Mutation Of Motif Research Articles

Clinical Manifestations Are Evolving and Progressive in Patients with Vexas Syndrome

Introduction Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) syndrome is caused by somatic mutations in UBA1 and characterized by systemic autoinflammation and hematologic manifestations. Clinically, disease progression appears heterogenous, with differing degrees of organ involvement seen in individual patients over time. Disease mortality is high, and to date only UBA1 variant, presence of MDS or significant cytopenia, and clonal hematopoiesis have been associated with worse overall survival. Prospective data are currently lacking on evolution of symptoms and organ involvement. We characterized disease evolution over time and looked at the benefit of prescribed therapies in our prospective cohort of VEXAS patients. Methods Forty-four VEXAS patients with a confirmed UBA1 mutation have been enrolled in our study at the National Institutes of Health (NIH) since 2019. We collected demographic data and clinical history, and performed standardized evaluations including peripheral blood counts, inflammatory markers, serum protein electrophoresis, and bone marrow biopsies. All patients were screened for eligibility and suitability for hematopoietic stem cell transplant (HSCT) on a phase II clinical trial (NCT05027945). Patients who did not undergo HSCT received recommendations for medical therapy. Results Subjects were male with median age of 67 years (50 - 79) and 95% were of White ethnicity. The most common UBA1 variant was M41T (n=19, 43%) followed by M41L (n=15, 34%), M41V (n=7, 16%), and splice motif mutations (n=4, 7%). Median variant allele fraction (VAF) of the cohort was 73%; M41T (76%), M41L (70%), M41V (58%), splice (61%). Thirty-six (82%) patients initially presented with skin lesions and/or arthralgias. Other initial manifestations included fever (27%), fatigue (25%), chondritis (14%), pulmonary (11%) and ocular/orbital (9%). Interestingly, only one patient presented with symptomatic anemia. At the time of first NIH clinic visit patients had manifestations of VEXAS disease for median of 6 years (range 1 - 11). Only 20% (9/44) had no active symptoms. Fatigue (39%) was most common followed by skin rash, arthralgias, pulmonary, and neurologic manifestations (Figure). Twenty-three percent had developed transfusion dependent anemia. Eleven patients (25%) had bone marrow findings meeting criteria for myelodysplastic syndrome (MDS); one patient had MDS-EB1. Venous thromboembolism was documented in 18 patients (40%). Thirteen patients (30%) had plasma cell disorder, four of whom had multiple myeloma (MM). All except one patient were receiving prednisone at NIH evaluation, with median dose of 20mg (range 5mg-50mg). Sixteen patients (36%) had elevated CRP despite glucocorticoids and steroid-sparing agents. In 66% of patients, concomitant steroid-sparing agents were used, most frequently IL-6 inhibitors (n=9), or JAK inhibitors (n=6). Other agents included erythropoietin stimulating agents (n=2), canakinumab (n=2), adalimumab (n=2), methotrexate (n=2), and azacytidine, azathioprine, colchicine, dapsone, ustekinumab in one patient each. Two patients were on MM directed therapies. At last follow-up, 8 patients (18%) had died, 6 patients (14%) were post HSCT, and 9 patients (20%) were pursuing HSCT. The indication for HSCT was refractory inflammation in two patients and progressive marrow failure in four. Twenty-one patients continued medical management: 15 (35%) with controlled inflammation on their respective regimens and 6 (14%) with progressive hemato-inflammatory manifestations. Of these six patients, one was non-compliant with therapy other than prednisone and other five were deemed unfit for HSCT due to poor performance status (n=2), age (n=2), or kidney disease (n=1). Conclusion Clinical manifestations of VEXAS syndrome evolve and progress with time. Severe cytopenia is rare at disease onset. Our observations indicate that biologics directed at JAK-STAT pathway, IL-6, IL-1 and hypomethylating agents may provide qualitative benefit in one-third of cases evidenced by controlled disease, but are not steroid sparing. HSCT is a curative approach, but, the timing is not well established. Most challenging are patients who have exhausted all effective steroid-sparing agents, require high dose of glucocorticoids, and are not a HSCT candidate. Development of effective therapies for VEXAS is a research priority to improve outcomes.

Uracil-DNA glycosylase of murine gammaherpesvirus 68 binds cognate viral replication factors independently of its catalytic residues.

