1016/S0021-9258(17)36859-X.8175783
Ortiz P. A. ; Ulloque R. ; Kihara G. K. ; Zheng H. ; Kinzy T. G. Translation Elongation Factor 2 Anticodon Mimicry Domain Mutants Affect Fidelity and Diphtheria Toxin Resistance. J. Biol. Chem. 2006, 281 (43 ), 32639–32648. 10.1074/jbc.M607076200.16950777
Bär C. ; Zabel R. ; Liu S. ; Stark M. J. R. ; Schaffrath R. A Versatile Partner of Eukaryotic Protein Complexes That Is Involved in Multiple Biological Processes: Kti11/Dph3. Mol. Microbiol. 2008, 69 (5 ), 1221–1233. 10.1111/j.1365-2958.2008.06350.x.18627462
Liu S. ; Bachran C. ; Gupta P. ; Miller-Randolph S. ; Wang H. ; Crown D. ; Zhang Y. ; Wein A. N. ; Singh R. ; Fattah R. ; Leppla S. H. Diphthamide Modification on Eukaryotic Elongation Factor 2 Is Needed to Assure Fidelity of mRNA Translation and Mouse Development. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (34 ), 13817–13822. 10.1073/pnas.1206933109.22869748
Uthman S. ; Bär C. ; Scheidt V. ; Liu S. ; ten Have S. ; Giorgini F. ; Stark M. J. R. ; Schaffrath R. The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network. PLoS Genet. 2013, 9 (2 ), e1003334 10.1371/journal.pgen.1003334.23468660
Collier R. J. Diphtheria Toxin: Mode of Action and Structure. Bacteriol. Rev. 1975, 39 (1 ), 54–85. 10.1128/br.39.1.54-85.1975.164179
Iglewski B. H. ; Liu P. V. ; Kabat D. Mechanism of Action of Pseudomonas Aeruginosa Exotoxin Aiadenosine Diphosphate-Ribosylation of Mammalian Elongation Factor 2 in Vitro and in Vivo. Infect. Immun. 1977, 15 (1 ), 138–144. 10.1128/iai.15.1.138-144.1977.188760
Wilson B. A. ; Collier R. J. Diphtheria Toxin and Pseudomonas Aeruginosa Exotoxin A: Active-Site Structure and Enzymic Mechanism. Curr. Top. Microbiol. Immunol. 1992, 175 , 27–41. 10.1007/978-3-642-76966-5_2.1628498
Jørgensen R. ; Purdy A. E. ; Fieldhouse R. J. ; Kimber M. S. ; Bartlett D. H. ; Merrill A. R. Cholix Toxin, a Novel ADP-Ribosylating Factor from Vibrio Cholerae. J. Biol. Chem. 2008, 283 (16 ), 10671–10678. 10.1074/jbc.M710008200.18276581
Schaffrath R. ; Abdel-Fattah W. ; Klassen R. ; Stark M. J. R. The Diphthamide Modification Pathway from Saccharomyces Cerevisiae – Revisited. Mol. Microbiol. 2014, 94 (6 ), 1213–1226. 10.1111/mmi.12845.25352115
Tsuda-Sakurai K. ; Miura M. The Hidden Nature of Protein Translational Control by Diphthamide: The Secrets under the Leather. J. Biochem. 2019, 165 (1 ), 1–8. 10.1093/jb/mvy071.30204891
Chen J. Y. ; Bodley J. W. ; Livingston D. M. Diphtheria Toxin-Resistant Mutants of Saccharomyces Cerevisiae. Mol. Cell. Biol. 1985, 5 (12 ), 3357–3360. 10.1128/MCB.5.12.3357.3915773
Liu S. ; Milne G. T. ; Kuremsky J. G. ; Fink G. R. ; Leppla S. H. Identification of the Proteins Required for Biosynthesis of Diphthamide, the Target of Bacterial ADP-Ribosylating Toxins on Translation Elongation Factor 2. Mol. Cell. Biol. 2004, 24 (21 ), 9487–9497. 10.1128/MCB.24.21.9487-9497.2004.15485916
