Tetratricopeptide Repeat Domain Research Articles

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.

Expanding the clinical phenotype and genetic spectrum of GEMIN5 disorders: Early-infantile developmental and epileptic encephalopathies.

Several biallelic truncating and missense variants of the gem nuclear organelle-associated protein 5 (GEMIN5) gene have been reported to cause neurodevelopmental disorders characterized by cerebellar atrophy, intellectual disability, and motor dysfunction. However, the association between biallelic GEMIN5 variants and early-infantile developmental and epileptic encephalopathies (EIDEEs) has not been reported. This study aimed to expand the phenotypic spectrum of GEMIN5 and explore the correlations between epilepsy and molecular sub-regional locations. We performed whole-exome sequencing in two patients with EIDEE with unexplained etiologies. The damaging effects of variants were predicted using multiple in silico tools and modeling. All reported patients with GEMIN5 pathogenic variants and detailed neurological phenotypes were analyzed to evaluate the genotype-phenotype relationship. Novel biallelic GEMIN5 variants were identified in two unrelated female patients with EIDEE, including a frameshift variant (Hg19, chr5:154284147-154284148delCT: NM_015465: c.2551_c.2552delCT: p.(Leu851fs*30)), a nonsense mutation (Hg19, chr5:154299603-154299603delTinsAGA: NM_015465: c.1523delTinsAGA: p.(Leu508*)), and two missense variants (Hg19, chr5:154282663T>A: NM_015465: c.2705T>A: p.(Leu902Gln) and Hg19, chr5:154281002C>G: NM_015465: c.2911C>G: p.(Gln971Glu)), which were inherited from asymptomatic parents and predicted to be damaging or probably damaging using in silico tools. Except p.Leu508*, all these mutations are located in tetratricopeptide repeat (TPR) domain. Our two female patients presented with seizures less than 1 month after birth, followed by clusters of spasms. Brain magnetic resonance imaging suggests dysgenesis of the corpus callosum and cerebellar hypoplasia. Video electroencephalogram showed suppression-bursts. Through a literature review, we found 5 published papers reporting 48 patients with biallelic variants in GEMIN5. Eight of 48 patients have epilepsy, and 5 patients started before 1 year old, which reminds us of the relevance between GEMIN5 variants and EIDEE. Further analysis of the 49 GEMIN5 variants in those 50 patients demonstrated that variants in TPR-like domain or RBS domain were more likely to be associated with epilepsy. We found novel biallelic variants of GEMIN5 in two individuals with EIDEE and expanded the clinical phenotypes of GEMIN5 variants. It is suggested that the GEMIN5 gene should be added to the EIDEE gene panel to aid in the clinical diagnosis of EIDEE and to help determine patient prognosis.

Identification of Leishmania donovani PEX5-PTS1 Interaction Inhibitors through Fluorescence Polarization-Based High-Throughput Screening.

Leishmaniasis, an infectious disease caused by pathogenic Leishmania parasites, affects millions of people in developing countries, and its re-emergence in developed countries, particularly in Europe, poses a growing public health concern. The limitations of current treatments and the absence of effective vaccines necessitate the development of novel therapeutics. In this study, we focused on identifying small molecule inhibitors which prevents the interaction between peroxin 5 (PEX5) and peroxisomal targeting signal 1 (PTS1), pivotal for kinetoplastid parasite survival. The Leishmania donovani PEX5, containing a C-terminal tetratricopeptide repeat (TPR) domain, was expressed and purified, followed by the quantification of kinetic parameters of PEX5-PTS1 interactions. A fluorescence polarization-based high-throughput screening assay was developed and small molecules inhibiting the LdPEX5-PTS1 interaction were discovered through the screening of a library of 51,406 compounds. Based on the confirmatory assay, nine compounds showed half maximal inhibitory concentration (IC50) values ranging from 3.89 to 24.50 µM. In silico docking using a homology model of LdPEX5 elucidated that the molecular interactions between LdPEX5 and the inhibitors share amino acids critical for PTS1 binding. Notably, compound P20 showed potent activity against the growth of L. donovani promastigotes, L. major promastigotes, and Trypanosoma brucei blood stream form, with IC50 values of 12.16, 19.21, and 3.06 μM, respectively. The findings underscore the potential of targeting LdPEX5-PTS1 interactions with small molecule inhibitors as a promising strategy for the discovery of new anti-parasitic compounds.

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1.

