Heterologous Expression of Stress-Responsive DUF538 Domain Containing Protein and its Morpho-Biochemical Consequences

As a usual response, plants induce/activate various proteins which are thought to be involved in defense mechanisms against the biotic and abiotic stresses they may be confronted with. The novel DUF538 domain containing proteins with unknown functions have been found to be induced/activated in response to different environmental stress stimuli in plants. In order to perform biochemical studies with these new plant stress-responsive proteins, a cDNA containing DUF538 domain was amplified from Celosia cristata full-length leaf expression library using a specific primer set. The isolated cDNA was subsequently expressed in Escherichia coli as a part of maltose-binding fusion protein (MBP-DUF538 construct) and purified at the yield of about 32 mg per liter of cell culture by affinity chromatography without affecting the recombinant bacterial cell growth. The purified fusion product was exogenously applied (10 μg per 4 cm²) on the leaves of Nicotiana tobaccum L. The results revealed that fused DUF538 protein does not induce morphological reposes, but elevates redox enzyme activities including catalase, peroxidase, polyphenol oxidase and phenyalanine ammonia lyase. This is the first time ever time report with respect to the heterologous expression of a plant stress-responsive DUF538 domain that may provide a basis to study its physiological roles and biochemical activities in vitro and in vivo.     

Pectin methylesterase activity of plant DUF538 protein superfamily

DUF538 (domain of unknown function 538) proteins are known as a group of putative hypothetical proteins in a wide range of plant species. They have been identified from some plants challenged with various environmental stresses. However, a little is known about their functional properties. They have been newly predicted to have binding capacity and esterase-type hydrolytic activity towards bacterial lipopolysaccharides and chlorophyll molecules as carboxylic compounds in plants. In the present study, the binding ability and the methylesterase activity of DUF538 proteins towards pectin molecules were also predicted. Their similarities to pectin methylesterases and their binding ability to pectin molecule were predicted using bioinformatic tools as well as the experimental method. A probable cooperation was speculated between DUF538 and pectin methylesterase protein families in cell wall associated defense responses in plants.     

DUF538 Protein Super Family is Predicted to be the Potential Homologue of Bactericidal/Permeability-Increasing Protein in Plant System

DUF538 protein super family includes a number of plant proteins that their role is not yet clear. These proteins have been frequently reported to be expressed in plants under various stressful stimuli such as bacteria and elicitors. In order to further understand about this protein family we utilized bioinformatics tools to analyze its structure in details. As a result, plants DUF538 was predicted to be the partial structural homologue of BPI (bactericidal/permeability increasing) proteins in mammalian innate immune system that provides the first line of defense against different pathogens including bacteria, fungi, viruses and parasites. Moreover, on the base of the experimental data, it was identified that exogenously applied purified fused product of Celosia DUF538 affects the bacterial growth more possibly similar to BPI through the binding to the bacterial membranes. In conclusion, as the first ever time report, we nominated DUF538 protein family as the potential structural and functional homologue of BPI protein in plants, providing a basis to study the novel functions of this protein family in the biological systems in the future.     

DUF538 protein superfamily is predicted to be chlorophyll hydrolyzing enzymes in plants

The possible hydrolytic activity towards chlorophyll molecules was predicted for DUF538 protein superfamily in plants. It was examined by using computational as well as experimental tools including in vitro chlorophyll degradation, antioxidant compounds production and in vivo real-time gene expression tests. Comparison of the computational data with the experimental results indicated that DUF538 proteins might be chlorophyll hydrolyzing enzyme (most probably carboxyesterase) which degrade chlorophyll molecules (66 % per 12 hrs) to produce new compounds (1.8 fold per 12 hrs) with antioxidant properties. The relevance of DUF538 gene expression level with the chlorophyll contents (2.8 fold increase per chlorophyll content of 50 %) of the drought-stressed leaves showed that chlorophyll degradation by DUF538 is most probably induced in response to stress stimuli. Despite membranous chlorophyll catabolic pathways, DUF538-dependent reactions is predicted to be occurred in the cytosol of the under stressed plants. We addressed as to whether chlorophyll breakdown to antioxidant compounds by DUF538 is a defense mechanism of plants against stress stimuli, in vivo? This question is going to be investigated in our next research project.     

The photoconvertible water-soluble chlorophyll-binding protein of Chenopodium album is a member of DUF538, a superfamily that distributes in Embryophyta

Various plants possess hydrophilic chlorophyll (Chl) proteins known as water-soluble Chl-binding proteins (WSCPs). WSCPs exist in two forms: Class I and Class II, of which Class I alone exhibits unique photoconvertibility. Although numerous genes encoding Class II WSCPs have been identified and the molecular properties of their recombinant proteins have been well characterized, no Class I WSCP gene has been identified to date. In this study, we cloned the cDNA and a gene encoding the Class I WSCP of Chenopodium album (CaWSCP). Sequence analyses revealed that CaWSCP comprises a single exon corresponding to 585 bp of an open reading frame encoding 195 amino acid residues. The CaWSCP protein sequence possesses a signature of DUF538, a protein superfamily of unknown function found almost exclusively in Embryophyta. The recombinant CaWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (CaWSCP-His) that removes Chls from the thylakoid. Under visible light illumination, the reconstituted CaWSCP-His was successfully photoconverted into a different pigment with an absorption spectrum identical to that of native CaWSCP. Interestingly, while CaWSCP-His could bind both Chl a and Chl b, photoconversion occurred only in CaWSCP-His reconstituted with Chl a.     

Identification of <Emphasis Type="Italic">DUF538</Emphasis> cDNA clone from <Emphasis Type="Italic">Celosia cristata</Emphasis> expressed sequences of nonstressed and stressed leaves

DUF538 domain-containing protein family consists of several plant proteins of unknown functions. This protein family has already been discovered by genome annotation tools and cloned as an inducible gene product under various environmental stress conditions. For the first time, we presented a full length DUF538 cDNA (encoding 170 amino acid residues) clone, which was randomly isolated from Celosia cristata leaf cDNA library constructed under normal growth conditions and consistently amplified from leaf cDNA populations prepared from nonstressed and drought-stressed leaves. We predicted that a DUF538 gene product can be a putative candidate for common stress-related protein (regulatory factor) in the plant system. The nucleotide and deduced amino acid sequences of the isolated clone have been submitted to EMBL data bases under accession no. AJ535713.     

A Thermoacidophile-Specific Protein Family,DUF3211, Functions as a Fatty Acid Carrier with Novel Binding Mode

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/β structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C₁₄) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode.     

The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding

The dynamic responses of microtubules (MTs) to internal and external signals are modulated by a plethora of microtubule-associated proteins (MAPs). In higher plants, many plant-specific MAPs have emerged during evolution as advantageous to their sessile lifestyle. Some members of the IQ67 domain (IQD) protein family have been shown to be plant-specific MAPs. However, the mechanisms of interaction between IQD proteins and MTs remain elusive. Here we demonstrate that the domain of unknown function 4005 (DUF4005) of the Arabidopsis IQD family protein ABS6/AtIQD16 is a novel MT-binding domain. Cosedimentation assays showed that the DUF4005 domain binds directly to MTs in vitro. GFP-labeled DUF4005 also decorates all types of MT arrays tested in vivo. Furthermore, we showed that a conserved stretch of 15 amino acid residues within the DUF4005 domain, which shares sequence similarity with the C-terminal MT-binding domain of human MAP Kif18A, is required for the binding to MTs. Transgenic lines overexpressing the DUF4005 domain displayed a spectrum of developmental defects, including spiral growth and stunted growth at the organismal level. At the cellular level, DUF4005 overexpression caused defects in epidermal pavement cell and trichome morphogenesis, as well as abnormal anisotropic cell elongation in the hypocotyls of dark-grown seedlings. These data establish that the DUF4005 domain of ABS6/AtIQD16 is a new MT-binding domain, overexpression of which perturbs MT homeostasis in plants. Our findings provide new insights into the MT-binding mechanisms of plant IQD proteins.     

Characterization of DUF724 gene family in Arabidopsis thaliana

Eighteen genes that encode the proteins with highly conserved Domain of Unknown Function 724 (DUF724) and Agenet domains were identified in plant taxa but not in animals and fungi. They are actively expressed in many different plant tissues, implying that they may play important roles in plants. Here we report the characterization of their structural organizations, expression patterns and protein–protein interactions. In Arabidopsis, the DUF724 genes were expressed in roots, leaves, shoot apical meristems, anthers and pollen grains. At least seven of the ten Arabidopsis DUF724 proteins (AtDuf1 to AtDuf10) were localized in nucleus. Three of them (AtDuf3, AtDuf5 and AtDuf7) may form homodimers or homopolymers, but did not interact with other members of the same family. Together with the significant similarity between DUF724 proteins and FMRP in the fundamental and characteristic molecular architecture, the results implies the DUF724 gene family may be involved in the polar growth of plant cells via transportation of RNAs.     

Caenorhabditis elegans BAH-1 Is a DUF23 Protein Expressed in Seam Cells and Required for Microbial Biofilm Binding to the Cuticle

The cuticle of Caenorhabditis elegans, a complex, multi-layered extracellular matrix, is a major interface between the animal and its environment. Biofilms produced by the bacterial genus Yersinia attach to the cuticle of the worm, providing an assay for surface characteristics. A C. elegans gene required for biofilm attachment, bah-1, encodes a protein containing the domain of unknown function DUF23. The DUF23 domain is found in 61 predicted proteins in C. elegans, which can be divided into three distinct phylogenetic clades. bah-1 is expressed in seam cells, which are among the hypodermal cells that synthesize the cuticle, and is regulated by a TGF-β signaling pathway.     

Structural and Functional Characterization of DUF1471 Domains of Salmonella Proteins SrfN,YdgH/SssB,and YahO

Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21–91), and the C-terminal domain III (residues 244–314)). Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.     

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

Background

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats.

Results

ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development.

Conclusions

We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.     

Structural diversity in the Mycobacteria DUF3349 superfamily

A protein superfamily with a “Domain of Unknown Function,”, DUF3349 (PF11829), is present predominately in Mycobacterium and Rhodococcus bacterial species suggesting that these proteins may have a biological function unique to these bacteria. We previously reported the inaugural structure of a DUF3349 superfamily member, Mycobacterium tuberculosis Rv0543c. Here, we report the structures determined for three additional DUF3349 proteins: Mycobacterium smegmatis MSMEG_1063 and MSMEG_1066 and Mycobacterium abscessus MAB_3403c. Like Rv0543c, the NMR solution structure of MSMEG_1063 revealed a monomeric five α‐helix bundle with a similar overall topology. Conversely, the crystal structure of MSMEG_1066 revealed a five α‐helix protein with a strikingly different topology and a tetrameric quaternary structure that was confirmed by size exclusion chromatography. The NMR solution structure of a fourth member of the DUF3349 superfamily, MAB_3403c, with 18 residues missing at the N‐terminus, revealed a monomeric α‐helical protein with a folding topology similar to the three C‐terminal helices in the protomer of the MSMEG_1066 tetramer. These structures, together with a GREMLIN‐based bioinformatics analysis of the DUF3349 primary amino acid sequences, suggest two subfamilies within the DUF3349 family. The division of the DUF3349 into two distinct subfamilies would have been lost if structure solution had stopped with the first structure in the DUF3349 family, highlighting the insights generated by solving multiple structures within a protein superfamily. Future studies will determine if the structural diversity at the tertiary and quaternary levels in the DUF3349 protein superfamily have functional roles in Mycobacteria and Rhodococcus species with potential implications for structure‐based drug discovery.     

Crystal structure of cce_0566 from Cyanothece 51142, a protein associated with nitrogen fixation in the DUF269 family

The crystal structure for cce_0566 (171 aa, 19.4 kDa), a DUF269 annotated protein from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142, was determined to 1.60 Å resolution. Cce_0566 is a homodimer with each molecule composed of eight α-helices folded on one side of a three strand anti-parallel β-sheet. Hydrophobic interactions between the side chains of largely conserved residues on the surface of each β-sheet hold the dimer together. The fold observed for cce_0566 may be unique to proteins in the DUF269 family, hence, the protein may also have a function unique to nitrogen fixation. A solvent accessible cleft containing conserved charged residues near the dimer interface could represent the active site or ligand-binding surface for the protein’s biological function.Structured summary of protein interactionsDUF269 and DUF269 bind by x-ray crystallography (View interaction)     

巴西橡胶树胶乳中DUF1262结构域蛋白结构与基因表达分析

为了揭示天然橡胶生物合成酶互作蛋白结构及其在天然橡胶生物合成过程中的功能。本研究以橡胶树胶乳橡胶粒子总蛋白为研究对象,采用免疫共沉淀实验技术以天然橡胶合成关键酶顺式-异戊二烯基转移酶（CPT）抗体从胶乳中捕获了1个含DUF1262结构域的未知功能蛋白。生物信息学分析表明橡胶树基因组中包含50个编码含DUF1262结构域蛋白的基因序列;蛋白质相互作用网络分析表明DUF1262结构域蛋白可能参与调节信号转导或转录调控等过程;荧光定量PCR结果表明编码该蛋白基因的转录本在根、叶、花、枝和胶乳等组织中广泛分布,但在胶乳中表达较低,在树皮表达较高;水杨酸、脱落酸、过氧化氢及干旱处理可增强该基因在叶片中的转录水平。本研究证明DUF1262参与橡胶树逆境反应等生理过程,为揭示胶乳生物合成调控机制提供新线索。     

Backbone resonance assignments of the monomeric DUF59 domain of human Fam96a

Proteins containing a domain of unknown function 59 (DUF59) appear to have a variety of physiological functions, ranging from iron-sulfur cluster assembly to DNA repair. DUF59 proteins have been found in bacteria, archaea and eukaryotes, however Fam96a and Fam96b are the only mammalian proteins predicted to contain a DUF59 domain. Fam96a is an 18 kDa protein comprised primarily of a DUF59 domain (residues 31–157) and an N-terminal signal peptide (residues 1–27). Interestingly, the DUF59 domain of Fam96a exists as monomeric and dimeric forms in solution, and X-ray crystallography studies of both forms unexpectedly revealed two different domain-swapped dimer structures. Here we report the backbone resonance assignments and secondary structure of the monomeric form of the 127 residue DUF59 domain of human Fam96a. This study provides the basis for further understanding the structural variability exhibited by Fam96a and the mechanism for domain swapping.