MdTPR16, an apple tetratricopeptide repeat (TPR)-like superfamily gene, positively regulates drought stress in apple.

The Tetratricopeptide repeat (TPR)-like superfamily with TPR conserved domains is widely involved in the growth and abiotic stress in many plants. In this report, the gene MdTPR16 belongs to the TPR family in apple (Malus domestica). Promoter analysis reveal that MdTPR16 incorporated various stress response elements, including the drought stress response elements. And different abiotic stress treatments, drought especially, significantly induce the response of MdTPR16. Overexpression of MdTPR16 result in better drought tolerance in apple and Arabidopsis by up-regulating the expression levels of drought stress-related genes, achieving a higher chlorophyll content level, more material accumulation, and overall better growth compared to WT in the drought. Under drought stress, the overexpressed MdTPR16 also mitigate the oxidative damage in cells by reducing the electrolyte leakage, malondialdehyde content, and the H2O2 and O2- accumulation in apples and Arabidopsis. In conclusion, MdTPR16 act as a beneficial regulator of drought stress response by regulating the expression of related genes and the cumulation of reactive oxygen species (ROS).

An Analytical Review of the Structural Features of Pentatricopeptide Repeats: Strategic Amino Acids, Repeat Arrangements and Superhelical Architecture.

Tricopeptide repeats are common in natural proteins, and are exemplified by 34- and 35-residue repeats, known respectively as tetratricopeptide repeats (TPRs) and pentatricopeptide repeats (PPRs). In both classes, each repeat unit forms an antiparallel bihelical structure, so that multiple such units in a polypeptide are arranged in a parallel fashion. The primary structures of the motifs are nonidentical, but amino acids of similar properties occur in strategic positions. The focus of the present work was on PPR, but TPR, its better-studied cousin, is often included for comparison. The analyses revealed that critical amino acids, namely Gly, Pro, Ala and Trp, were placed at distinct locations in the higher order structure of PPR domains. While most TPRs occur in repeats of three, the PPRs exhibited a much greater diversity in repeat numbers, from 1 to 30 or more, separated by spacers of various sequences and lengths. Studies of PPR strings in proteins showed that the majority of PPR units are single, and that the longer tandems (i.e., without space in between) occurred in decreasing order. The multi-PPR domains also formed superhelical vortices, likely governed by interhelical angles rather than the spacers. These findings should be useful in designing and understanding the PPR domains.

Identification and Functional Analysis of Tomato TPR Gene Family.

Tomato (Solanum lycopersicum) as an important vegetable grown around the world is threatened by many diseases, which seriously affects its yield. Therefore, studying the interaction between tomato and pathogenic bacteria is biologically and economically important. The TPR (Tetratricopeptide repeat) gene family is a class of genes containing TPR conserved motifs, which are widely involved in cell cycle regulation, gene expression, protein degradation and other biological processes. The functions of TPR gene in Arabidopsis and wheat plants have been well studied, but the research on TPR genes in tomato is not well studied. In this study, 26 TPR gene families were identified using bioinformatics based on tomato genome data, and they were analyzed for subcellular localization, phylogenetic evolution, conserved motifs, tissue expression, and GO (Gene Ontology) analysis. The qRT-PCR was used to detect the expression levels of each member of the tomato TPR gene family (SlTPRs) under biological stress (Botrytis cinerea) and abiotic stress such as drought and abscisic acid (ABA). The results showed that members of the tomato TPR family responded to various abiotic stresses and Botrytis cinerea stress, and the SlTPR2 and SlTPR4 genes changed significantly under different stresses. Using VIGS (Virus-induced gene silencing) technology to silence these two genes, the silenced plants showed reduced disease resistance. It was also shown that TPR4 can interact with atpA which encodes a chloroplast ATP synthase CF1 α subunit. The above results provide a theoretical basis for further exploring the molecular mechanism of TPR-mediated resistance in disease defense, and also provide a foundation for tomato disease resistance breeding.

Novel genes associated with folic acid-mediated metabolism in mouse: A bioinformatics study.