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

The cryo-EM structure of ASK1 reveals an asymmetric architecture allosterically modulated by TRX1

Apoptosis signal-regulating kinase 1 (ASK1) is a crucial stress sensor, directing cells toward apoptosis, differentiation, and senescence via the p38 and JNK signaling pathways. ASK1 dysregulation has been associated with cancer and inflammatory, cardiovascular, and neurodegenerative diseases, among others. However, our limited knowledge of the underlying structural mechanism of ASK1 regulation hampers our ability to target this member of the MAP3K protein family towards developing therapeutic interventions for these disorders. Nevertheless, as a multidomain Ser/Thr protein kinase, ASK1 is regulated by a complex mechanism involving dimerization and interactions with several other proteins, including thioredoxin 1 (TRX1). Thus, the present study aims at structurally characterizing ASK1 and its complex with TRX1 using several biophysical techniques. As shown by cryo-EM analysis, in a state close to its active form, ASK1 is a compact and asymmetric dimer, which enables extensive interdomain and interchain interactions. These interactions stabilize the active conformation of the ASK1 kinase domain. In turn, TRX1 functions as a negative allosteric effector of ASK1, modifying the structure of the TRX1-binding domain and changing its interaction with the tetratricopeptide repeats domain. Consequently, TRX1 reduces access to the activation segment of the kinase domain. Overall, our findings not only clarify the role of ASK1 dimerization and inter-domain contacts but also provide key mechanistic insights into its regulation, thereby highlighting the potential of ASK1 protein-protein interactions as targets for anti-inflammatory therapy.

P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA–LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.

Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.

Identification of TTC21A as a Potential Prognostic Marker in Head and Neck Squamous Cell Carcinoma: In Silico Analysis.

Tetratricopeptide repeat domain 21A (TTC21A) plays a crucial role in ciliary function and has been associated with various pathogenic processes, including carcinogenesis. However, its role in head and neck squamous cell carcinoma (HNSCC) has not been elucidated. Based on the sequencing and microarray data of HNSCC from publicly available databases, the expression of TTC21A was compared between different subgroups based on clinical and molecular parameters. The survival analysis and regression analysis were conducted using the Kaplan-Meier method and the Cox method, respectively. Functional analysis was performed by the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and gene set enrichment analysis (GSEA) tools. Immune infiltration analysis was performed based on the expression of TTC21A. TTC21A decreased in tumor tissues and was associated with N stage, histologic grade, HPV infection, and TP53 mutation in HNSCC. TTC21A was an independent indicator of overall survival for patients with HNSCC. A high level of TTC21A expression indicated a favorable prognosis. The TTC21A expression level was involved with immune-related signaling regulation, immune-related gene expression, and immune cell infiltration. TTC21A expression was potent in predicting immunotherapeutic benefits. TTC21A, as a potential predictor of favorable outcomes and immunotherapy response for HNSCC, is related to immune-related signaling regulation, immune-related gene expression, and immune cell infiltration.

ZmTDM1 encodes a tetratricopeptide repeat domain protein and is required for meiotic exit in maize.

Elaborate cell-cycle control must be adopted to ensure the continuity of the meiotic second division and termination after that. Despite its importance, however, the genetic controls underlying the meiotic cell cycle have not been reported in maize. Here, we characterized a meiotic cell-cycle controller ZmTDM1, which is a homolog of Arabidopsis TDM1 and encodes a canonical tetratricopeptide repeat domain protein in maize. The Zmtdm1 homozygous plants exhibited complete male sterility and severe female abortion. In Zmtdm1 mutants, cell-cycle progression was almost identical to that of wild type from leptotene to anaphase II. However, chromosomes in the tetrad failed meiotic termination at the end of the second division and underwent additional divisions in succession without DNA replication, reducing the ploidy to less than haploid in the product. In addition, two ZmTDM1-like homologs (ZmTDML1 and ZmTDML2) were not functional in meiotic cell-cycle control. Moreover, ZmTDM1 interacted with RING-type E3 ubiquitin ligase, revealing that it acts as a subunit of the APC/C E3 ubiquitin ligase complex. Overall, our results identified a regulator of meiotic cell cycle in maize and demonstrated that ZmTDM1 is essential for meiotic exit after meiosis II.

