Tetratricopeptide Research Articles

The essential role of TTC28 in maintaining chromosomal stability via HSPA8 chaperone-mediated autophagy

There are three distinct forms of autophagy, namely, macroautophagy, microautophagy, and HSPA8 chaperone-mediated autophagy (CMA). While macroautophagy is widely recognized as a regulator of chromosomal instability (CIN) through various pathways, the contributions of CMA and microautophagy to CIN remain uncertain. TTC28, a conserved gene in vertebrates, is frequently mutated and down-regulated in numerous human cancers. This study presents findings demonstrating the interaction between human tetratricopeptide repeat domain 28 (TTC28) and heat shock protein member 8 (HSPA8) and lysosomal-associated membrane protein 2A proteins. The tetratricopeptide repeat domains of TTC28 bind to the C-terminal motif (PTIEEVD) in HSPA8, resulting in the subsequent degradation of TTC28 via CMA/microautophagy. Notably, the baseline frequency of micronuclei (FMN) in human cancer cells with TTC28 knockout cells was three times greater than that in cells with wild-type TTC28 (7.7% vs. 2.3%, P = 4.86E-09). Furthermore, the overexpression of Ttc28 mitigated the impact of TTC28 knockout on FMN (11.9% vs. 4.8%, P = 2.83E-11). Our findings also demonstrate that CMA has a protective effect on genome stability and that TTC28 plays an essential role in the effect of CMA. These results were further supported by the quantification of γH2AX and comet analyses and the analysis of The Cancer Genome Atlas data via bioinformatics. Mechanistically, TTC28 regulates mitosis and cytokinesis, which are involved in the maintenance of genome integrity by CMA. In conclusion, our study demonstrated that TTC28 is not only an HSPA8-mediated CMA/microautophagy substrate but also essential for maintaining chromosomal stability via CMA. Comprehensive TTC28 downregulation may lead to CIN in cancer cells.

Potential protective role of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in COVID-19

The COVID-19 pandemic has prompted a quest to understand why certain individuals remain uninfected or asymptomatic despite repetitive exposure to SARS-CoV-2. Here, we focused on six exposed females residing with their symptomatic and reinfected SARS-CoV-2 PCR-positive COVID-19 partners. Peripheral blood mononuclear cell samples from couples were analysed for poly (I:C)-induced mRNA expression of type I/III interferons and interferon-stimulated genes (ISGs). Remarkably, we found a significant upregulation of the ISG interferon-inducible protein with tetrapeptide repeats 3 (IFIT3) gene exclusively in exposed uninfected or asymptomatic females, suggesting a potential role in protective immunity against symptomatic COVID-19.

Photoactivatable O-GlcNAc Transferase Library Enables Covalent Chemical Capture of Solvent-Exposed TPR Domain Interactions.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential, stress-sensing enzyme responsible for adding the O-GlcNAc monosaccharide to thousands of nuclear and cytoplasmic proteins to regulate cellular homeostasis. OGT substrates are found in almost all intracellular processes, and perturbations in protein O-GlcNAc levels have been implicated in proteostatic diseases, such as cancers, metabolic disorders, and neurodegeneration. This broad disease activity makes OGT an attractive therapeutic target; however, the substrate diversity makes pan-inhibition as a therapeutic strategy unfeasible. Rather, a substrate-specific approach to targeting is more advantageous, but how OGT chooses its substrates remains poorly understood. Substrate specificity is controlled by the interactions between OGT's non-catalytic tetratricopeptide repeat (TPR) domain, rather than its glycosyltransferase domain. OGT's TPR domain forms a 100 Å superhelical structure, containing a lumenal surface, known as the substrate-binding surface, and a solvent-exposed surface. To date, there are no tools to site-selectively target regions of the domain and differentiate between the two binding surfaces. Here, we developed a library of recombinant OGT constructs containing site-specifically incorporated photoactivatable unnatural amino acids (UAAs) along the solvent-exposed surface of the TPR domain to covalently capture and map OGT's interactome.

AAV1.tMCK.NT-3 gene therapy improves phenotype in Sh3tc2-/- mouse model of Charcot-Marie-Tooth Type 4C.