Herpesviruses are large double-stranded DNA viruses that encode core replication proteins and accessory factors involved in nucleotide metabolism and DNA repair. Mammalian uracil-DNA glycosylases (UNG) excise deleterious uracil residues from their genomic DNA. Each herpesvirus UNG studied to date has demonstrated conservation of the enzymatic function to excise uracil residues from DNA. We previously reported that a murine gammaherpesvirus (MHV68) with a stop codon in ORF46 (ORF46.stop) that encodes for vUNG was defective in lytic replication and latency in vivo. However, a mutant virus that expressed a catalytically inactive vUNG (ORF46.CM) had no replication defect unless coupled with additional mutations in the catalytic motif of the viral dUTPase (ORF54.CM). The disparate phenotypes observed in the vUNG mutants led us to explore the non-enzymatic properties of vUNG. Immunoprecipitation of vUNG followed by mass spectrometry in MHV68-infected fibroblasts identified a complex comprising the cognate viral DNA polymerase, vPOL, encoded by ORF9, and the viral DNA polymerase processivity factor, vPPF, encoded by ORF59. MHV68 vUNG co-localized with vPOL and vPPF in subnuclear structures consistent with viral replication compartments. In reciprocal co-immunoprecipitations, the vUNG formed a complex with the vPOL and vPPF upon transfection with either factor alone or in combination. Lastly, we determined that key catalytic residues of vUNG are not required for interactions with vPOL and vPPF upon transfection or in the context of infection. We conclude that the vUNG of MHV68 associates with vPOL and vPPF independently of its catalytic activity. IMPORTANCE Gammaherpesviruses encode a uracil-DNA glycosylase (vUNG) that is presumed to excise uracil residues from viral genomes. We previously identified the vUNG enzymatic activity, but not the protein itself, as dispensable for gammaherpesvirus replication in vivo. In this study, we report a non-enzymatic role for the viral UNG of a murine gammaherpesvirus in forming a complex with two key components of the viral DNA replication machinery. Understanding the role of the vUNG in this viral DNA replication complex may inform the development of antiviral drugs that combat gammaherpesvirus-associated cancers.

Amine-recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor

Bacteria possess various receptors that sense different signals and transmit information to enable an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Signals recognized by sensor domains are poorly reflected in overall sequence identity, and therefore, the identification of signals from the amino acid sequence of the sensor alone presents a challenge. Biogenic amines are of great physiological importance for microorganisms and humans. They serve as substrates for aerobic and anaerobic growth and play a role of neurotransmitters and osmoprotectants. Here, we report the identification of a sequence motif that is specific for amine-sensing sensor domains that belong to the Cache superfamily of the most abundant extracellular sensors in prokaryotes. We identified approximately 13,000 sensor histidine kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases from 8,000 bacterial and archaeal species that contain the amine-recognizing motif. The screening of compound libraries and microcalorimetric titrations of selected sensor domains confirmed their ability to specifically bind biogenic amines. Mutants in the amine-binding motif or domains that contain a single mismatch in the binding motif had either no or a largely reduced affinity for amines. We demonstrate that the amine-recognizing domain originated from the universal amino acid-sensing Cache domain, thus providing insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and therefore holds a strong promise to enable the identification of signals stimulating numerous receptors.

The Helix-Loop-Helix motif of human EIF3A regulates translation of proliferative cellular mRNAs.

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5' UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

Short tandem repeats bind transcription factors to tune eukaryotic gene expression.

Short tandem repeats (STRs) are enriched in eukaryotic cis-regulatory elements and alter gene expression, yet how they regulate transcription remains unknown. We found that STRs modulate transcription factor (TF)-DNA affinities and apparent on-rates by about 70-fold by directly binding TF DNA-binding domains, with energetic impacts exceeding many consensus motif mutations. STRs maximize the number of weakly preferred microstates near target sites, thereby increasing TF density, with impacts well predicted by statistical mechanics. Confirming that STRs also affect TF binding in cells, neural networks trained only on in vivo occupancies predicted effects identical to those observed in vitro. Approximately 90% of TFs preferentially bound STRs that need not resemble known motifs, providing a cis-regulatory mechanism to target TFs to genomic sites.

Structure-Based Optimization of the Third Generation Type II Macrocycle TRK Inhibitors with Improved Activity against Solvent-Front, xDFG, and Gatekeeper Mutations.