A risk variant in TTC3 modifies the actin cytoskeleton organization and PI3K‐Akt signaling in iPSC‐derived forebrain neurons

AbstractBackgroundWe identified a rare, nonsynonymous variant in the tetratricopeptide repeat domain 3 (TTC3) gene that segregated in a non‐Hispanic white late onset Alzheimer disease (LOAD) family (Kohli, et al, 2016). This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious and is extremely rare. Studies have reported that cortical TTC3 expression is reduced in LOAD patients and negatively correlated with AD neuropathology.MethodTo understand the mechanism by which the TTC3 p.S1038C may contribute to LOAD risk, CRISPR/Cas9 genome edited induced pluripotent stem cells (iPSCs) were developed that were homozygous for the variant to examine cellular and transcriptional consequences in iPSC‐derived neuronal cells (Laverde‐Paz, et al, 2021).ResultQuantitative PCR and western blot analysis demonstrated that TTC3 levels were decreased in edited compared to unedited iPSCs, as well as differentiated neurons. In growing neuronal precursor cells (NPCs), cells with the TTC3 variant recovered more quickly from a scratch wound. Since there is evidence that modulation of TTC3 affects neurite growth, morphological measures of axon formation were assessed using the Incucyte Zoom. Studies demonstrate an increase in neurite outgrowth, which phenotypically corresponds with previous studies of a decrease in TTC3 function. This phenotype was tempered by treatment with Cytochalasin D, an inhibitor of actin polymerization. Additionally, TTC3 ubiquitinates phosphorylated AKT and regulates AKT signaling. The edited cells were found to have an increase in phosphorylated AKT (pAKT) relative to total AKT. RNA‐seq of day 70 neurons identified 979 genes that were differentially expressed (FDR<0.05). This included known AD genes (BACE1) and genes in AD GWAS loci (ADAMTS1, MAF, NCK2). KEGG pathway analysis identified differential expression in PI3K‐Akt signaling pathway components, in which TTC3 has been previously implicated, as well as the axon guidance pathway, the GABAergic synapse pathway, and the Wnt signaling pathway.ConclusionCombined, these results suggest that the TTC3 p.S1038C variant causes a loss of function. Utilizing a CRISPR genome edited iPSC carrying a homozygous alteration in TTC3, we were able to identify potential mechanisms by which TTC3 may contribute to LOAD risk.

TTC22 as a potential prognostic marker and therapeutic target in pancreatic cancer: Insights into immune infiltration and epithelial‑mesenchymal transition.

The mortality rate of pancreatic adenocarcinoma is high, and the effect of traditional treatment is unsatisfactory, thus novel biomarkers are required. Although the important role of tetratricopeptide repeat domain 22 (TTC22) in colon cancer is well established, its precise role in pancreatic cancer remains unclear and requires further investigation. Pan-cancer analysis and single-cell sequencing revealed TTC22 was differentially expressed in various tumors, especially in pancreatic adenocarcinoma. Additionally, clinical data for pancreatic cancer showed a negative association between TTC22 expression and clinical parameters, including survival prognosis. The correlation between TTC22 and immune infiltration in pancreatic cancer was validated by functional enrichment analysis. ESTIMATE and single sample Gene Set Enrichment Analysis algorithms were used to further analyze immune infiltration of TTC22 in pancreatic cancer, and the results suggested that TTC22 inhibited tumor immunity and was negatively correlated with plasmacytoid dendritic cells. Reverse transcription-quantitative PCR further confirmed the differential expression of TTC22 in pancreatic cancer cell lines. Wound healing, Transwell and colony formation assays showed that TTC22 affected the migration and invasion of pancreatic cancer cells. These findings demonstrate that TTC22 may serve as a potential prognostic marker and therapeutic target for the management of pancreatic cancer.

Roles of GR Isoforms and Hsp90-binding Immunophilins in the Modulation of Glucocorticoid Biological Responses.

Glucocorticoid steroids play cardinal roles during the life span of an individual, modulating almost all aspects of the physiology, including the metabolism of carbohydrates, lipids and amino acids, as well as the immune response, neurological biology, stress adaptation, apoptosis, cell division, cell fate, inflammatory responses, etc. Glucocorticoids exert their biological effects by activation of the glucocorticoid receptor (GR), a bona fide ligand-activated transcription factor belonging to the nuclear receptor superfamily. The GR is expressed in virtually all cells of the human body showing isoformic versions and also transcription variants. GR forms oligomeric heterocomplexes that include the 90-kDa heat-shock protein (Hsp90) as an essential hub of the chaperone oligomer. The nature of chaperones associated with this heterocomplex is responsible for the modulation of the subcellular localization of the GR and its biological actions in a given tissue or cell type. In this sense, the discovery that immunophilins containing tetratricopeptide repeats (TPR) domains are responsible for the GR cytoplasmic transport mechanism and the nuclear retention half-time of the receptor opened new trends in our understanding of its complex mechanism of action. Because the properties of GR ligands influence these protein-protein interactions, specific steroid•receptor complexes may confer the GR different features providing new therapeutic opportunities to manage the disease. In this article, we analyze multiple aspects of the GR mechanism of action, some properties of the GR isoforms, and the latest findings revealing the roles of Hsp90-binding immunophilins to manage the glucocorticoid biological response.