Charcot-Marie-Tooth Type 4C (CMT4C) is associated with mutations in the SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene, primarily expressed in Schwann cells (SCs). Neurotrophin-3 (NT-3) is an important autocrine factor for SC survival and differentiation, and it stimulates neurite outgrowth and myelination. In this study, scAAV1.tMCK.NT-3 was delivered intramuscularly to 4-week-old Sh3tc2-/- mice, a model for CMT4C, and treatment efficacy was assessed at 6-month post-gene delivery. Efficient transgene production was verified with the detection of NT-3 in serum from the treated cohort. NT-3 gene therapy improved functional and electrophysiological outcomes including rotarod, grip strength and nerve conduction velocity. Qualitative and quantitative histopathological studies showed that hypomyelination of peripheral nerves and denervated status of neuromuscular junctions at lumbrical muscles were also improved in the NT-3-treated mice. Morphometric analysis in mid-sciatic and tibial nerves showed treatment-induced distally prominent regenerative activity in the nerve and an increase in the estimated SC density. This indicates that SC proliferation and differentiation, including the promyelination stage, are normal in the Sh3tc2-/- mice, consistent with the previous findings that Sh3tc2 is not involved in the early stages of myelination. Moreover, in size distribution histograms, the number of myelinated axons within the 3- to 6-µm diameter range increased, suggesting that treatment resulted in continuous radial growth of regenerating axons over time. In conclusion, this study demonstrates the efficacy of AAV1.NT-3 gene therapy in the Sh3tc2-/- mouse model of CMT4C, the most common recessively inherited demyelinating CMT subtype.

Diversity and structural-functional insights of alpha-solenoid proteins.

Alpha-solenoids are a significant and diverse subset of structured tandem repeat proteins (STRPs) that are important in various domains of life. This review examines their structural and functional diversity and highlights their role in critical cellular processes such as signaling, apoptosis, and transcriptional regulation. Alpha-solenoids can be classified into three geometric folds: low curvature, high curvature, and corkscrew, as well as eight subfolds: ankyrin repeats; Huntingtin, elongation factor 3, protein phosphatase 2A, and target of rapamycin; armadillo repeats; tetratricopeptide repeats; pentatricopeptide repeats; Pumilio repeats; transcription activator-like; and Sel-1 and Sel-1-like repeats. These subfolds represent distinct protein families with unique structural properties and functions, highlighting the versatility of alpha-solenoids. The review also discusses their association with disease, highlighting their potential as therapeutic targets and their role in protein design. Advances in state-of-the-art structure prediction methods provide new opportunities and challenges in the functional characterization and classification of this kind of fold, emphasizing the need for continued development of methods for their identification and proper data curation and deposition in the main databases.

Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.

How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend Myxococcus xanthus against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ2 and σ4 domains, undergoing σ4-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.

Mitochondrial Bioenergetics of Functional Wound Closure is Dependent on Macrophage-Keratinocyte Exosomal Crosstalk.

Tissue nanotransfection (TNT)-based fluorescent labeling of cell-specific exosomes has shown that exosomes play a central role in physiological keratinocyte-macrophage (mϕ) crosstalk at the wound-site. Here, we report that during the early phase of wound reepithelialization, macrophage-derived exosomes (Exomϕ), enriched with the outer mitochondrial membrane protein TOMM70, are localized in leading-edge keratinocytes. TOMM70 is a 70 kDa adaptor protein anchored in the mitochondrial outer membrane and plays a critical role in maintaining mitochondrial function and quality. TOMM70 selectively recognizes cytosolic chaperones by its tetratricopeptide repeat (TPR) domain and facilitates the import of preproteins lacking a positively charged mitochondrial targeted sequence. Exosomal packaging of TOMM70 in mϕ was independent of mitochondrial fission. TOMM70-enriched Exomϕ compensated for the hypoxia-induced depletion of epidermal TOMM70, thereby rescuing mitochondrial metabolism in leading-edge keratinocytes. Thus, macrophage-derived TOMM70 is responsible for the glycolytic ATP supply to power keratinocyte migration. Blockade of exosomal uptake from keratinocytes impaired wound closure with the persistence of proinflammatory mϕ in the wound microenvironment, pointing toward a bidirectional crosstalk between these two cell types. The significance of such bidirectional crosstalk was established by the observation that in patients with nonhealing diabetic foot ulcers, TOMM70 is deficient in keratinocytes of wound-edge tissues.