The solvent-front (SF), gatekeeper, and xDFG motif mutations of tropomyosin receptor kinase (TRK) mediating acquired resistance of larotrectinib and entrectinib represent an unmet clinical need. To date, no effective drugs are being approved to overcome these mutants. Thus, a series of macrocycle compounds were designed and synthesized as new type II TRK inhibitors to combat clinically relevant mutations. The representative compound 10g exhibited excellent potency against wide type TRKA/C, TRKAG595R, TRKAG667C, and TRKAF589L with IC50 values of 5.21, 4.51, 6.77, 1.42, and 6.13 nM, respectively, and a good kinome selectivity against 378 kinases. 10g also strongly suppressed the proliferation of Ba/F3 cells transfected with SF, GK, xDFG, and others (Val to Met) single mutants with IC50 values of 1.43-47.56 nM. Moreover, 10g demonstrated ideal antitumor efficacy in both BaF3-CD74-NTRK1G595R and BaF3-CD74-NTRK1G667C xenograft models. The study provides a promising lead compound for pan-anticancer drug discovery.

Double gene mutations of LRSAM1 and REEP1 and a new REEP1 mutation site found in a patient with amyotrophic lateral sclerosis with subjective paresthesia: A case report

AbstractAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective degeneration of upper and lower motor neurons. Although dyskinesia is the most prominent clinical manifestation of ALS, with an in‐depth understanding of disease pathogenesis and clinical detection, more and more ALS patients are found to have nonmotor symptoms, such as sensory impairment. Genetic testing technology has developed rapidly in recent years. New genes have been proven to be involved in the pathogenesis of ALS. However, according to the existing research evidence, no literature has reported that patients with ALS have leucine‐rich repeats and sterility α mutations in motif 1 (LRSAM1) and receptor expression accessory protein 1 (REEP1). The mutation sites of REEP1 gene have not been reported, and the simultaneous mutations of two genes have not been reported. In the largest human gene mutation frequency database gnomad, the mutation sites of two genes are currently defined as new heterozygous variants with unclear clinical significance. Therefore, this article reports the clinical data of this case to further deepen the clinicians' understanding of the disease, and may provide evidence for further study of the new genotype–phenotype of LRSAM1 and REEP1.

Abstract P2041: Identification Of The Nuclear Localization Signal In RBM20 And Its Function In The Pathogenesis Of Dilated Cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop an aggressive and arrhythmogenic form of dilated cardiomyopathy (DCM). Five of over 900 genetic mutations in RBM20 have recently been validated in animal models. Four of these five mutations are located in the arginine/serine-rich (RS) domain with the fifth in the RNA recognition motif (RRM). Interestingly, only mutations in the RS domain cause severe DCM, which suggests that disruption of RS domain function is crucial for the pathogenesis of DCM. To test this hypothesis, we generated mice expressing RBM20 with an in-frame deletion of the RS domain ( Rbm20 ΔRS ). We show that Rbm20 ΔRS mice manifest DCM with RBM20 mis-localization and granule formation, similar to that in RS domain mutation knock-in (KI) animals. Conversely, mice expressing RBM20 lacking the RRM do not exhibit RBM20 nucleocytoplasmic transport or granule formation, and, similar to RRM mutation KI mice, do not develop DCM. These data suggest that the critical nuclear localization signal (NLS) in RBM20 is located within the RS domain and that disruption of this sequence leads to RBM20 mis-localization and granule formation. In vitro experiments employing sequence deletion plasmids and immunocytochemical staining revealed that the first nine amino acids in the RS domain constitute the core NLS in RBM20, which is further supported by the fact that all three validated DCM-causing mutations are located in this segment. Analysis of plasmids containing DCM-associated mutations in other regions of RBM20, as well as in the non-NLS RS domain sequence, further confirmed that only mutations in the NLS facilitate re-localization and granule formation. Phosphorylation of residues within the NLS has been shown play an important role in protein nucleocytoplasmic transport. To test this, we mutated all phosphorylatable serine residues in the NLS to unphosphorylatable alanine or phosphomimetic aspartate and provide evidence that phosphorylation is dispensable for nucleocytoplasmic transport. Collectively, our findings identify the critical NLS in RBM20 and demonstrate that mutations in this NLS cause severe DCM through disruption of RS domain-mediated nuclear localization and sarcoplasmic granule formation.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knock-in (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We show that Rbm20ΔRS mice manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized to the sarcoplasm in Rbm20ΔRS mice, which led to the formation of RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif (RRM) show similar mis-splicing of RBM20 target genes, but do not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrate that only DCM-associated mutations in the RS domain facilitate RBM20 nucleocytoplasmic transport and promote granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain. Mutation analysis of phosphorylation sites in the RS domain indicate that this modification is dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

Clustering of phosphatase RPTPα promotes Src signaling and the arthritogenic action of synovial fibroblasts.

