The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding ß-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.

Dissecting OGT's TPR domain to identify determinants of cellular function.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.

The Tetratricopeptide repeat protein TaTPR-B1 regulates spike compactness in bread wheat.

Spike compactness (SC) is strongly associated with wheat (Triticum aestivum L.) grain yield. In this study, we conducted a quantitative trait locus (QTL) analysis using a doubled haploid (DH) population derived from a cross between two common wheat varieties with contrasting spike morphology, revealing 16 stable QTLs associated with SC. The effect of a major QTL, QSc.cau-6B.1, was validated in 231 F7 recombinant inbred lines (RILs) derived from the same cross as the DH population. Using two residual heterozygous lines (RHLs), we delimited QSc.cau-6B.1 to an approximately 0.5-Mbp physical interval containing four high-confidence genes. The tetratricopeptide repeat-TraesCS6B03G1214400 (TaTPR-B1) was the priority candidate gene according to sequence and expression variations between near-isogenic lines. Accordingly, TaTPR-B1 knockout in the common wheat variety 'CB037' significantly increased SC compared to the wild type (WT). Conversely, TaTPR-B1 overexpression in the common wheat variety 'Fielder' significantly decreased SC compared to the WT. Moreover, we developed a PCR-based marker targeting the 32-bp insertion/deletion (InDel) between the two TaTPR-B1 alleles, which could be practical and valuable in modern wheat breeding programs for diagnostic purposes. Collectively, these findings provide insight into the genetic basis of SC in common wheat and present a valuable target with a breeder-friendly diagnostic marker for gene pyramid breeding.

Metal cofactor stabilization by a partner protein is a widespread strategy employed for amidase activation.

Construction and remodeling of the bacterial peptidoglycan (PG) cell wall must be carefully coordinated with cell growth and division. Central to cell wall construction are hydrolases that cleave bonds in peptidoglycan. These enzymes also represent potential new antibiotic targets. One such hydrolase, the amidase LytH in Staphylococcus aureus, acts to remove stem peptides from PG, controlling where substrates are available for insertion of new PG strands and consequently regulating cell size. When it is absent, cells grow excessively large and have division defects. For activity, LytH requires a protein partner, ActH, that consists of an intracellular domain, a large rhomboid protease domain, and three extracellular tetratricopeptide repeats (TPRs). Here, we demonstrate that the amidase-activating function of ActH is entirely contained in its extracellular TPRs. We show that ActH binding stabilizes metals in the LytH active site and that LytH metal binding in turn is needed for stable complexation with ActH. We further present a structure of a complex of the extracellular domains of LytH and ActH. Our findings suggest that metal cofactor stabilization is a general strategy used by amidase activators and that ActH houses multiple functions within a single protein.

A conserved, noncanonical insert in FIS1 mediates TBC1D15 and DRP1 recruitment for mitochondrial fission.

Mitochondrial fission protein 1 (FIS1) is conserved in all eukaryotes, yet its function in metazoans is thought divergent. Structure-based sequence alignments of FIS1 revealed a conserved, but noncanonical, three-residue insert in its first tetratricopeptide repeat (TPR) suggesting a conserved function. In vertebrates, this insert is serine (S45), lysine (K46), and tyrosine (Y47). To determine the biological role of the "SKY insert," three variants were tested in HCT116 cells for altered mitochondrial morphology and recruitment of fission mechanoenzyme DRP1 and mitophagic adaptor TBC1D15. Similar to ectopically expressed wildtype FIS1, substitution of the SKY insert with alanine (AAA) fragmented mitochondria into perinuclear clumps associated with increased mitochondrial DRP1. In contrast, deletion variants (either ∆SKY or ∆SKYD49G) elongated mitochondrial networks with reduced mitochondrial recruitment of DRP1, despite DRP1 coimmunoprecipitates being highly enriched with ΔSKY variants. Ectopic wildtype FIS1 drove co-expressed YFP-TBC1D15 entirely from the cytoplasm to mitochondria as punctate structures concomitant with enhanced mitochondrial DRP1 recruitment. YFP-TBC1D15 co-expressed with the AAA variant further enhanced mitochondrial DRP1 recruitment, indicating a gain of function. In contrast, YFP-TBC1D15 co-expressed with deletion variants impaired mitochondrial DRP1 and YFP-TBC1D15 recruitment; however, mitochondrial fragmentation was restored. These phenotypes were not due to misfolding or poor expression of FIS1 variants, although ∆SKYD49G induced conformational heterogeneity that is lost upon deletion of the regulatory Fis1 arm, indicating SKY-arm interactions. Collectively, these results support a unifying model whereby FIS1 activity is effectively governed by intramolecular interactions between its regulatory arm and a noncanonical TPR insert that is conserved across eukaryotes.