Folic acid plays an essential role in the central nervous system and cancer. This study aimed to screen genes related to folic acid metabolism. Datasets (GSE80587, GSE65267 and GSE116299) correlated to folic acid were screened in the Gene Expression Omnibus. Weighed gene co-expression network analysis was performed to identify modules associated with sample traits of folic acid and organs (brain, prostate and kidney). Functional enrichment analysis was performed for the eigengenes in modules that were significantly correlated with sample traits. Accordingly, the hub genes and key nodes in the modules were identified using the protein interaction network. A total of 17,252 genes in three datasets were identified. One module, which included 97 genes that were highly correlated with sample traits (including folic acid treatment [cor = -0.57, P = 3e-04] and kidney [cor = -0.68, p = 4e-06]), was screened out. Hub genes, including tetratricopeptide repeat protein 38 (Ttc38) and miR-185, as well as those (including Sema3A, Insl3, Dll1, Msh4 and Snai1) associated with "neuropilin binding", "regulation of reproductive process" and "vitamin D metabolic process", were identified. Genes, including Ttc38, Sema3A, Insl3, Dll1, Msh4 and Snai1, were the novel factors that may be associated with the development of the kidneys and related to folic acid treatment.

Cancer-derived UTX TPR mutations G137V and D336G impair interaction with MLL3/4 complexes and affect UTX subcellular localization.

The ubiquitously transcribed tetratricopeptide repeat on X chromosome (UTX) is a major histone H3 lysine 27 (H3K27) demethylase and the mixed-lineage leukemia (MLL) proteins are the H3K4 methyltransferases. UTX is one of the major components of MLL3- and MLL4-containing (MlLL3/4) complexes and likely has functions within the complexes. Although UTX is frequently mutated in various types of cancer and is thought to play a crucial role as a tumor suppressor, the importance of UTX interaction with MLL3/4 complexes in cancer formation is poorly understood. Here, we analyzed the ability of cancer-derived UTX mutant proteins to interact with ASH2L, which is a common core component of all the MLL complexes, and MLL3/4-specific components PTIP and PA1, and found that several single-amino acid substitution mutations in the tetratricopeptide repeat (TPR) affect UTX interaction with these components. Interaction-compromised mutants G137V and D336G and a TPR-deleted mutant Δ80-397 were preferentially localized to the cytoplasm, suggesting that UTX is retained in the nucleus by MLL3/4 complexes through their interaction with the TPR. Intriguingly, WT UTX suppressed colony formation in soft agar, whereas G137V failed. This suggests that interaction of UTX with MLL3/4 complex plays a crucial role in their tumor suppressor function. Preferential cytoplasmic localization was also observed for endogenous proteins of G137V and another mutant G137VΔ138 in HCT116 created by CRISPR-Cas9 gene editing. Interestingly, expression levels of these mutants were low and MG312 stabilized both endogenous as well as exogenous G137V proteins. These results reveal a novel mechanism of UTX regulation and reinforce the importance of UTX interaction with MLL3/4 complexes in cancer formation.

Biallelic Mutations in Tetratricopeptide Repeat Domain 26 (Intraflagellar Transport 56) Cause Severe Biliary Ciliopathy in Humans.

BACKGROUND AND AIMS: The clinical consequences of defective primary cilium (ciliopathies) are characterized by marked phenotypic and genetic heterogeneity. Although fibrocystic liver disease is an established ciliopathy phenotype, severe neonatal cholestasis is rarely recognized as such. APPROACH AND RESULTS: We describe seven individuals from seven families with syndromic ciliopathy clinical features, including severe neonatal cholestasis (lethal in one and necessitating liver transplant in two). Positional mapping revealed a single critical locus on chromosome 7. Whole-exome sequencing revealed three different homozygous variants in Tetratricopeptide Repeat Domain 26 (TTC26) that fully segregated with the phenotype. TTC26 (intraflagellar transport [IFT] 56/DYF13) is an atypical component of IFT-B complex, and deficiency of its highly conserved orthologs has been consistently shown to cause defective ciliary function in several model organisms. We show that cilia in TTC26-mutated patient cells display variable length and impaired function, as indicated by dysregulated sonic hedgehog signaling, abnormal staining for IFT-B components, and transcriptomic clustering with cells derived from individuals with closely related ciliopathies. We also demonstrate a strong expression of Ttc26 in the embryonic mouse liver in a pattern consistent with its proposed role in the normal development of the intrahepatic biliary system. CONCLUSIONS: In addition to establishing a TTC26-related ciliopathy phenotype in humans, our results highlight the importance of considering ciliopathies in the differential diagnosis of severe neonatal cholestasis even in the absence of more typical features.