Webb T. R. ; Cross S. H. ; McKie L. ; Edgar R. ; Vizor L. ; Harrison J. ; Peters J. ; Jackson I. J. Diphthamide Modification of eEF2 Requires a J-Domain Protein and Is Essential for Normal Development. J. Cell Sci. 2008, 121 (19 ), 3140–3145. 10.1242/jcs.035550.18765564
Stahl S. ; da Silva Mateus Seidl A. R. ; Ducret A. ; Kux van Geijtenbeek S. ; Michel S. ; Racek T. ; Birzele F. ; Haas A. K. ; Rueger R. ; Gerg M. ; Niederfellner G. ; Pastan I. ; Brinkmann U. Loss of Diphthamide Pre-Activates NF-κB and Death Receptor Pathways and Renders MCF7 Cells Hypersensitive to Tumor Necrosis Factor. Proc. Natl. Acad. Sci. U.S.A. 2015, 112 (34 ), 10732–10737. 10.1073/pnas.1512863112.26261303
Cinà D. P. ; Ketela T. ; Brown K. R. ; Chandrashekhar M. ; Mero P. ; Li C. ; Onay T. ; Fu Y. ; Han Z. ; Saleem M. ; Moffat J. ; Quaggin S. E. Forward Genetic Screen in Human Podocytes Identifies Diphthamide Biosynthesis Genes as Regulators of Adhesion. Am. J. Physiol.: Renal Physiol. 2019, 317 (6 ), F1593–F1604. 10.1152/ajprenal.00195.2019.31566424
Gillies A. T. ; Taylor R. ; Gestwicki J. E. Synthetic Lethal Interactions in Yeast Reveal Functional Roles of J Protein Co-Chaperones. Mol. BioSyst. 2012, 8 (11 ), 2901 10.1039/c2mb25248a.22851130
Zhang Y. ; Lin Z. ; Zhu J. ; Wang M. ; Lin H. Diphthamide Promotes TOR Signaling by Increasing the Translation of Proteins in the TORC1 Pathway. Proc. Natl. Acad. Sci. U.S.A. 2021, 118 (37 ), e2104577118 10.1073/pnas.2104577118.34507998
Hawer H. ; Ütkür K. ; Arend M. ; Mayer K. ; Adrian L. ; Brinkmann U. ; Schaffrath R. Importance of Diphthamide Modified EF2 for Translational Accuracy and Competitive Cell Growth in Yeast. PLoS One 2018, 13 (10 ), e0205870 10.1371/journal.pone.0205870.30335802
Kampinga H. H. ; Andreasson C. ; Barducci A. ; Cheetham M. E. ; Cyr D. ; Emanuelsson C. ; Genevaux P. ; Gestwicki J. E. ; Goloubinoff P. ; Huerta-Cepas J. ; Kirstein J. ; Liberek K. ; Mayer M. P. ; Nagata K. ; Nillegoda N. B. ; Pulido P. ; Ramos C. ; De los Rios P. ; Rospert S. ; Rosenzweig R. ; Sahi C. ; Taipale M. ; Tomiczek B. ; Ushioda R. ; Young J. C. ; Zimmermann R. ; Zylicz A. ; Zylicz M. ; Craig E. A. ; Marszalek J. Function, Evolution, and Structure of J-Domain Proteins. Cell Stress Chaperones 2019, 24 (1 ), 7–15. 10.1007/s12192-018-0948-4.30478692
Kampinga H. H. ; Craig E. A. The HSP70 Chaperone Machinery: J Proteins as Drivers of Functional Specificity. Nat. Rev. Mol. Cell Biol. 2010, 11 (8 ), 579–592. 10.1038/nrm2941.20651708
Verma A. K. ; Tamadaddi C. ; Tak Y. ; Lal S. S. ; Cole S. J. ; Hines J. K. ; Sahi C. The Expanding World of Plant J-Domain Proteins. Crit. Rev. Plant Sci. 2019, 38 (5–6 ), 382–400. 10.1080/07352689.2019.1693716.33223602