Receptor-type protein phosphatase α (RPTPα) promotes fibroblast-dependent arthritis and fibrosis, in part, by enhancing the activation of the kinase SRC. Synovial fibroblasts lining joint tissue mediate inflammation and tissue damage, and their infiltration into adjacent tissues promotes disease progression. RPTPα includes an ectodomain and two intracellular catalytic domains (D1 and D2) and, in cancer cells, undergoes inhibitory homodimerization, which is dependent on a D1 wedge motif. Through single-molecule localization and labeled molecule interaction microscopy of migrating synovial fibroblasts, we investigated the role of RPTPα dimerization in the activation of SRC, the migration of synovial fibroblasts, and joint damage in a mouse model of arthritis. RPTPα clustered with other RPTPα and with SRC molecules in the context of actin-rich structures. A known dimerization-impairing mutation in the wedge motif (P210L/P211L) and the deletion of the D2 domain reduced RPTPα-RPTPα clustering; however, it also unexpectedly reduced RPTPα-SRC association. The same mutations also reduced recruitment of RPTPα to actin-rich structures and inhibited SRC activation and cellular migration. An antibody against the RPTPα ectodomain that prevented the clustering of RPTPα also inhibited RPTPα-SRC association and SRC activation and attenuated fibroblast migration and joint damage in arthritic mice. A catalytically inactivating RPTPα-C469S mutation protected mice from arthritis and reduced SRC activation in synovial fibroblasts. We conclude that RPTPα clustering retains it to actin-rich structures to promote SRC-mediated fibroblast migration and can be modulated through the extracellular domain.

G-protein couple receptor (GPER1) plays an important role during ovarian folliculogenesis and early development of the Chinese Alligator

The critical role of the G protein-coupled receptor 1 (GPER1), a member of the seven-transmembrane G protein-coupled receptor family, in the functional regulation of oocytes accumulated abundant theories in the early research on model animals. However, the full-length cDNA encoding GPER1 and its role in the folliculogenesis has not been illustrated in crocodilians. 0.5, 3, and 12 months old Alligator sinensis cDNA samples were used to clone the full-length cDNA encoding GPER1. Immunolocalization and quantitative analysis were performed using Immunofluorescence technique, RT-PCR and Western blot. Simultaneously, studies on GPER1's promoter deletion and cis-acting transcriptional regulation mechanism were conducted. Immunolocalization staining for the germline marker DDX4 and GPER1 demonstrated that DDX4-positive oocytes were clustered tightly together within the nests, whereas scarcely any detectable GPER1 was present in the oocytes nest in Stage I. After that, occasionally GPER1-positive immunosignal was observed in oocytes and somatic cells additional with the primordial follicles, and it was mainly located at the granulosa cells or thecal cells within the early PFs in the Stage III. The single mutation of the putative SP1 motif, double mutating of Ets/SP1 and SP1/CRE binding sites all depressed promoter activities. This result will help to investigate the role of GPER1 in the early folliculogenesis of A. sinensis.

The inner mitochondrial membrane fission protein MTP18 serves as a mitophagy receptor to prevent apoptosis in oral cancer.

MTP18 (also known as MTFP1), an inner mitochondrial membrane protein, plays a vital role in maintaining mitochondrial morphology by regulating mitochondrial fission. Here, we found that MTP18 functions as a mitophagy receptor that targets dysfunctional mitochondria into autophagosomes for elimination. Interestingly, MTP18 interacts with members of the LC3 (also known as MAP1LC3) family through its LC3-interacting region (LIR) to induce mitochondrial autophagy. Mutation in the LIR motif (mLIR) inhibited that interaction, thus suppressing mitophagy. Moreover, Parkin or PINK1 deficiency abrogated mitophagy in MTP18-overexpressing human oral cancer-derived FaDu cells. Upon exposure to the mitochondrial oxidative phosphorylation uncoupler CCCP, MTP18[mLIR]-FaDu cells showed decreased TOM20 levels without affecting COX IV levels. Conversely, loss of Parkin or PINK1 resulted in inhibition of TOM20 and COX IV degradation in MTP18[mLIR]-FaDu cells exposed to CCCP, establishing Parkin-mediated proteasomal degradation of outer mitochondrial membrane as essential for effective mitophagy. We also found that MTP18 provides a survival advantage to oral cancer cells exposed to cellular stress and that inhibition of MTP18-dependent mitophagy induced cell death in oral cancer cells. These findings demonstrate that MTP18 is a novel mitophagy receptor and that MTP18-dependent mitophagy has pathophysiologic implications for oral cancer progression, indicating inhibition of MTP18-mitophagy could thus be a promising cancer therapy strategy.