Functional Characterization of Small Alarmone Synthetase and Small Alarmone Hydrolase Proteins from Treponema denticola.

The stringent response enables bacteria to survive nutrient starvation, antibiotic challenge, and other threats to cellular survival. Two alarmone (magic spot) second messengers, guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp), which are synthesized by RelA/SpoT homologue (RSH) proteins, play central roles in the stringent response. The pathogenic oral spirochete bacterium Treponema denticola lacks a long-RSH homologue but encodes putative small alarmone synthetase (Tde-SAS, TDE1711) and small alarmone hydrolase (Tde-SAH, TDE1690) proteins. Here, we characterize the respective in vitro and in vivo activities of Tde-SAS and Tde-SAH, which respectively belong to the previously uncharacterized RSH families DsRel and ActSpo2. The tetrameric 410-amino acid (aa) Tde-SAS protein preferentially synthesizes ppGpp over pppGpp and a third alarmone, pGpp. Unlike RelQ homologues, alarmones do not allosterically stimulate the synthetic activities of Tde-SAS. The ~180 aa C-terminal tetratricopeptide repeat (TPR) domain of Tde-SAS acts as a brake on the alarmone synthesis activities of the ~220-aa N-terminal catalytic domain. Tde-SAS also synthesizes "alarmone-like" nucleotides such as adenosine tetraphosphate (ppApp), albeit at considerably lower rates. The 210-aa Tde-SAH protein efficiently hydrolyzes all guanosine and adenosine-based alarmones in a Mn(II) ion-dependent manner. Using a growth assays with a ΔrelAΔspoT strain of Escherichia coli that is deficient in pppGpp/ppGpp synthesis, we demonstrate that Tde-SAS can synthesize alarmones in vivo to restore growth in minimal media. Taken together, our results add to our holistic understanding of alarmone metabolism across diverse bacterial species. IMPORTANCE The spirochete bacterium Treponema denticola is a common component of the oral microbiota. However, it may play important pathological roles in multispecies oral infectious diseases such as periodontitis: a severe and destructive form of gum disease, which is a major cause of tooth loss in adults. The operation of the stringent response, a highly conserved survival mechanism, is known to help many bacterial species cause persistent or virulent infections. By characterizing the biochemical functions of the proteins putatively responsible for the stringent response in T. denticola, we may gain molecular insight into how this bacterium can survive within harsh oral environments and promote infection. Our results also expand our general understanding of proteins that synthesize nucleotide-based intracellular signaling molecules in bacteria.

An Alzheimer’s disease risk variant in TTC3 modifies the actin cytoskeleton organization and the PI3K-Akt signaling pathway in iPSC-derived forebrain neurons

A missense variant in the tetratricopeptide repeat domain 3 (TTC3) gene (rs377155188, p.S1038C, NM_003316.4:c 0.3113C>G) was found to segregate with disease in a multigenerational family with late-onset Alzheimer’s disease. This variant was introduced into induced pluripotent stem cells (iPSCs) derived from a cognitively intact individual using CRISPR genome editing, and the resulting isogenic pair of iPSC lines was differentiated into cortical neurons. Transcriptome analysis showed an enrichment for genes involved in axon guidance, regulation of actin cytoskeleton, and GABAergic synapse. Functional analysis showed that the TTC3 p.S1038C iPSC-derived neuronal progenitor cells had altered 3-dimensional morphology and increased migration, while the corresponding neurons had longer neurites, increased branch points, and altered expression levels of synaptic proteins. Pharmacological treatment with small molecules that target the actin cytoskeleton could revert many of these cellular phenotypes, suggesting a central role for actin in mediating the cellular phenotypes associated with the TTC3 p.S1038C variant.