Rosenzweig R. ; Nillegoda N. B. ; Mayer M. P. ; Bukau B. The Hsp70 Chaperone Network. Nat. Rev. Mol. Cell Biol. 2019, 20 (11 ), 665–680. 10.1038/s41580-019-0133-3.31253954
Craig E. A. ; Marszalek J. How Do J-Proteins Get Hsp70 to Do So Many Different Things?. Trends Biochem. Sci. 2017, 42 (5 ), 355–368. 10.1016/j.tibs.2017.02.007.28314505
Thakur A. ; Chitoor B. ; Goswami A. V. ; Pareek G. ; Atreya H. S. ; D’Silva P. Structure and Mechanistic Insights into Novel Iron-Mediated Moonlighting Functions of Human J-Protein Cochaperone, Dph4. J. Biol. Chem. 2012, 287 (16 ), 13194–13205. 10.1074/jbc.M112.339655.22367199
Dong M. ; Su X. ; Dzikovski B. ; Dando E. E. ; Zhu X. ; Du J. ; Freed J. H. ; Lin H. Dph3 Is an Electron Donor for Dph1-Dph2 in the First Step of Eukaryotic Diphthamide Biosynthesis. J. Am. Chem. Soc. 2014, 136 (5 ), 1754–1757. 10.1021/ja4118957.24422557
Verma A. K. ; Diwan D. ; Raut S. ; Dobriyal N. ; Brown R. E. ; Gowda V. ; Hines J. K. ; Sahi C. Evolutionary Conservation and Emerging Functional Diversity of the Cytosolic Hsp70:J Protein Chaperone Network of Arabidopsis Thaliana. G3: Genes, Genomes, Genet. 2017, 7 (6 ), 1941–1954. 10.1534/g3.117.042291.
Sahi C. ; Craig E. A. Network of General and Specialty J Protein Chaperones of the Yeast Cytosol. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (17 ), 7163–7168. 10.1073/pnas.0702357104.17438278
Boratyn G. M. ; Camacho C. ; Cooper P. S. ; Coulouris G. ; Fong A. ; Ma N. ; Madden T. L. ; Matten W. T. ; McGinnis S. D. ; Merezhuk Y. ; Raytselis Y. ; Sayers E. W. ; Tao T. ; Ye J. ; Zaretskaya I. BLAST: A More Efficient Report with Usability Improvements. Nucleic Acids Res. 2013, 41 (W1 ), W29–W33. 10.1093/nar/gkt282.23609542
Cherry J. M. ; Hong E. L. ; Amundsen C. ; Balakrishnan R. ; Binkley G. ; Chan E. T. ; Christie K. R. ; Costanzo M. C. ; Dwight S. S. ; Engel S. R. ; Fisk D. G. ; Hirschman J. E. ; Hitz B. C. ; Karra K. ; Krieger C. J. ; Miyasato S. R. ; Nash R. S. ; Park J. ; Skrzypek M. S. ; Simison M. ; Weng S. ; Wong E. D. Saccharomyces Genome Database: The Genomics Resource of Budding Yeast. Nucleic Acids Res. 2012, 40 (D1 ), D700–D705. 10.1093/nar/gkr1029.22110037
Berardini T. Z. ; Reiser L. ; Li D. ; Mezheritsky Y. ; Muller R. ; Strait E. ; Huala E. The Arabidopsis Information Resource: Making and Mining the “Gold Standard” Annotated Reference Plant Genome. Genesis 2015, 53 (8 ), 474–485. 10.1002/dvg.22877.26201819
Gabaldón T. ; Koonin E. V. Functional and Evolutionary Implications of Gene Orthology. Nat. Rev. Genet. 2013, 14 (5 ), 360–366. 10.1038/nrg3456.23552219
Katoh K. MAFFT: A Novel Method for Rapid Multiple Sequence Alignment Based on Fast Fourier Transform. Nucleic Acids Res. 2002, 30 (14 ), 3059–3066. 10.1093/nar/gkf436.12136088