Functionalized Virus Nanoparticles Alleviates Osteoporosis via Targeting the Function of RANK-Specific Motifs.

Osteoporosis is a common skeletal disease characterized by excessive osteoclast-induced bone loss. RANKL/RANK signaling pathway is essential for osteoclastogenesis and is a key target for osteoporosis. However, regarding the fact that RANKL/RANK also functions beyond bone, the total block of RANKL/RANK will have unwanted impact on other organs. Our previous study revealed that mutation of RANK-specific motifs inhibited osteoclastogenesis without effects on other organs in mice. However, the instability and low cellular uptake efficiency limited the application of the therapeutic peptide originating from the amino acid sequence of RANK-specific motifs (RM). To this end, in this study, the peptide RM (SRPVQEQGGA (C to N terminal)) was chemically modified onto the surface of the plant virus-based nanoparticles cowpea chlorotic mottle virus (CCMV). Subsequent experiments showed that the novel virus nanoparticles RM-CCMV had excellent biocompatibility and stability, which ultimately facilitated its cellular uptake efficiency and improved its inhibitive effects on osteoclastogenesis. Moreover, RM-CCMV achieved bone enrichment and suppressed bone resorption by inhibiting osteoclastogenesis and improving the parameters of bone histomorphology in murine femurs. To be mentioned, the effective dose of CCMV conjugated RM was only 6.25% of free RM. In summary, these results have provided a promising therapeutic strategy for osteoporosis.

Somatic gain-of-function mutations in BUD13 promote oncogenesis by disrupting Fbw7 function.

Somatic mutations occurring on key enzymes are extensively studied and targeted therapies are developed with clinical promises. However, context-dependent enzyme function through distinct substrates complicated targeting a given enzyme. Here, we develop an algorithm to elucidate a new class of somatic mutations occurring on enzyme-recognizing motifs that cancer may hijack to facilitate tumorigenesis. We validate BUD13-R156C and -R230Q mutations evading RSK3-mediated phosphorylation with enhanced oncogenicity in promoting colon cancer growth. Further mechanistic studies reveal BUD13 as an endogenous Fbw7 inhibitor that stabilizes Fbw7 oncogenic substrates, while cancerous BUD13-R156C or -R230Q interferes with Fbw7Cul1 complex formation. We also find this BUD13 regulation plays a critical role in responding to mTOR inhibition, which can be used to guide therapy selections. We hope our studies reveal the landscape of enzyme-recognizing motif mutations with a publicly available resource and provide novel insights for somatic mutations cancer hijacks to promote tumorigenesis with the potential for patient stratification and cancer treatment.

Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin

Proteins containing a CAAX motif at the C-terminus undergo prenylation for localization and activity and include a series of key regulatory proteins, such as RAS superfamily members, heterotrimeric G proteins, nuclear lamina protein, and several protein kinases and phosphatases. However, studies of prenylated proteins in esophageal cancer are limited. Here, through research on large-scale proteomic data of esophageal cancer in our laboratory, we found that paralemmin-2 (PALM2), a potential prenylated protein, was upregulated and associated with poor prognosis in patients. Low-throughput verification showed that the expression of PALM2 in esophageal cancer tissues was higher than that in their paired normal esophageal epithelial tissues, and it was generally expressed in the membrane and cytoplasm of esophageal cancer cells. PALM2 interacted with the two subunits of farnesyl transferase (FTase), FNTA and FNTB. Either the addition of an FTase inhibitor or mutation in the CAAX motif of PALM2 (PALM2C408S) impaired its membranous localization and reduced the membrane location of PALM2, indicating PALM2 was prenylated by FTase. Overexpression of PALM2 enhanced the migration of esophageal squamous cell carcinoma cells, whereas PALM2C408S lost this ability. Mechanistically, PALM2 interacted with the N-terminal FERM domain of ezrin of the ezrin/radixin/moesin (ERM) family. Mutagenesis indicated that lysine residues K253/K254/K262/K263 in ezrin’s FERM domain and C408 in PALM2’s CAAX motif were important for PALM2/ezrin interaction and ezrin activation. Knockout of ezrin prevented enhanced cancer cell migration by PALM2 overexpression. PALM2, depending on its prenylation, increased both ezrin membrane localization and phosphorylation of ezrin at Y146. In summary, prenylated PALM2 enhances the migration of cancer cells by activating ezrin.