The E3 Ubiquitin Ligase, CHIP/STUB1, Inhibits Aggregation of Phosphorylated Proteoforms of Microtubule-associated Protein Tau (MAPT)

Hyper-phosphorylated tau accumulates as insoluble fibrils in Alzheimer’s disease (AD) and related dementias. The strong correlation between phosphorylated tau and disease has led to an interest in understanding how cellular factors discriminate it from normal tau. Here, we screen a panel of chaperones containing tetratricopeptide repeat (TPR) domains to identify those that might selectively interact with phosphorylated tau. We find that the E3 ubiquitin ligase, CHIP/STUB1, binds 10-fold more strongly to phosphorylated tau than unmodified tau. The presence of even sub-stoichiometric concentrations of CHIP strongly suppresses aggregation and seeding of phosphorylated tau. We also find that CHIP promotes rapid ubiquitination of phosphorylated tau, but not unmodified tau, in vitro. Binding to phosphorylated tau requires CHIP’s TPR domain, but the binding mode is partially distinct from the canonical one. In cells, CHIP restricts seeding by phosphorylated tau, suggesting that it could be an important barrier in cell-to-cell spreading. Together, these findings show that CHIP recognizes a phosphorylation-dependent degron on tau, establishing a pathway for regulating the solubility and turnover of this pathological proteoform.

Novel homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia owing to dynein arm complex and mitochondrial sheath defects in flagella.

Introduction: Tracing the genetic causes for male infertility due to asthenoteratozoospermia has revealed at least 40 causative genes, which provides valuable reference for the genetic testing of asthenoteratozoospermia in clinical practice. To identify deleterious variants in the human tetratricopeptide repeat domain 12 (TTC12) gene in a large cohort of infertile Chinese males with asthenoteratozoospermia. Methods: A total of 314 unrelated asthenoteratozoospermia-affected men were recruited for whole exome sequencing. The effects of the identified variants were evaluated by in silico analysis, and confirmed by in vitro experiments. Intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of assisted reproduction technique therapy. Results and Discussion: Novel homozygous TTC12 variants (c.1467_1467delG (p.Asp490Thrfs*14), c.1139_1139delA (p.His380Profs*4), and c.1117G>A (p.Gly373Arg)) were identified in three (0.96%) of the 314 cases. Three mutants were indicated to be damaging using in silico prediction tools, and were further confirmed by in vitro functional analysis. Hematoxylin and eosin staining and ultrastructural observation of the spermatozoa revealed multiple morphological abnormalities of flagella, with the absence of outer and inner dynein arms. Notably, significant mitochondrial sheath malformations were also observed in the sperm flagella. Immunostaining assays indicated that TTC12 is present throughout the flagella, and was strongly concentrated in the mid-piece in control spermatozoa. However, spermatozoa from TTC12-mutated individuals exhibited almost no staining intensity of TTC12 and outer and inner dynein arms components. The three men accepted ICSI treatment using their ejaculated spermatozoa, and two female partners successfully delivered healthy babies. Our findings provide direct genetic evidence that homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia by causing dynein arm complex defects and mitochondrial sheath malformations in the flagellar. We also demonstrated that TTC12 deficiency-mediated infertility could be overcome by ICSI technology.

A newborn with multiple intestinal atresias found to have variants in tetratricopeptide repeat domain 7A gene

We report a male infant born with multiple intestinal atresias (MIA) prompting early genetic testing. Bi-allelic, compound heterozygous variants in tetratricopeptide repeat domain 7A (TTC7A) gene were identified. TTC7A is involved in transporting phosphatidylinositol 4-kinase IIIα into the plasma membrane (1). Mutations in this gene disrupt intestinal development and polarity, causing intestinal diseases such as atresia, enteropathy, and very early onset inflammatory bowel disease (VEO-IBD), as well as variable degrees of immunodeficiency (1).The identification of variants in TTC7A gene prompted an immune evaluation since as many as 75% of patients with TTC7A mutations reported in the literature have an underlying immunodeficiency (2). Flow cytometry was relatively unremarkable except for a slightly low memory T-cell count and percentage. NK-cell and B-cell numbers and percentages were appropriate for age. His phytohemagglutinin response was relatively low, but still significant, suggesting a possible component of combined immunodeficiency. To further characterize his T-cell function, we assessed his T-cell proliferation response to anti-CD3, which showed moderately decreased response of CD45+ lymphocytes and CD3+ T-cells to soluble anti-CD3 alone. However, co-stimulation with anti-CD3+anti-CD28 and anti-CD3+IL-2 normalized his T-cell proliferative response. Immunoglobulins were all low. Given his subtle T-cell defect and hypogammaglobulinemia, he was classified as having MIA with combined immunodeficiency (MIA-CID) phenotype of TTC7A.Cobalamin was tried in this patient as it has a low side effect profile and showed benefits in gut motility and intestinal tract luminal narrowing in a TTC7A zebrafish model (3). We considered investigational use of leflunomide, which showed the most promising results in the same zebrafish model (3). Bowel and hematopoietic stem-cell transplantation were considered too. Enterectomy was also offered, as this was shown to be effective in a case report of two patients with similar MIA-CID TTC7A phenotype (4). Unfortunately, the patient did not respond to steroids or cobalamin. His clinical condition worsened, and he passed away at four months of age.Mutations in the TTC7A gene result in various gastrointestinal pathologies with varying degrees of immunodeficiency in approximately 75% of patients (2). All patients with MIA or VEO-IBD should have an immune evaluation and genetic testing.