The application of plasmid DNA constructs encoding antiviral proteins as potential therapeutics against viral infections in salmonid aquaculture

Treatments to control infectious diseases in aquacultured fish commonly focus on the use of antibiotics (which are ineffective against viruses) and inorganic disinfectants. Currently, antiviral treatments are not available for use in aquaculture. The use of plasmid vectors to overexpress antiviral proteins in fish has been reported, with positive results against viral infections. The aim of this work was to overexpress the antiviral proteins interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) and natural killer enhancing factor (NKEF) using plasmid vectors (pIFIT5 and pNKEF) as novel therapeutic agents against viral infections. The antiviral activity of pIFIT5 against viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) was evaluated in vitro in epithelioma papulosum cyprinid and Chinook salmon embryo cell lines. Moreover, pNKEF antiviral activity against IPNV was also evaluated. Results showed that pIFIT5 had antiviral activity against both viruses, but pNKEF did not protect against IPNV. In addition, we evaluated the use of pIFIT5 and pNKEF constructs as therapeutics in rainbow trout. Biodistribution assays demonstrated systemic distribution of the IFIT5-TFP1 and NKEF-TFP1 proteins in rainbow trout injected with the plasmids, and pIFIT5 and pNKEF triggered an immune response in the injected fish. In a pilot study, we evaluated the level of protection conferred by pIFIT5 and pNKEF against VHSV in rainbow trout, and we observed 10 % survival for pIFIT5 compared to pTFP. Together, these results suggest the potential of pIFIT5 as therapeutic agent in aquaculture.

Peripheral mononuclear cells and systemic lupus erythematosus association: Integrated study of single-cell sequencing and mendelian randomization analysis.

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease predominantly affecting women. Despite advances in treatment, recent developments in single-cell RNA sequencing (scRNA-seq) and Mendelian randomization (MR) continue to facilitate the need for precision medicine. Data obtained from the GSE135779 dataset underwent quality control, normalization, and dimensionality reduction using Seurat and MonacoImmuneData. Marker genes identified subgroups for analysis with CellChat and ClusterProfilerR. MR analysis of these genes' eQTLs was performed to establish causal relationships with SLE using IEU Open GWAS project data. Single-cell analysis revealed distinct cellular subtypes and highlighted increased monocyte levels in patients with SLE. MR analysis revealed 12 genes, particularly interferon induced protein with tetratricopeptide repeats 3 (IFIT3), causally related to SLE. Gene ontology and the Kyoto encyclopedia of genes and genomes analyses identified pathways significant to SLE pathogenesis. Visualization of these genes at the single-cell level revealed their role in disease progression. Cell communication differences between IFIT3-positive and -negative groups were also observed. This study demonstrates the potential of scRNA-seq and MR in identifying critical factors in SLE pathogenesis, thereby supporting the need for targeted therapies. Identifying IFIT3, among other genes, as central to SLE progression opens new avenues for precision medicine approaches in SLE management.

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.

Flow cytometry FRET reveals post-translational modifications drive Protein Phosphatase-5 conformational changes in mammalian cells.

The serine/threonine Protein Phosphatase-5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other Protein Phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5-TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity have yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predicts its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms.

B-089 Pseudo-clonal Increased IgG4 with Polyclonal Distribution Demonstrated by MALDI-TOF-MS in a Pediatric Patient with Tricho-Hepato-Enteric Syndrome (THES)