Exon shuffling potentiates a diverse repertoire of brown algal NB-ARC-TPR candidate immune receptor proteins via alternative splicing.

Like other organisms, brown algae are subject to diseases caused by bacteria, fungi, and viruses. Brown algal immunity mechanisms are not well characterized; however, there is evidence suggesting that pathogen receptors exist in brown algae. One key protein family likely associated with brown algal innate immunity possesses an NB-ARC domain analogous to innate immune proteins in plants and animals. In this study, we conducted an extensive survey of NB-ARC genes in brown algae and obtained insights into the domain organization and evolutionary history of the encoded proteins. Our data show that brown algae possess an ancient NB-ARC-tetratricopeptide repeat (NB-TPR) domain architecture. We identified an N-terminal effector domain, the four-helix bundle, which was not previously found associated with NB-ARC domains. The phylogenetic tree including NB-ARC domains from all kingdoms of life suggests the three clades of brown algal NB-TPRs are likely monophyletic, whereas their TPRs seem to have distinct origins. One group of TPRs exhibit intense exon shuffling, with various alternative splicing and diversifying selection acting on them, suggesting exon shuffling is an important mechanism for evolving ligand-binding specificities. The reconciliation of gene duplication and loss events of the NB-ARC genes reveals that more independent gene gains than losses have occurred during brown algal evolution, and that tandem duplication has played a major role in the expansion of NB-ARC genes. Our results substantially enhance our understanding of the evolutionary history and exon shuffling mechanisms of the candidate innate immune repertoire of brown algae.

Engineering Proteins for PEDOT Dispersions: A New Horizon for Highly Mixed Ionic-Electronic Biocompatible Conducting Materials.

Poly (3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS) is the most used conducting polymer from energy to biomedical applications. Despite its exceptional properties, there is a need for developing new materials that can improve some of its inherent limitations, e.g., biocompatibility. In this context, doping PEDOT is propose with a robust recombinant protein with tunable properties, the consensus tetratricopeptide repeated protein (CTPR). The doping consists of an oxidative polymerization, where the PEDOT chains are stabilized by the negative charges of the CTPR protein. CTPR proteins are evaluated with three different lengths (3, 10, and 20 identical CTPR units) and optimized varied synthetic conditions. These findings revealed higher doping rate and oxidized state of the PEDOT chains when doped with the smallest scaffold (CTPR3). These PEDOT:CTPR hybrids possess ionic and electronic conductivity. Notably, PEDOT:CTPR3 displayed an electronic conductivity of 0.016 S cm-1, higher than any other reported protein-doped PEDOT. This result places PEDOT:CTPR3 at the level of PEDOT-biopolymer hybrids, and brings it closer in performance to PEDOT:PSS gold standard. Furthermore, PEDOT:CTPR3 dispersion is successfully optimized for inkjet printing, preserving its electroactivity properties after printing. This approach opens the door to the use of these novel hybrids for bioelectronics.

A novel tetratricopeptide-repeat protein, TTP1, forms complexes with glutamyl-tRNA reductase and protochlorophyllide oxidoreductase during tetrapyrrole biosynthesis.

The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.

Versatile allosteric properties in Pex5-like tetratricopeptide repeat proteins to induce diverse downstream function.