Architecture of the Cellulose Synthase Outer Membrane Channel and Its Association with the Periplasmic TPR Domain.

Extracellular bacterial cellulose contributes to biofilm stability and to the integrity of the bacterial cell envelope. In Gram-negative bacteria, cellulose is synthesized and secreted by a multi-component cellulose synthase complex. The BcsA subunit synthesizes cellulose and also transports the polymer across the inner membrane. Translocation across the outer membrane occurs through the BcsC porin, which extends into the periplasm via 19 tetra-tricopeptide repeats (TPR). We present the crystal structure of a truncated BcsC, encompassing the last TPR repeat and the complete outer membrane channel domain, revealing a 16-stranded, ß barrel pore architecture. The pore is blocked by an extracellular gating loop, while the extended C terminus inserts deeply into the channel and positions a conserved Trp residue near its extracellular exit. The channel is lined with hydrophilic and aromatic residues suggesting a mechanism for facilitated cellulose diffusion based on aromatic stacking and hydrogen bonding.

The Bub1-TPR Domain Interacts Directly with Mad3 to Generate Robust Spindle Checkpoint Arrest.

The spindle checkpoint monitors kinetochore-microtubule interactions and generates a "wait anaphase" delay when any defects are apparent [1-3]. This provides time for cells to correct chromosome attachment errors and ensure high-fidelity chromosome segregation. Checkpoint signals are generated at unattached chromosomes during mitosis. To activate the checkpoint, Mps1Mph1 kinase phosphorylates the kinetochore component KNL1Spc105/Spc7 on conserved MELT motifs to recruit Bub3-Bub1 complexes [4-6] via a direct Bub3 interaction with phospho-MELT motifs [7, 8]. Mps1Mph1 then phosphorylates Bub1, which strengthens its interaction with Mad1-Mad2 complexes to produce a signaling platform [9, 10]. The Bub1-Mad1 platform is thought to recruit Mad3, Cdc20, and Mad2 to produce the mitotic checkpoint complex (MCC), which is the diffusible wait anaphase signal [9, 11, 12]. The MCC binds and inhibits the mitotic E3 ubiquitin ligase, known as Cdc20-anaphase promoting complex/cyclosome (APC/C), and stabilizes securin and cyclin to delay anaphase onset [13-17]. Here we demonstrate, in both budding and fission yeast, that kinetochores and KNL1Spc105/Spc7 can be bypassed; simply inducing heterodimers of Mps1Mph1 kinase and Bub1 is sufficient to trigger metaphase arrest that is dependent on Mad1, Mad2, and Mad3. We use this to dissect the domains of Bub1 necessary for arrest, highlighting the need for Bub1-CD1, which binds Mad1 [9], and Bub1's highly conserved N-terminal tetratricopeptide repeat (TPR) domain [18, 19]. We demonstrate that the Bub1 TPR domain is both necessary and sufficient to bind and recruit Mad3. We propose that this brings Mad3 into close proximity to Mad1-Mad2 and Mps1Mph1 kinase, enabling efficient generation of MCC complexes.

Structure of the periplasmic domain of SflA involved in spatial regulation of the flagellar biogenesis of Vibrio reveals a TPR/SLR-like fold.

Bacteria have evolved various types of flagellum, an organella for bacterial motility, to adapt to their habitat environments. The number and the spatial arrangement of the flagellum are precisely controlled to optimize performance of each type of the flagellar system. Vibrio alginolyticus has a single sheathed flagellum at the cell pole for swimming. SflA is a regulator protein to prevent peritrichous formation of the sheathed flagellum, and consists of an N-terminal periplasmic region, a transmembrane helix, and a C-terminal cytoplasmic region. Whereas the cytoplasmic region has been characterized to be essential for inhibition of the peritrichous growth, the role of the N-terminal region is still unclear. We here determined the structure of the N-terminal periplasmic region of SflA (SflAN) at 1.9-Å resolution. The core of SflAN forms a domain-swapped dimer with tetratricopeptide repeat (TPR)/Sel1-like repeat (SLR) motif, which is often found in the domains responsible for protein-protein interaction in various proteins. The structural similarity and the following mutational analysis based on the structure suggest that SflA binds to unknown partner protein by SflAN and the binding signal is important for the precise control of the SflA function.