Robert X. ; Gouet P. Deciphering Key Features in Protein Structures with the New ENDscript Server. Nucleic Acids Res. 2014, 42 (W1 ), W320–W324. 10.1093/nar/gku316.24753421
Lu S. ; Wang J. ; Chitsaz F. ; Derbyshire M. K. ; Geer R. C. ; Gonzales N. R. ; Gwadz M. ; Hurwitz D. I. ; Marchler G. H. ; Song J. S. ; Thanki N. ; Yamashita R. A. ; Yang M. ; Zhang D. ; Zheng C. ; Lanczycki C. J. ; Marchler-Bauer A. CDD/SPARCLE: The Conserved Domain Database in 2020. Nucleic Acids Res. 2020, 48 (D1 ), D265–D268. 10.1093/nar/gkz991.31777944
Liu W. ; Xie Y. ; Ma J. ; Luo X. ; Nie P. ; Zuo Z. ; Lahrmann U. ; Zhao Q. ; Zheng Y. ; Zhao Y. ; Xue Y. ; Ren J. IBS: An Illustrator for the Presentation and Visualization of Biological Sequences. Bioinformatics 2015, 31 (20 ), 3359–3361. 10.1093/bioinformatics/btv362.26069263
Jumper J. ; Evans R. ; Pritzel A. ; Green T. ; Figurnov M. ; Ronneberger O. ; Tunyasuvunakool K. ; Bates R. ; Žídek A. ; Potapenko A. ; Bridgland A. ; Meyer C. ; Kohl S. A. A. ; Ballard A. J. ; Cowie A. ; Romera-Paredes B. ; Nikolov S. ; Jain R. ; Adler J. ; Back T. ; Petersen S. ; Reiman D. ; Clancy E. ; Zielinski M. ; Steinegger M. ; Pacholska M. ; Berghammer T. ; Bodenstein S. ; Silver D. ; Vinyals O. ; Senior A. W. ; Kavukcuoglu K. ; Kohli P. ; Hassabis D. Highly Accurate Protein Structure Prediction with AlphaFold. Nature 2021, 596 (7873 ), 583–589. 10.1038/s41586-021-03819-2.34265844
Liu H. ; Naismith J. H. An Efficient One-Step Site-Directed Deletion, Insertion, Single and Multiple-Site Plasmid Mutagenesis Protocol. BMC Biotechnol. 2008, 8 (1 ), 91 10.1186/1472-6750-8-91.19055817
Bryksin A. V. ; Matsumura I. Overlap Extension PCR Cloning: A Simple and Reliable Way to Create Recombinant Plasmids. BioTechniques 2010, 48 (6 ), 463–465. 10.2144/000113418.20569222
Mumberg D. ; Müller R. ; Funk M. Yeast Vectors for the Controlled Expression of Heterologous Proteins in Different Genetic Backgrounds. Gene 1995, 156 (1 ), 119–122. 10.1016/0378-1119(95)00037-7.7737504
Gietz R. D. ; Schiestl R. H. Quick and Easy Yeast Transformation Using the LiAc/SS Carrier DNA/PEG Method. Nat. Protoc. 2007, 2 (1 ), 35–37. 10.1038/nprot.2007.14.17401335
Shindo D. ; Oikawa T. Energy Dispersive X-Ray Spectroscopy. Anal. Electron Microsc. Mater. Sci. 2002, 81–102. 10.1007/978-4-431-66988-3_4.
Newbury D. E. ; Ritchie N. W. M. Is Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDS) Quantitative?. Scanning 2013, 35 (3 ), 141–168. 10.1002/sca.21041.22886950
Villahermosa D. ; Christensen O. ; Knapp K. ; Fleck O. Schizosaccharomyces pombeMutSα and MutLα Maintain Stability of Tetra-Nucleotide Repeats and Msh3 of Hepta-Nucleotide Repeats. G3: Genes, Genomes, Genet. 2017, 7 (5 ), 1463–1473. 10.1534/g3.117.040816.