Proteins composed of tetratricopeptide repeat (TPR) arrays belong to the α-solenoid tandem-repeat family that have unique properties in terms of their overall conformational flexibility and ability to bind to multiple protein ligands. The peroxisomal matrix protein import receptor Pex5 comprises two TPR triplets that recognize protein cargos with a specific C-terminal Peroxisomal Targeting Signal (PTS) 1 motif. Import of PTS1-containing protein cargos into peroxisomes through a transient pore is mainly driven by allosteric binding, coupling and release mechanisms, without a need for external energy. A very similar TPR architecture is found in the functionally unrelated TRIP8b, a regulator of the hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel. TRIP8b binds to the HCN ion channel via a C-terminal sequence motif that is nearly identical to the PTS1 motif of Pex5 receptor cargos. Pex5, Pex5-related Pex9, and TRIP8b also share a less conserved N-terminal domain. This domain provides a second protein cargo-binding site and plays a distinct role in allosteric coupling of initial cargo loading by PTS1 motif-mediated interactions and different downstream functional readouts. The data reviewed here highlight the overarching role of molecular allostery in driving the diverse functions of TPR array proteins, which could form a model for other α-solenoid tandem-repeat proteins involved in translocation processes across membranes.

Structural studies of human fission protein FIS1 reveal a dynamic region important for GTPase DRP1 recruitment and mitochondrial fission.

Fission protein 1 (FIS1) and dynamin-related protein 1 (DRP1) were initially described as being evolutionarily conserved for mitochondrial fission, yet in humans the role of FIS1 in this process is unclear and disputed by many. In budding yeast where Fis1p helps to recruit the DRP1 ortholog from the cytoplasm to mitochondria for fission, an N-terminal "arm" of Fis1p is required for function. The yeast Fis1p arm interacts intramolecularly with a conserved tetratricopeptide repeat core and governs in vitro interactions with yeast DRP1. In human FIS1, NMR and X-ray structures show different arm conformations, but its importance for human DRP1 recruitment is unknown. Here, we use molecular dynamics simulations and comparisons to experimental NMR chemical shifts to show the human FIS1 arm can adopt an intramolecular conformation akin to that observed with yeast Fis1p. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human FIS1 with and without the arm. Using NMR, we observed the human FIS1 arm is also sensitive to environmental changes. We reveal the importance of these findings in cellular studies where removal of the FIS1 arm reduces DRP1 recruitment and mitochondrial fission similar to the yeast system. Moreover, we determined that expression of mitophagy adapter TBC1D15 can partially rescue arm-less FIS1 in a manner reminiscent of expression of the adapter Mdv1p in yeast. These findings point to conserved features of FIS1 important for its activity in mitochondrial morphology. More generally, other tetratricopeptide repeat-containing proteins are flanked by disordered arms/tails, suggesting possible common regulatory mechanisms.

Two distinct classes of cochaperones compete for the EEVD motif in heat shock protein 70 to tune its chaperone activities.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

Inner membrane YfgM-PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation.

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM-Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD-YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon.

The Arabidopsis TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE (TTL) family members are involved in root system formation via their interaction with cytoskeleton and cell wall remodeling.

Lateral roots (LR) are essential components of the plant edaphic interface; contributing to water and nutrient uptake, biotic and abiotic interactions, stress survival, and plant anchorage. We have identified the TETRATRICOPEPTIDE-REPEAT THIOREDOXIN-LIKE 3 (TTL3) gene as being related to LR emergence and later development. Loss of function of TTL3 leads to a reduced number of emerged LR due to delayed development of lateral root primordia (LRP). This trait is further enhanced in the triple mutant ttl1ttl3ttl4. TTL3 interacts with microtubules and endomembranes, and is known to participate in the brassinosteroid (BR) signaling pathway. Both ttl3 and ttl1ttl3ttl4 mutants are less sensitive to BR treatment in terms of LR formation and primary root growth. The ability of TTL3 to modulate biophysical properties of the cell wall was established under restrictive conditions of hyperosmotic stress and loss of root growth recovery, which was enhanced in ttl1ttl3ttl4. Timing and spatial distribution of TTL3 expression is consistent with its role in development of LRP before their emergence and subsequent growth of LR. TTL3 emerged as a component of the root system morphogenesis regulatory network.

The TPR- and J-domain-containing proteins DJC31 and DJC62 are involved in abiotic stress responses in Arabidopsis thaliana.