Impact of FKBP52 on cell proliferation and hormone-dependent cancers.

FK506 binding protein 52 (FKBP52) (gene name FKBP4) is a 52 kDa protein that belongs to the FKBP family; it binds to the immunosuppressant FK506 and has proline isomerase activity. In addition to its FK domain-containing peptidylprolyl isomerase activity, FKBP52 also acts as a cochaperone through the tetratricopeptide repeat domain that mediates binding to heat shock protein 90. Previous studies have reported that FKBP52 is associated with hormone-dependent, stress-related, and neurodegenerative diseases, revealing its diverse functions. In particular, the effects of FKBP52 on cancer have attracted considerable attention. FKBP52 promotes the growth of hormone-dependent cancers by activating steroid hormone receptors. Recent studies have shown that the expression of FKBP52 is increased not only in steroid hormone-dependent cancer cells but also in colorectal, lung, and liver cancers, revealing its diverse functions that contribute to cancer growth. This review summarizes reports related to hormone-dependent cancer and cell proliferation in terms of the structure of FKBP52 and its function on interacting molecules.

Abstract 6507: E3 ubiquitin-protein ligase, tetratricopeptide repeat domain 3 (TTC3), H4 clustered histone 5 (H4C5), and epithelial cell adhesion molecule (EpCAM) are novel urine-enriched liquid biopsy biomarkers to detect prostate cancer in men

Abstract Background: Prostate Cancer (PCa) is one of the leading causes of cancer deaths among American men; however, the estimated number of diagnoses represents a small fraction of disease-related biopsies performed yearly. Although the Prostate-Specific Antigen (PSA) test is widely used for screening, several advisory groups recommend against using PSA because of its suboptimal performance. Thus, there is an urgent unmet need for novel and more accurate diagnostic biomarkers for PCa detection and to differentiate aggressive cancer from its indolent form. Methods: In our study, we used voided urine (50ml) from pre and post-prostatectomy men with PCa and urine from normal healthy men as control. We isolated RNA from exfoliated cells and debris shed into urine and performed RNA-sequencing using the Illumina Next-seq 550 platform. Advanced computational and machine-learning approaches were employed to identify candidate biomarkers in men with PCa. We examined the TCGA database to validate the PCa-specific expression of the identified RNA in tumor tissues. Two RNA markers were further tested by qPCR, and one urinary soluble protein marker was measured by immunoassays. Results: Our study included 106 men with PCa and 88 control men. We identified and validated the presence of ≥1 RNA markers (TTC3, H4C5) and a protein marker (EpCAM) in urine as potential candidate biomarkers for PCa detection. We tested and developed these markers using qPCR for TTC3, H4C5, and ELISA assay for EpCAM with higher specificity and sensitivity (Table 1). Our results outperformed known urinary markers, PCA3 and SPDEF (Figure 1). TTC3, H4C5, and EpCAM markers diminished to low or undetectable levels in post-prostatectomy compared to pre-prostatectomy men with PCa. shRNA knockdown of TTC3 and EpCAM in androgen-sensitive and insensitive cells induced biological changes, suggesting their relevance to Prostate Cancer. Conclusions: We report a highly accurate panel of 3 urine-based biomarkers that detect PCa consisting of EpCAM (protein) and TTC3 and H4C5 (RNA). To identify the advantage of using individual or combinatorial markers is an ongoing study in our laboratory. Citation Format: Ranjan Joseph Perera, Marcio Moschovas, Roshane Perera, Bongyong Lee, Alexandra Miller, Ernest K. Amankwah, Rudramani Pokhrel, Christian Pavlovich, Guru Sonpavde, Vipul Patel. E3 ubiquitin-protein ligase, tetratricopeptide repeat domain 3 (TTC3), H4 clustered histone 5 (H4C5), and epithelial cell adhesion molecule (EpCAM) are novel urine-enriched liquid biopsy biomarkers to detect prostate cancer in men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6507.