Zhang H. ; Quintana J. ; Ütkür K. ; Adrian L. ; Hawer H. ; Mayer K. ; Gong X. ; Castanedo L. ; Schulten A. ; Janina N. ; Peters M. ; Wirtz M. ; Brinkmann U. ; Schaffrath R. ; Krämer U. Translational Fidelity and Growth of Arabidopsis Require Stress-Sensitive Diphthamide Biosynthesis. Nat. Commun. 2022, 13 (1 ), 4009 10.1038/s41467-022-31712-7.35817801
Sun J. ; Zhang J. ; Wu F. ; Xu C. ; Li S. ; Zhao W. ; Wu Z. ; Wu J. ; Zhou C.-Z. ; Shi Y. Solution Structure of Kti11p from Saccharomyces Cerevisiae Reveals a Novel Zinc-Binding Module. Biochemistry 2005, 44 (24 ), 8801–8809. 10.1021/bi0504714.15952786
Zimmermann B. ; Schweinsberg S. ; Drewianka S. ; Herberg F. W. Effect of Metal Ions on High-Affinity Binding of Pseudosubstrate Inhibitors to PKA. Biochem. J. 2008, 413 (1 ), 93–101. 10.1042/BJ20071665.18373497
Säbel C. E. ; Neureuther J. M. ; Siemann S. A Spectrophotometric Method for the Determination of Zinc, Copper, and Cobalt Ions in Metalloproteins Using Zincon. Anal. Biochem. 2010, 397 (2 ), 218–226. 10.1016/j.ab.2009.10.037.19854146
Taiwo F. A. Electron Paramagnetic Resonance Spectroscopic Studies of Iron and Copper Proteins. Spectroscopy 2003, 17 (1 ), 53–63. 10.1155/2003/673567.
Mayer K. ; Mundigl O. ; Kettenberger H. ; Birzele F. ; Stahl S. ; Pastan I. ; Brinkmann U. Diphthamide Affects Selenoprotein Expression: Diphthamide Deficiency Reduces Selenocysteine Incorporation, Decreases Selenite Sensitivity and Pre-Disposes to Oxidative Stress. Redox Biol. 2019, 20 , 146–156. 10.1016/j.redox.2018.09.015.30312900
Argüelles S. ; Camandola S. ; Cutler R. G. ; Ayala A. ; Mattson M. P. Elongation Factor 2 Diphthamide Is Critical for Translation of Two IRES-Dependent Protein Targets, XIAP and FGF2, under Oxidative Stress Conditions. Free Radical Biol. Med. 2014, 67 , 131–138. 10.1016/j.freeradbiomed.2013.10.015.24140707
Kroczynska B. ; Blond S. Y. Cloning and Characterization of a New Soluble Murine J-Domain Protein That Stimulates BiP, Hsc70 and DnaK ATPase Activity with Different Efficiencies. Gene 2001, 273 (2 ), 267–274. 10.1016/S0378-1119(01)00583-2.11595173
Zhang J. ; Bai Z. ; Ouyang M. ; Xu X. ; Xiong H. ; Wang Q. ; Grimm B. ; Rochaix J. ; Zhang L. The DnaJ Proteins DJA6 and DJA5 Are Essential for Chloroplast Iron–Sulfur Cluster Biogenesis. EMBO J. 2021, 40 (13 ), e106742 10.15252/embj.2020106742.33855718
Xu X. M. ; Lin H. ; Latijnhouwers M. ; Møller S. G. Dual Localized AtHscB Involved in Iron Sulfur Protein Biogenesis in Arabidopsis. PLoS One 2009, 4 (10 ), e7662 10.1371/journal.pone.0007662.19865480
Leaden L. ; Busi M. V. ; Gomez-Casati D. F. The Mitochondrial Proteins AtHscB and AtIsu1 Involved in Fe–S Cluster Assembly Interact with the Hsp70-Type Chaperon AtHscA2 and Modulate Its Catalytic Activity. Mitochondrion 2014, 19 , 375–381. 10.1016/j.mito.2014.11.002.25462017
Couturier J. ; Touraine B. ; Briat J.-F. ; Gaymard F. ; Rouhier N. The Iron-Sulfur Cluster Assembly Machineries in Plants: Current Knowledge and Open Questions. Front. Plant Sci. 2013, 4 , 259 10.3389/fpls.2013.00259.23898337