Molecular chaperones play an important role during the response to different stresses. Since plants are sessile organisms, they need to be able to adapt quickly to different conditions. To do so, plants possess a complex chaperone machinery, composed of HSP70, HSP90, J proteins and other factors. In this study we characterized DJC31 (also known as TPR16) and DJC62 (also known as TPR15) of Arabidopsis thaliana, two J proteins that additionally carry clamp-type tetratricopeptide repeat domains. Using cell fractionation and split GFP, we could show that both proteins are attached to the cytosolic side of the endoplasmic reticulum membrane. Moreover, an interaction with cytosolic HSP70.1 and HSP90.2 could be shown using bimolecular fluorescence complementation. Knockout of both DJC31 and DJC62 caused severe defects in growth and development, which affected almost all organs. Furthermore, it could be shown that the double mutant is more sensitive to osmotic stress and treatment with abscisic acid, but surprisingly exhibited enhanced tolerance to drought. Taken together, these findings indicate that DJC31 and DJC62 might act as important regulators of chaperone-dependent signaling pathways involved in plant development and stress responses.

Interaction of the tetratricopeptide repeat domain of aryl hydrocarbon receptor-interacting protein-like 1 with the regulatory Pγ subunit of phosphodiesterase 6.

Phosphodiesterase-6 (PDE6) is key to both phototransduction and health of rods and cones. Proper folding of PDE6 relies on the chaperone activity of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and mutations in both PDE6 and AIPL1 can cause a severe form of blindness. Although AIPL1 and PDE6 are known to interact via the FK506-binding protein domain of AIPL1, the contribution of the tetratricopeptide repeat (TPR) domain of AIPL1 to its chaperone function is poorly understood. Here, we demonstrate that AIPL1-TPR interacts specifically with the regulatory Pγ subunit of PDE6. Use of NMR chemical shift perturbation (CSP) mapping technique revealed the interface between the C-terminal portion of Pγ and AIPL1-TPR. Our solution of the crystal structure of the AIPL1-TPR domain provided additional information, which together with the CSP data enabled us to generate a model of this interface. Biochemical analysis of chimeric AIPL1-AIP proteins supported this model and also revealed a correlation between the affinity of AIPL1-TPR for Pγ and the ability of Pγ to potentiate the chaperone activity of AIPL1. Based on these results, we present a model of the larger AIPL1-PDE6 complex. This supports the importance of simultaneous interactions of AIPL1-FK506-binding protein with the prenyl moieties of PDE6 and AIPL1-TPR with the Pγ subunit during the folding and/or assembly of PDE6. This study sheds new light on the versatility of TPR domains in protein folding by describing a novel TPR-protein binding partner, Pγ, and revealing that this subunit imparts AIPL1 selectivity for its client.

An EMS-induced mutation in a tetratricopeptide repeat-like superfamily protein gene (Ghir_A12G008870) on chromosome A12 is responsible for the liy short fiber phenotype in cotton.

KEY MESSAGE: The EMS-induced threonine/isoleucine substitution in a tetratricopeptide repeat-like superfamily protein encoded by gene Ghir_A12G008870 is responsible for the Ligon-lintless-y (liy) short fiber phenotype in cotton. A short fiber mutant Ligon-lintless-y was created through treating the seeds of the cotton line MD15 with ethyl methanesulfonate. Genetic analysis indicated that the short fiber phenotype is controlled by a single recessive locus designated liy. From F2 populations derived from crosses between the mutant and its wild type (WT), we selected 132 short fiber progeny (liy/liy) and made two DNA bulks. We sequenced these DNA bulks along with the two parents of the population. The liy locus was located on chromosome A12. Using multiple F2 populations and F3 progeny plants, we mapped the liy locus within a genomic region of 1.18 Mb. In this region, there is only one gene, i.e., Ghir_A12G008870 encoding a tetratricopeptide repeat-like superfamily protein that has a non-synonymous mutation between the liy mutant and its WT. Analysis of a SNP marker representing this gene in the F2 and F3 progeny plants demonstrated its complete linkage with the liy short fiber phenotype. We further analyzed this SNP marker in a panel of 384 cotton varieties. The mutant allele is absent in all varieties analyzed. RNAseq and RT-qPCR analysis of the gene Ghir_A12G008870 during fiber development showed a significant expression difference between the liy mutant and its WT in developing fiber cells beginning at 12 days post-anthesis. Virus-induced gene silencing of the gene Ghir_A12G008870 significantly reduced the fiber length of the WT cotton line MD15. Taken together, our results suggest that the gene Ghir_A12G008870 is involved in the cotton fiber cell elongation process and is a promising candidate gene responsible for the liy short fiber phenotype.

Intrinsic Disorder in Tetratricopeptide Repeat Proteins.