PsbZIP1 and PsbZIP10 induce anthocyanin synthesis in plums (Prunus salicina cv. Taoxingli) via PsUFGT by methyl salicylate treatment during postharvest

‘Taoxingli’ plum is a characteristic fruit in Shengzhou, Zhejiang Province, China. It is favored by consumers for its vibrant color and high nutrition, closely related to anthocyanin synthesis and accumulation. Anthocyanin synthase (ANS) and uridine diphosphate flavonoid 3-O-glucosyltransferase (UFGT) are two key enzymes in the downstream portion of anthocyanin synthetic pathways. 100 μM methyl salicylate (MeSA) enhanced the expression of five PsbZIPs (bZIP1, 2, 3, 4, 10), which was positively correlated with the activities of PsANS and PsUFGT, their encoding gene expression, and anthocyanin content. The gene expression validated by qRT-PCR was correspond to FPKM value obtained from the transcriptome data. Yeast one-hybrid assays and motif mutation demonstrated that PsbZIP1 and PsbZIP10 can directly bind to the PsUFGT promoter via the G-box motif (5′-CACGTG-3′). These results indicated that PsbZIP1 and PsbZIP10 triggered anthocyanin synthesis in ‘Taoxingli’ plum fruit by binding to the G-box of the PsUFGT promoter after MeSA treatment during postharvest.

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology.

The U6 promoter, a typical RNA polymerase III promoter, is widely used to transcribe small RNAs in vector-based siRNA systems. The RNAi efficiency is mainly dependent on the transcriptional activity of the U6 promoter. However, studies have found that U6 promoters isolated from some fishes do not work well in distantly related species. To isolate a U6 promoter with high transcriptional efficiency from fish, in this study, we cloned five U6 promoters in orange-spotted grouper, of which only the grouper U6-1 (GU6-1) promoter contains the OCT element in the distant region. Functional studies revealed that the GU6-1 promoter has high transcriptional ability, which could efficiently transcribe shRNA and result in target gene knockdown in vitro and in vivo. Subsequently, the deletion or mutation of the OCT motif resulted in a significant decrease in promoter transcriptional activity, demonstrating that the OCT element plays an important role in enhancing the grouper U6 promoter transcription. Moreover, the transcriptional activity of the GU6-1 promoter showed little species specificity. It not only works in the grouper but also possesses high transcriptional activity in the zebrafish. Knockdown of the mstn gene in zebrafish and grouper through shRNA driven by the GU6-1 promoter could promote fish growth, suggesting that the GU6-1 promoter can be used as a potential molecular tool in aquaculture practice.

Determinants of tumor immune evasion: the role of T cell exposed motif frequency and mutant amino acid exposure.

Few neoepitopes detected in tumor biopsies are immunogenic. Tumor-specific T cell responses require both the presentation of an epitope that differs from wildtype and the presence of T cells with neoepitope-cognate receptors. We show that mutations detected in tumor biopsies result in an increased frequency of rare amino acid combinations compared to the human proteome and gastrointestinal microorganisms. Mutations in a large data set of oncogene and tumor suppressor gene products were compared to wildtype, and to the count of corresponding amino acid motifs in the human proteome and gastrointestinal microbiome. Mutant amino acids in T cell exposed positions of potential neoepitopes consistently generated amino acid motifs that are less common in both proteome reference datasets. Approximately 10% of the mutant amino acid motifs are absent from the human proteome. Motif frequency does not change when mutants were positioned in the MHC anchor positions hidden from T cell receptors. Analysis of neoepitopes in GBM and LUSC cases showed less common T cell exposed motifs, and HLA binding preferentially placing mutant amino acids in an anchor position for both MHC I and MHC II. Cross-presentation of mutant exposed neoepitopes by MHC I and MHC II was particularly uncommon. Review of a tumor mutation dataset known to generate T cell responses showed immunogenic epitopes were those with mutant amino acids exposed to the T cell receptor and with exposed pentamer motifs present in the human and microbiome reference databases. The study illustrates a previously unrecognized mechanism of tumor immune evasion, as rare T cell exposed motifs produced by mutation are less likely to have cognate T cells in the T cell repertoire. The complex interactions of HLA genotype, binding positions, and mutation specific changes in T cell exposed motif underscore the necessity of evaluating potential neoepitopes in each individual patient.