Abstract Background THES is a rare autosomal recessive syndromic enteropathy caused by pathogenic variants in two genes, the Tetratricopeptide Repeat Domain 37 (TTC37) and Ski2 Like RNA Helicase (SKIV2L), that encode components of the human SKI complex involved in RNA degradation. This disease is characterized by several clinical features with variable penetrance including intractable congenital diarrhea, wooly hair abnormalities, intrauterine growth restriction, facial dysmorphism, immune dysfunction, liver abnormalities, and skin hyperpigmentation. Hypogammaglobulinemia with low IgG and normal to low IgM levels have been described in these cases which may require intravenous immunoglobulin (IVIG) replacement therapy. Some cases have a reduced population of switched memory B cells, T cells with reduced IFN-γ secretion, and hyporesponsive NK cells. Our patient is a 4-year-old female reported to have a homozygous TTC37 mutation with clinical manifestations of TPN dependence, liver fibrosis with portal hypertension, splenomegaly, developmental delay, thrombocytopenia and anemia requiring transfusion, and a history of hypogammaglobinemia requiring IVIG replacement. Patient would go on to develop elevated immunoglobin levels leading to additional testing described below. Methods Serum total protein analysis performed using the using Siemens Atellica CH Total Protein II test (Weichselbaum method using biuret reagent). Serum immunoglobin (IgA, IgG and IgG), IgG subclass, and kappa/lambda free light chain level analysis performed by turbidimetric photometry assay using a Binding Site Optilite analyzer. Serum protein electrophoresis and immunotyping performed using CAPILLARYS 2 capillary zone electrophoresis and immunosubtraction with antisera. Serum protein immunofixation performed using Sebia Hydrasys 2 with Hydragel 9 IF Maxi kit. Confirmation and final determination of serum immunoproteins performed at our reference laboratory using MASS-FIX (MALDI-TOF MS) matrix-assisted laser desorption/ionization-time of flight mass spectrometry after patient serum applied to 5 separate immunoaffinity resins specific to immunoglobulin G, A, M, K, and L for purification. Results Initial testing showed a broadly migrating beta-gamma region IgG kappa monotypic restriction measuring 1.7 g/dL, with predominantly kappa subtraction and trivial lambda subtraction by immunosubtraction; serum free kappa/lambda light chain ratio was increased at 22.94. Serum immunoglobins and free light chains measured (mg/dL): IgA 738 (15 - 160), IgM 48 (50 - 240), IgG 2,805 (405 - 1,160), free kappa 20.42 (0.33 - 1.94), and free lambda 0.89 (0.57 - 2.63). IgG subclasses measured (mg/dL): IgG1 533 (320 - 950), IgG2 174 (50 - 340), IgG3 16.9 (10.0 - 120.0), IgG4 1,221.1 (2.0 - 110.0). Additional serum protein values included (g/dL): total protein 8.10 (6.00 - 8.30), albumin 2.97 (3.43 - 4.84), alpha-1 0.32 (0.21 - 0.44), alpha-2 0.52 (0.54 - 0.97), beta globulin 3.42 (0.65 - 1.03), and gamma globulin 0.87 (0.70 - 2.30). The polyclonal nature of the IgG4 spike was later demonstrated by the MASS-FIX mass spectrograph. Conclusions MASS-FIX is a useful addition to the toolset used in the diagnosis of possible monoclonal gammopathies and protein disorders, especially where broad monotypic restrictions are observed or in patients with abnormal presentations for monoclonal gammopathies. Using current standard techniques, the above case could have been incorrectly classified as a monoclonal process resulting in unnecessary cost and incorrect treatment.

Transcriptome-wide Identification of Nine Tandem Repeat Protein Families in Roselle (Hibiscus sabdariffa L.).

Plants are rich in tandem repeats-containing proteins. It is postulated that the occurrence of tandem repeat gene families facilitates the adaptation and survival of plants in adverse environmental conditions. This study intended to identify the tandem repeats in the transcriptome of a high potential tropical horticultural plant, roselle (Hibiscus sabdariffa L.). A total of 92,974 annotated de novo assembled transcripts were analysed using in silico approach, and 6,541 transcripts that encoded proteins containing tandem repeats with length of 20-60 amino acid residues were identified. Domain analysis revealed a total of nine tandem repeat protein families in the transcriptome of roselle, which are the Ankyrin repeats (ANK), Armadillo repeats (ARM), elongation factor-hand domain repeats (EF-hand), Huntingtin, elongation factor 3, protein phosphatase 2A, yeast kinase TOR1 repeats (HEAT), Kelch repeats (Kelch), leucine rich repeats (LRR), pentatricopeptide repeats (PPR), tetratricopeptide repeats (TPR) and WD40 repeats (WD40). Functional annotation analysis further matched 6,236 transcripts to 1,045 known proteins that contained tandem repeats including proteins implicated in plant development, protein-protein interaction, immunity and abiotic stress responses. The findings provide new insights into the occurrence of tandem repeats in the transcriptome and lay the foundation to elucidate the functional associations between tandem peptide repeats (TRs) and proteins in roselle and facilitate the identification of novel biotic and abiotic response related tandem repeats genes that may be useful in breeding improved varieties.

Desipramine reverses remote memory deficits by activating calmodulin-CaMKII pathway in a UTX knockout mouse model of Kabuki syndrome.

Kabuki syndrome (KS) is a rare developmental disorder characterised by multiple congenital anomalies and intellectual disability. UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), which encodes a histone demethylase, is one of the two major pathogenic risk genes for KS. Although intellectual disability is a key phenotype of KS, the role of UTX in cognitive function remains unclear. Currently, no targeted therapies are available for KS. This study aimed to investigate how UTX regulates cognition, to explore the mechanisms underlying UTX dysfunction and to identify potential molecular targets for treatment. We generated UTX conditional knockout mice and found that UTX deletion downregulated calmodulin transcription by disrupting H3K27me3 (trimethylated histone H3 at lysine 27) demethylation. UTX-knockout mice showed decreased phosphorylation of calcium / calmodulin-dependent protein kinase II, impaired long-term potentiation and deficit in remote contextual fear memory. These effects were reversed by an Food and Drug Administration-approved drug desipramine. Our results reveal an epigenetic mechanism underlying the important role of UTX in synaptic plasticity and cognitive function, and suggest that desipramine could be a potential treatment for KS.