Among the realm of repeat containing proteins that commonly serve as "scaffolds" promoting protein-protein interactions, there is a family of proteins containing between 2 and 20 tetratricopeptide repeats (TPRs), which are functional motifs consisting of 34 amino acids. The most distinguishing feature of TPR domains is their ability to stack continuously one upon the other, with these stacked repeats being able to affect interaction with binding partners either sequentially or in combination. It is known that many repeat-containing proteins are characterized by high levels of intrinsic disorder, and that many protein tandem repeats can be intrinsically disordered. Furthermore, it seems that TPR-containing proteins share many characteristics with hybrid proteins containing ordered domains and intrinsically disordered protein regions. However, there has not been a systematic analysis of the intrinsic disorder status of TPR proteins. To fill this gap, we analyzed 166 human TPR proteins to determine the degree to which proteins containing TPR motifs are affected by intrinsic disorder. Our analysis revealed that these proteins are characterized by different levels of intrinsic disorder and contain functional disordered regions that are utilized for protein-protein interactions and often serve as targets of various posttranslational modifications.

Ttc39c is upregulated during skeletal muscle atrophy and modulates ERK1/2 MAP kinase and hedgehog signaling.

Tetratricopeptide repeat domain containing 39c (Ttc39c) is expressed in skeletal muscle and is transcriptionally activated in response to neurogenic atrophy in mice. Expression analysis using quantitative polymerase chain reaction and Western blots revealed that Ttc39c is expressed in both proliferating and differentiated muscle cells, peaking during early differentiation and then decreasing as cells progress further through the differentiation process. To further analyze the transcriptional regulation of Ttc39c, promoter fragments of the gene were cloned and fused with the secreted alkaline phosphatase reporter gene. The Ttc39c reporter plasmids were then transfected into cultured mouse muscle cells and found to have transcriptional activity. Furthermore, overexpression of MyoD and myogenin resulted in significant transcriptional repression of the Ttc39c reporter genes. To determine subcellular localization, an expression plasmid with the Ttc39c complementary DNA fused with green fluorescent protein was transfected into muscle cells and analyzed by confocal fluorescent microscopy showing that Tct39c localizes exclusively to the cytoplasm of cultured cells. To assess potential function in muscle, Ttc39c was overexpressed leading to vitiated muscle cell differentiation, impaired ERK1/2 MAP Kinase and Hedgehog signaling, and increased expression of IFT144 and IFT43, which are part of the IFT-A complex involved in retrograde transport in primary cilia. Interestingly, Ttc39c knockdown also resulted in inhibition of muscle cell differentiation and impaired Hedgehog and MAP Kinase signaling but did not affect IFT144 or IFT433 expression. The results of this study demonstrate that muscle cell differentiation is sensitive to abnormal Ttc39c expression and that normal Ttc39c expression appears to be necessary for proper MAP Kinase and Hedgehog signal transduction in developing muscle cells.

Functional Mechanisms Underlying the Antimicrobial Activity of the Oryza sativa Trx-like Protein.

Plants are constantly subjected to a variety of environmental stresses and have evolved regulatory responses to overcome unfavorable conditions that might reduce or adversely change a plant's growth or development. Among these, the regulated production of reactive oxygen species (ROS) as a signaling molecule occurs during plant development and pathogen defense. This study demonstrates the possible antifungal activity of Oryza sativa Tetratricopeptide Domain-containing thioredoxin (OsTDX) protein against various fungal pathogens. The transcription of OsTDX was induced by various environmental stresses known to elicit the generation of ROS in plant cells. OsTDX protein showed potent antifungal activity, with minimum inhibitory concentrations (MICs) against yeast and filamentous fungi ranging between 1.56 and 6.25 and 50 and 100 µg/mL, respectively. The uptake of SYTOX-Green into fungal cells and efflux of calcein from artificial fungus-like liposomes suggest that its killing mechanism involves membrane permeabilization and damage. In addition, irregular blebs and holes apparent on the surfaces of OsTDX-treated fungal cells indicate the membranolytic action of this protein. Our results suggest that the OsTDX protein represents a potentially useful lead for the development of pathogen-resistant plants.

Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aß) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria-destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP-1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP-1 treatment of SH-SY5Y cells results in decrease in mitochondria-associated APP and protects SH-SY5Y cells from toxic effect of Aß1-42 exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP-1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.