Fasolo J. ; Sboner A. ; Sun M. G. F. ; Yu H. ; Chen R. ; Sharon D. ; Kim P. M. ; Gerstein M. ; Snyder M. Diverse Protein Kinase Interactions Identified by Protein Microarrays Reveal Novel Connections between Cellular Processes. Genes Dev. 2011, 25 (7 ), 767–778. 10.1101/gad.1998811.21460040
Popescu S. C. ; Popescu G. V. ; Bachan S. ; Zhang Z. ; Gerstein M. ; Snyder M. ; Dinesh-Kumar S. P. MAPK Target Networks in Arabidopsis Thaliana Revealed Using Functional Protein Microarrays. Genes Dev. 2009, 23 (1 ), 80–92. 10.1101/gad.1740009.19095804
Butković A. ; González R. ; Rivarez M. P. S. ; Elena S. F. A Genome-Wide Association Study Identifies Arabidopsis Thaliana Genes That Contribute to Differences in the Outcome of Infection with Two Turnip Mosaic Potyvirus Strains That Differ in Their Evolutionary History and Degree of Host Specialization. Virus Evolution 2021, 7 (2 ), veab063 10.1093/ve/veab063.34532063
Li G. ; He Y. ; Liu H. ; Liu D. ; Chen L. ; Luo Y. ; Chen L. ; Qi L. ; Wang Y. ; Wang Y. ; Wang Y. ; Zhan L. ; Zhang N. ; Zhu X. ; Song T. ; Guo H. DNAJC24 Is a Potential Therapeutic Target in Hepatocellular Carcinoma through Affecting Ammonia Metabolism. Cell Death Dis. 2022, 13 (5 ), 490 10.1038/s41419-022-04953-z.35606363
Wei H. ; Xiang L. ; Wayne A. S. ; Chertov O. ; FitzGerald D. J. ; Bera T. K. ; Pastan I. Immunotoxin Resistance via Reversible Methylation of the DPH4 Promoter Is a Unique Survival Strategy. Proc. Natl. Acad. Sci. U.S.A. 2012, 109 (18 ), 6898–6903. 10.1073/pnas.1204523109.22509046
Hruz T. ; Laule O. ; Szabo G. ; Wessendorp F. ; Bleuler S. ; Oertle L. ; Widmayer P. ; Gruissem W. ; Zimmermann P. Genevestigator V3: A Reference Expression Database for the Meta-Analysis of Transcriptomes. Adv. Bioinf. 2008, 2008 , 1–5. 10.1155/2008/420747.
Winter D. ; Vinegar B. ; Nahal H. ; Ammar R. ; Wilson G. V. ; Provart N. J. An “Electronic Fluorescent Pictograph” Browser for Exploring and Analyzing Large-Scale Biological Data Sets. PLoS One 2007, 2 (8 ), e718 10.1371/journal.pone.0000718.17684564
Lill R. ; Mühlenhoff U. Iron-Sulfur Protein Biogenesis in Eukaryotes: Components and Mechanisms. Annu. Rev. Cell Dev. Biol. 2006, 22 (1 ), 457–486. 10.1146/annurev.cellbio.22.010305.104538.16824008
Raut S. ; Yadav K. ; Verma A. K. ; Tak Y. ; Waiker P. ; Sahi C. Co-Evolution of Spliceosomal Disassembly Interologs: Crowning J-Protein Component with Moonlighting RNA-Binding Activity. Curr. Genet. 2019, 65 (2 ), 561–573. 10.1007/s00294-018-0906-9.30467716
Mossessova E. ; Lima C. D. Ulp1-SUMO Crystal Structure and Genetic Analysis Reveal Conserved Interactions and a Regulatory Element Essential for Cell Growth in Yeast. Mol. Cell 2000, 5 (5 ), 865–876. 10.1016/S1097-2765(00)80326-3.10882122
Cao Y. ; Dhahad H. A. ; El-Shorbagy M. A. ; et al. Green synthesis of bimetallic ZnO–CuO nanoparticles and their cytotoxicity properties. Sci. Rep. 2011, 11 , 23479 10.1038/s41598-021-02937-1.