Prognostic value of TMTC1 in pan-cancer analysis

BackgroundTransmembrane and tetratricopeptide repeat containing 1 (TMTC1) is a recently discovered enzyme involved in the O-mannosylation of cadherins and protocadherins. It has been implicated in various types of cancer, but the overall prognostic significance of TMTC1 in pan-cancer and its potential as an immunotherapeutic target remain unclear. MethodsWe applied various bioinformatics methods to investigate the potential oncogenic roles of TMTC1 using public databases. This analysis involved examining the expression, prognosis, genetic alterations, immune infiltration, immunotherapy response, drug sensitivity, and regulatory mechanisms of the TMTC1 gene in diverse cancer types. ResultsIn this study, we observed that TMTC1 expression is reduced in 19 types of cancer (ACC, BLCA, BRCA, CESC, COAD, ESCA, GBM, KICH, KIRC, KIRP, LAML, LUAD, LUSC, PRAD, READ, STAD, THCA, UCEC, and UCS) compared to normal tissues. Conversely, TMTC1 expression is elevated in OV and PAAD relative to normal tissues. Moreover, our analysis revealed that high expression of TMTC1 was associated with worse overall survival (OS) outcomes in patients with ACC, BLCA, COAD, GBM, KIRP, OV, STAD, and UCEC, but better OS outcomes in patients with CESC, KIRC, LUSC, and PAAD. Notably, patients with TMTC1 mutations or deep deletions demonstrated longer OS, while those with TMTC1 amplification showed shorter OS. There was a significant correlation between the expression level of TMTC1 and the infiltration of cancer-associated fibroblasts (CAFs) and endothelial cells. Using data from six real-world immunotherapy cohorts of BLCA, SKCM and RCC, we discovered that high TMTC1 expression was associated with better OS or progression-free survival (PFS). Lastly, through TMTC1-related gene enrichment analysis, some biological processes and pathways were found to be significantly enriched, such as vascular endothelial growth factor receptor signaling pathway and ECM-receptor interaction. ConclusionsOur study demonstrates the prognostic significance of TMTC1 in pan-cancer and highlights its potential as an immunotherapeutic target.

Structural dynamics of the TPR domain of the peroxisomal cargo receptor Pex5 in Trypanosoma

Peroxisomal protein import has been identified as a valid target in trypanosomiases, an important health threat in Central and South America. The importomer is built of multiple peroxins (Pex) and structural characterization of these proteins facilitates rational inhibitor development. We report crystal structures of the Trypanosoma brucei and T. cruzi tetratricopeptide repeat domain (TPR) of the cytoplasmic peroxisomal targeting signal 1 (PTS1) receptor Pex5. The structure of the TPR domain of TbPex5 represents an apo-form of the receptor which, together with the previously determined structure of the complex of TbPex5 TPR and PTS1 demonstrate significant receptor dynamics associated with signal peptide recognition. The structure of the complex of TPR domain of TcPex5 with PTS1 provided in this study details the molecular interactions that guide signal peptide recognition at the atomic level in the pathogenic species currently perceived as the most relevant among Trypanosoma. Small – angle X – ray scattering (SAXS) data obtained in solution supports the crystallographic findings on the compaction of the TPR domains of TbPex5 and TcPex5 upon interaction with the cargo.

Molecular characterisation and expression analysis of an interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) homologue from Asian seabass (Lates calcarifer)

The interferon-induced protein with tetratricopeptide repeats 1(IFIT1) is an interferon-inducible protein that acts downstream of the interferon pathway in response to viral infection or an interferon-stimulation. Our study describes the isolation and characterization of an IFIT1 homologue from Lates calcarifer (named LcIFIT1), as well as its expression profile in response to viral mimetic poly(I:C) and Red-spotted grouper nervous necrosis virus (RGNNV). The complete ORF of LcIFIT1 encodes a 441 amino acid protein with nine tetratricopeptide repeat (TPR) domains. The protein-coding region of the LcIFIT1 gene was encoded by two exons separated by a small 157 bp intron. The phylogenetic analysis revealed that LcIFIT1 is evolutionarily conserved and forms a distinct clade with homologues from other teleosts. LcIFIT1 mRNA was constitutively expressed in all tissues examined, with predominant expression in blood. LcIFIT1 expression was upregulated following poly(I:C) and RGNNV challenges in different tissues examined. These results shed light on the antiviral role of LcIFIT1 in the host immune response against nodaviral infection.