Double autoinhibition mechanism of signal transduction ATPases with numerous domains (STAND) with a tetratricopeptide repeat sensor.

Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains.

miR663a­TTC22V1 axis inhibits colon cancer metastasis.

An increasing number of studies have demonstrated that microRNAs (miRs) may act as oncogenes or anti­oncogenes in various types of cancer, including colon cancer (CC). However, the clinical and biological significance of miR663a in the prognosis of CC and its underlying molecular mechanisms remain unknown. Using the reverse transcription­quantitative polymerase chain reaction on CC and surgical margin tissue samples from 172 patients with CC, it was identified that miR663a was significantly downregulated in CC (P<0.001), particularly in metastatic CC (P=0.044). miR663a overexpression inhibited the proliferation and migration/invasion of CC cells in vitro, and also tumor growth and metastasis of CC cells in vivo. Additionally, miR663a target genes were analyzed. Inverse changes in tetratricopeptide repeat domain 22 variant 1 (TTC22V1) in response to alterations in miR663a expression were observed. miR663a decreased the reporter activity of the wild­type TTC22V1­3' untranslated region (UTR), but did not decrease that of a 3'UTR mutant. miR663a completely abolished cell migration/invasion induced by TTC22V1 containing the wild­type 3'UTR sequence, but not that induced by TTC22V1 containing the 3'UTR mutant. An inverse correlation between miR663a and TTC22 mRNA levels was observed in CC tissues. These results suggest that TTC22V1 mRNA is a crucial miR663a target that directly promotes cell migration/invasion. TTC22, which, to the best of our knowledge, has rarely been investigated, is located in the nuclei of epithelial cells in colon stem cell niches at crypt bases, and is significantly downregulated in CC, particularly in non­metastatic CC. High TTC22V1 expression is a significant poor survival factor for patients with CC. Collectively, the results of the present study suggested that TTC22V1 may be a metastasis­associated gene and that the miR663a­TTC22V1 axis inhibited CC metastasis.

Identification of a novel gene regulating amygdala-mediated fear extinction.

Recent years have seen advances in our understanding of the neural circuits associated with trauma-related disorders, and the development of relevant assays for these behaviors in rodents. Although inherited factors are known to influence individual differences in risk for these disorders, it has been difficult to identify specific genes that moderate circuit functions to affect trauma-related behaviors. Here, we exploited robust inbred mouse strain differences in Pavlovian fear extinction to uncover quantitative trait loci (QTL) associated with this trait. We found these strain differences to be resistant to developmental cross-fostering and associated with anatomical variation in basolateral amygdala (BLA) perineuronal nets, which are developmentally implicated in extinction. Next, by profiling extinction-driven BLA expression of QTL-linked genes, we nominated Ppid (peptidylprolyl isomerase D, a member of the tetratricopeptide repeat (TPR) protein family) as an extinction-related candidate gene. We then showed that Ppid was enriched in excitatory and inhibitory BLA neuronal populations, but at lower levels in the extinction-impaired mouse strain. Using a virus-based approach to directly regulate Ppid function, we demonstrated that downregulating BLA-Ppid impaired extinction, while upregulating BLA-Ppid facilitated extinction and altered in vivo neuronal extinction encoding. Next, we showed that Ppid colocalized with the glucocorticoid receptor (GR) in BLA neurons and found that the extinction-facilitating effects of Ppid upregulation were blocked by a GR antagonist. Collectively, our results identify Ppid as a novel gene involved in regulating extinction via functional actions in the BLA, with possible implications for understanding genetic and pathophysiological mechanisms underlying risk for trauma-related disorders.

A tetratricopeptide repeat domain protein has profound effects on assembly of periplasmic flagella, morphology and motility of the lyme disease spirochete Borrelia burgdorferi.

Spirochetes possess a unique periplasmic flagellar motor component called the collar. However, little is known about the composition or function of the flagellar collar proteins. To identify a collar protein, we have inactivated almost all genes annotated as motility-related in the Borrelia burgdorferi genome and identified only FlbB, which comprises the base of the collar. Since the major components of the collar complex remained unidentified, we took advantage of a protein-protein interaction map developed in another spirochete, Treponema pallidum to identify proteins of unknown function that could be collar proteins. Subsequently, using various comprehensive approaches, we identified a tetratricopeptide repeat protein BB0236 as a potential candidate for the collar. Biochemical assays indicated that FlbB interacts with BB0236. Furthermore, ∆bb0236 mutant analyses indicated that BB0236 is crucial for collar structure assembly, cellular morphology, motility, orientation of periplasmic flagella and assembly of other flagellar structures. Moreover, using comparative motor analyses, we propose how the collar structure is assembled in B. burgdorferi. Together, our studies provide new insights into the organization and the complex assembly inherent to the unique spirochetal collar structure.