Identification of a Polypeptide Inhibitor of O-GlcNAc Transferase with Picomolar Affinity.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that binds thousands of different proteins, including substrates that it glycosylates and nonsubstrate interactors that regulate its biology. OGT also has one proteolytic substrate, the transcriptional coregulator host cell factor 1 (HCF-1), which it cleaves in a process initiated by glutamate side chain glycosylation at a series of central repeats. Although HCF-1 is OGT's most prominent binding partner, its affinity for the enzyme has not been quantified. Here, we report a time-resolved Förster resonance energy transfer assay to measure ligand binding to OGT and show that an HCF-1-derived polypeptide (HCF3R) binds with picomolar affinity to the enzyme (KD ≤ 85 pM). This high affinity is driven in large part by conserved asparagines in OGT's tetratricopeptide repeat domain, which form bidentate contacts to the HCF-1 peptide backbone; replacing any one of these asparagines with alanine reduces binding by more than 5 orders of magnitude. Because the HCF-1 polypeptide binds so tightly to OGT, we tested its ability to inhibit enzymatic function. We found that HCF3R potently inhibits OGT both in vitro and in cells and used this finding to develop a genetically encoded, inducible OGT inhibitor that can be degraded with a small molecule, allowing for reversible and tunable inhibition of OGT.

Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope

Phosphoethanolamine (pEtN) cellulose is a naturally occurring modified cellulose produced by several Enterobacteriaceae. The minimal components of the E. coli cellulose synthase complex include the catalytically active BcsA enzyme, a hexameric semicircle of the periplasmic BcsB protein, and the outer membrane (OM)-integrated BcsC subunit containing periplasmic tetratricopeptide repeats (TPR). Additional subunits include BcsG, a membrane-anchored periplasmic pEtN transferase associated with BcsA, and BcsZ, a periplasmic cellulase of unknown biological function. While cellulose synthesis and translocation by BcsA are well described, little is known about its pEtN modification and translocation across the cell envelope. We show that the N-terminal cytosolic domain of BcsA positions three BcsG copies near the nascent cellulose polymer. Further, the semicircle’s terminal BcsB subunit tethers the N-terminus of a single BcsC protein in a trans-envelope secretion system. BcsC’s TPR motifs bind a putative cello-oligosaccharide near the entrance to its OM pore. Additionally, we show that only the hydrolytic activity of BcsZ but not the subunit itself is necessary for cellulose secretion, suggesting a secretion mechanism based on enzymatic removal of translocation incompetent cellulose. Lastly, protein engineering introduces cellulose pEtN modification in orthogonal cellulose biosynthetic systems. These findings advance our understanding of pEtN cellulose modification and secretion.

Mutations in the N-domain of aryl hydrocarbon receptor interacting protein affect interactions with heat shock protein 90β and phosphodiesterase 4A5

The aryl hydrocarbon receptor interacting protein (AIP) is a cytoplasmic molecular co-chaperone and tumour suppressor that assists in protein stability and complex formation involving the aryl hydrocarbon receptor. Germline mutations in the AIP gene predispose to pituitary tumourigenesis with patients exhibiting an aggressive clinical phenotype. Full length AIP proteins harbouring N-domain mutations (R9Q, R16H, V49 M and K103R) were purified from E.coli utilizing a methodology that maintained structural integrity and monomeric stability. Mutations did not significantly affect the thermal stability of the protein and caused no overall disruptive effect in the protein structure. The mutations studied lowered the binding affinity of AIP towards two of its binding partners; heat shock protein 90β and phosphodiesterase 4A5 (PDE4A5). The inhibition of phosphodiesterase activity by AIP was also greatly reduced by all mutants. While previously published data has mainly concentrated on the tetratricopeptide repeats of the C-domain of AIP, we present clear evidence that AIP N-domain mutations play a significant role in two protein:protein interactions with partner proteins. The complex interactome of AIP suggests that any observable change in one or more of its binding partners cannot be disregarded as it may have repercussions on other biochemical pathways.