On the role, ecology, phylogeny, and structure of dual-family immunophilins.

The novel class of dual-family immunophilins (henceforth abbreviated as DFI) represents naturally occurring chimera of classical FK506-binding protein (FKBP) and cyclophilin (CYN), connected by a flexible linker that may include a three-unit tetratricopeptide (TPR) repeat. Here, I report a comprehensive analysis of all current DFI sequences and their host organisms. DFIs are of two kinds: CFBP (cyclosporin- and FK506-binding protein) and FCBP (FK506- and cyclosporin-binding protein), found in eukaryotes. The CFBP type occurs in select bacteria that are mostly extremophiles, such as psychrophilic, thermophilic, halophilic, and sulfur-reducing. Essentially all DFI organisms are unicellular. I suggest that DFIs are specialized bifunctional chaperones that use their flexible interdomain linker to associate with large polypeptides or multisubunit megacomplexes to promote simultaneous folding or renaturation of two clients in proximity, essential in stressful and denaturing environments. Analysis of sequence homology and predicted 3D structures of the FKBP and CYN domains as well as the TPR linkers upheld the modular nature of the DFIs and revealed the uniqueness of their TPR domain. The CFBP and FCBP genes appear to have evolved in parallel pathways with no obvious single common ancestor. The occurrence of both types of DFI in multiple unrelated phylogenetic clades supported their selection in metabolic and environmental niche roles rather than a traditional taxonomic relationship. Nonetheless, organisms with these rare immunophilins may define an operational taxonomic unit (OTU) bound by the commonality of chaperone function.

Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats.

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification found on hundreds of nucleocytoplasmic proteins in metazoa. Although a single enzyme, O-GlcNAc transferase (OGT), generates the entire cytosolic O-GlcNAc proteome, it is not understood how it recognizes its protein substrates, targeting only a fraction of serines/threonines in the metazoan proteome for glycosylation. We describe a trapped complex of human OGT with the C-terminal domain of TAB1, a key innate immunity-signalling O-GlcNAc protein, revealing extensive interactions with the tetratricopeptide repeats of OGT. Confirmed by mutagenesis, this interaction suggests that glycosylation substrate specificity is achieved by recognition of a degenerate sequon in the active site combined with an extended conformation C-terminal of the O-GlcNAc target site.

Expression of eukaryotic-like protein in the microbiome of sponges.

Eukaryotic-like proteins (ELPs) are classes of proteins that are found in prokaryotes, but have a likely evolutionary origin in eukaryotes. ELPs have been postulated to mediate host-microbiome interactions. Recent work has discovered that prokaryotic symbionts of sponges contain abundant and diverse genes for ELPs, which could modulate interactions with their filter-feeding and phagocytic host. However, the extent to which these ELP genes are actually used and expressed by the symbionts is poorly understood. Here, we use metatranscriptomics to investigate ELP expression in the microbiomes of three different sponges (Cymbastella concentrica, Scopalina sp. and Tedania anhelens). We developed a workflow with optimized rRNA removal and in silico subtraction of host sequences to obtain a reliable symbiont metatranscriptome. This showed that between 1.3% and 2.3% of all symbiont transcripts contain genes for ELPs. Two classes of ELPs (cadherin and tetratricopeptide repeats) were abundantly expressed in the C. concentrica and Scopalina sp. microbiomes, while ankyrin repeat ELPs were predominant in the T. anhelens metatranscriptome. Comparison with transcripts that do not encode ELPs indicated a constitutive expression of ELPs across a range of bacterial and archaeal symbionts. Expressed ELPs also contained domains involved in protein secretion and/or were co-expressed with proteins involved in extracellular transport. This suggests these ELPs are likely exported, which could allow for direct interaction with the sponge. Our study shows that ELP genes in sponge symbionts represent actively expressed functions that could mediate molecular interaction between symbiosis partners.

Origin of a folded repeat protein from an intrinsically disordered ancestor.

Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2-5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin.