The Fusicoccin story revisited.

In plant biology Fusicoccin (FC) is one of the most studied fungal metabolites to date. Since the structural identification in 1964, much has been learned about its effects on the physiology of plants, about the interference with the action of plant hormones, the molecular nature of the plant receptor(s) for FC and the biosynthetic pathway for FC in the fungus. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase|14-3-3 receptor has taken center stage in explaining all FC induced physiological effects. However, a more critical review shows that this is not at all evident for a number of FC induced effects. Examples of this are: the inhibition of outward rectifying K+-channels in guard cells, the phosphorylation/activation of PEP-carboxylase and malate accumulation, the antagonism with ABA induced production of H2O2 / NO and the effect on ethylene production. In addition, recently two other physiological processes were shown to be targeted by FC, viz. the activation of TORC1 and the interference of FC with the immune response to fungal elicitors. In this review, the notion will be challenged that all FC affected processes start with the binding to and activation of the PM-ATPase and the question is raised whether may be other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule; in analogy to the fungal product and plant hormone gibberellic acid. A relevant question in this respect is whether it is a coincidence that proteins that act in a coordinated fashion during stomatal opening (the ATPases and K+-channels) are targeted by FC? Or are the sites where FC binds in the plant, conserved during evolution because they serve a physiological role, namely the accommodation of a plant produced molecule? In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile to make a renewed attempt with current day improved technology to answer this question and may be upgrade FC or structural analogue(s) to a new level, the level of plant hormone.

Heterochromatin repeat organization at an individual level: Rex1BD and the 14-3-3 protein coordinate to shape the epigenetic landscape within heterochromatin repeats.

In eukaryotic cells, heterochromatin is typically composed of tandem DNA repeats and plays crucial roles in gene expression and genome stability. It has been reported that silencing at individual units within tandem heterochromatin repeats exhibits a position-dependent variation. However, how the heterochromatin is organized at an individual repeat level remains poorly understood. Using a novel genetic approach, our recent study identified a conserved protein Rex1BD required for position-dependent silencing within heterochromatin repeats. We further revealed that Rex1BD interacts with the 14-3-3 protein to regulate heterochromatin silencing by linking RNAi and HDAC pathways. In this review, we discuss how Rex1BD and the 14-3-3 protein coordinate to modulate heterochromatin organization at the individual repeat level, and comment on the biological significance of the position-dependent effect in heterochromatin repeats. We also identify the knowledge gaps that still need to be unveiled in the field.

Slow to Respond: A Rapidly Progressive Case of Sporadic Creutzfeldt-Jakob Disease.

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal neurodegenerative disorder caused by prion proteins. In about 85% of patients, CJD occurs as a sporadic disease with no recognizable pattern of transmission. Sporadic CJD (sCJD) can present with rapid cognitive and functional decline, memory deficits, myoclonus, pyramidal and extrapyramidal signs, and visual deficits. The large spectrum of phenotypic variability has made the recognition of prion diseases difficult, and given the rare incidence, it is not uncommon for it to be missed as a potential diagnosis. We present a highly unusual case of a 76-year-old woman with rapidly progressive sCJD who died within five weeks of presentation. Our case demonstrates a typical sequence of symptoms, with rapidly progressive dementia and cerebellar signs at disease onset and myoclonus later in the disease course.

14-3-3 proteins-a moonlight protein complex with therapeutic potential in neurological disorder: in-depth review with Alzheimer's disease.

Alzheimer's disease (AD) affects millions of people worldwide and is a gradually worsening neurodegenerative condition. The accumulation of abnormal proteins, such as tau and beta-amyloid, in the brain is a hallmark of AD pathology. 14-3-3 proteins have been implicated in AD pathology in several ways. One proposed mechanism is that 14-3-3 proteins interact with tau protein and modulate its phosphorylation, aggregation, and toxicity. Tau is a protein associated with microtubules, playing a role in maintaining the structural integrity of neuronal cytoskeleton. However, in the context of Alzheimer's disease (AD), an abnormal increase in its phosphorylation occurs. This leads to the aggregation of tau into neurofibrillary tangles, which is a distinctive feature of this condition. Studies have shown that 14-3-3 proteins can bind to phosphorylated tau and regulate its function and stability. In addition, 14-3-3 proteins have been shown to interact with beta-amyloid (Aß), the primary component of amyloid plaques in AD. 14-3-3 proteins can regulate the clearance of Aß through the lysosomal degradation pathway by interacting with the lysosomal membrane protein LAMP2A. Dysfunction of lysosomal degradation pathway is thought to contribute to the accumulation of Aß in the brain and the progression of AD. Furthermore, 14-3-3 proteins have been found to be downregulated in the brains of AD patients, suggesting that their dysregulation may contribute to AD pathology. For example, decreased levels of 14-3-3 proteins in cerebrospinal fluid have been suggested as a biomarker for AD. Overall, these findings suggest that 14-3-3 proteins may play an important role in AD pathology and may represent a potential therapeutic target for the disease. However, further research is needed to fully understand the mechanisms underlying the involvement of 14-3-3 proteins in AD and to explore their potential as a therapeutic target.

Calmodulin-Domain Protein Kinase PiCDPK1 Interacts with the 14-3-3-like Protein NtGF14 to Modulate Pollen Tube Growth.

Calcium-mediated signaling pathways are known to play important roles in the polar growth of pollen tubes. The calcium-dependent protein kinase, PiCDPK1, has been shown to be involved in regulating this process through interaction with a guanine dissociation inhibitor, PiRhoGDI1. To more fully understand the role of PiCDPK1 in pollen tube extension, we designed a pull-down study to identify additional substrates of this kinase. These experiments identified 123 putative interactors. Two of the identified proteins were predicted to directly interact with PiCDPK1, and this possibility was investigated in planta. The first, NtGF14, a 14-3-3-like protein, did not produce a noticeable phenotype when overexpressed in pollen alone but partially rescued the spherical tube phenotype caused by PiCDPK1 over-expression when co-over-expressed with the kinase. The second, NtREN1, a GTPase activating protein (GAP), severely inhibited pollen tube germination when over-expressed, and its co-over-expression with PiCDPK1 did not substantially affect this phenotype. These results suggest a novel in vivo interaction between NtGF14 and PiCDPK1 but do not support the direct interaction between PiCDPK1 and NtREN1. We demonstrate the utility of the methodology used to identify potential protein interactions while confirming the necessity of additional studies to confirm their validity. Finally, additional support was found for intersection between PiCDPK1 and RopGTPase pathways to control polar growth at the pollen tube tip.

Unveiling the functional significance of the 14.3.3 protein: A key player in Paracoccidioides brasiliensis biofilm formation.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. The interaction mediated by the presence of adhesins on the fungal surface and receptors in the extracellular matrix of the host, as well as the biofilm formation, is essential in its pathogenesis. Adhesins such as gp43, enolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and 14-3-3 have been demonstrated in the Paracoccidioides brasiliensis (Pb18) strain and recognized as necessary in the fungus-host interaction. The Pb 18 strain silenced to 14-3-3 showed changes in morphology, virulence, and adhesion capacity. The study aimed to evaluate the role of adhesin 14-3-3 in P. brasiliensis biofilm formation and the differential expression of genes related to adhesins, comparing planktonic and biofilm forms. The presence of biofilm was also verified in sutures in vitro and in vivo. The silenced strain (Pb14-3-3 aRNA) was compared with the wild type Pb18, determining the differential metabolic activity between the strains by the XTT reduction assay; the biomass by violet crystal and the polysaccharides by safranin, even as morphological differences by microscopic techniques. Differential gene expression for adhesins was also analyzed, comparing the relative expression of these in planktonic and biofilm forms at different times. The results suggested that the silencing of 14-3-3 protein altered the ability to form biofilm and its metabolism. The quantity of biomass was similar in both strains; however, the formation of exopolymeric substances and polysaccharide material was lower in the silenced strain. Our results showed increased expression of enolase, GAPDH, and 14-3-3 genes in the first periods of biofilm formation in the Pb18 strain. In contrast, the silenced strain showed a lower expression of these genes, indicating that gene silencing can influence the expression of other genes and be involved in the biofilm formation of P. brasiliensis. In vitro and in vivo assays using sutures confirmed this yeast's ability to form biofilm and may be implicated in the pathogenesis of paracoccidioidomycosis.

Rastonia solanacearum type â ¢ effectors target host 14-3-3 proteins to suppress plant immunity.

14-3-3 proteins play important roles in plant metabolism and stress response. Tomato 14-3-3 proteins, SlTFT4 and SlTFT7, serve as hubs of plant immunity and are targeted by some pathogen effectors. Ralstonia solanacearum with more than 70 type Ⅲ effectors (T3Es) is one of the most destructive plant pathogens. However, little is known on whether R. solanacearum T3Es target SlTFT4 and SlTFT7 and hence interfere with plant immunity. We first detected the associations of SlTFT4/SlTFT7 with R. solanacearum T3Es by luciferase complementation assay, and then confirmed the interactions by yeast two-hybrid approach. We demonstrated that 22 Ralstonia T3Es were associated with both SlTFT4 and SlTFT7, and five among them suppressed the hypersensitive response induced by MAPKKKα, a protein kinase which associated with SlTFT4/SlTFT7. We further demonstrated that suppression of MAPKKKα-induced HR and plant basal defense by the T3E RipAC depend on its association with 14-3-3 proteins. Our findings firstly demonstrate that R. solanacearum T3Es can manipulate plant immunity by targeting 14-3-3 proteins, SlTFT4 and SlTFT7, providing new insights into plant-R. solanacearum interactions.

Rapidly Progressive Dementia in an Elderly Male: Perseverance Becomes the Key to a Rare Diagnosis.

Cognitive impairment in a patient with rapidly progressive dementia (RPD) develops faster than expected for a known dementia syndrome. It poses as a diagnostic challenge for the physician who must identify the diagnosis among a broad spectrum of differentials. Here, we discuss the case of a 60-year-old male who presented with a four-month history of progressive gait disturbance, incoherent talking, dysarthria, hand tremors, and new-onset bladder incontinence. Neurological examination revealed fast saccades, cerebellar dysarthria, hypertonia, and normal power in all four limbs, brisk reflexes, past pointing, intentional tremors, resting myoclonic jerks, and ataxic gait. Initial differentials of progressive paraneoplastic encephalitis, infectious encephalitis, and toxic encephalopathy were considered. However, the results of lumbar puncture and blood investigations-voltage-gated potassium channel (VGKC) antibody and N-methyl-D-aspartate (NMDA) receptor antibody, tumor markers, viral markers being negative and ammonia and lactate levels being normal led us to think of another possibility. With such rapidly progressive dementia, myoclonic jerks, extrapyramidal signs, and cerebellar signs, a diagnosis of Creutzfeldt-Jakob disease (CJD) was taken into consideration. A cerebrospinal fluid (CSF) sample was sent for CSF protein 14-3-3 quantification by enzyme-linked immunosorbent assay (ELISA) and came out positive. During his stay in the hospital, our patient developed multiple complications, and his clinical state progressively worsened. With no signs of improvement and the known fatal nature of the disease, the goals of care were discussed with the family and we all agreed on providing palliative care. The patient passed away on day 15 of hospital admission.

Biofilm formation initiating rotifer-specific biopolymer and its predicted components.

The rotifer-specific biopolymer, namely Rotimer, is a recently discovered group of the biomolecule family. Rotimer has an active role in the biofilm formation initiated by rotifers (e.g., Euchlanis dilatata or Adineta vaga) or in the female-male sexual interaction of monogononts. To understand the Ca2+- and polarity-dependent formation of this multifunctional viscoelastic material, it is essential to explore its molecular composition. The investigation of the rotifer-enhanced biofilm and Rotimer-inductor conglomerate (RIC) formation yielded several protein candidates to predict the Rotimer-specific main components. The exudate of E. dilatata males was primarily applied from different biopolimer-containing samples (biofilm or RIC). The advantage of males over females lies in their degenerated digestive system and simple anatomy. Thus, their exudate is less contaminated with food and endosymbiont elements. The sequenced and annotated genome and transcriptome of this species opened the way for identifying Rotimer proteins by mass spectrometry. The predicted rotifer-biopolymer forming components are SCO-spondins and 14-3-3 protein. The characteristics of Rotimer are similar to Reissner's fiber, which is found in the central nervous system of vertebrates and is mainly formed from SCO-spondins. This molecular information serves as a starting point for its interdisciplinary investigation and application in biotechnology, biomedicine, or neurodegeneration-related drug development.

Assessment of the Immunoprotective Efficacy of Recombinant 14-3-3 Protein and Dense Granule Protein 10 (GRA10) as Candidate Antigens for Rabbit Vaccines against Eimeria intestinalis.

Eimeria intestinalis infects rabbits, causing severe intestinal coccidiosis. Prolonged anticoccidial drug use might lead to coccidia resistance and drug residues in food. Thus, vaccines are required to control rabbit coccidiosis. In this study, recombinant E. intestinalis 14-3-3 and GRA10 proteins (rEi-14-3-3 and rEi-GRA10) were obtained via prokaryotic expression and used as recombinant subunit vaccines. Fifty 30-day-old rabbits were randomly grouped as follows: PBS-uninfected group, PBS-infected group, Trx-His-S control group, and rEi-14-3-3 and rEi-GRA10 immunized groups. The rabbits were subcutaneously immunized twice at 2-week intervals, challenged with 7 × 104 sporulated oocysts, and sacrificed 14 days later. The protective effects were assessed via clinical signs, relative weight gain, oocyst reduction, mean intestinal lesion score, ACI (anticoccidial index), cytokine, and specific antibody levels in sera. The rEi-14-3-3 and rEi-GRA10 groups had higher relative weight gain rates of 81.94% and 73.61% (p < 0.05), and higher oocyst reduction rates of 86.13% and 84.87% (p < 0.05), respectively. The two immunized groups had fewer intestinal lesions (p < 0.05) and higher IgG levels (p < 0.05). Higher levels of IL-2, IL-4, and IFN-γ cytokines in the rEi-14-3-3 group (p < 0.05) and a higher level of IFN-γ in the rEi-GRA10 group (p < 0.05) were observed. The ACI values of the rEi-14-3-3 and rEi-GRA10 groups were 168.24 and 159.91, with good and moderate protective effects, respectively. Both rEi-14-3-3 and rEi-GRA10 induced humoral immunity in the rabbits. In addition, rEi-14-3-3 induced Th1- and Th2-type immune responses. Both recombinant proteins were protective against E. intestinalis infection in rabbits, with rEi-14-3-3 showing a better protective effect.

14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte.

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3ß interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, the precise localization and differential role of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3ß in glomeruli was restricted in podocytes, and 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. Although 14-3-3ß was dominantly co-localized with FKBP12 in the foot processes, a part of 14-3-3ß was co-localized with Par3 at the slit diaphragm. 14-3-3ß interacted with Par3, and FKBP12 bound to 14-3-3ß competitively with Par3. Deletion of 14-3-3ß enhanced the interaction of Par3 with Par6 in podocytes. Gene silencing for 14-3-3ß altered the structure of actin fibers and process formation. 14-3-3ß and synaptopodin expression was decreased in podocyte injury models. In contrast, 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and synaptopodin. Gene silencing for 14-3-3σ altered the structure of vimentin fibers and process formation. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury models but was decreased in the late stage. Together, the localization of 14-3-3ß at actin cytoskeleton plays a role in maintaining the foot processes and the Par complex in podocytes. In contrast, 14-3-3σ at vimentin cytoskeleton is essential for maintaining primary processes.

Ectopic over-expression of HaFT-1, a 14-3-3 protein from Haloxylon ammodendron, enhances acquired thermotolerance in transgenic Arabidopsis.

Haloxylon ammodendron, an important shrub utilized for afforestation in desert areas, can withstand harsh ecological conditions such as drought, high salt and extreme heat. A better understanding of the stress adaptation mechanisms of H. ammodendron is vital for ecological improvement in desert areas. In this study, the role of the H. ammodendron 14-3-3 protein HaFT-1 in thermotolerance was investigated. qRT-PCR analysis showed that heat stress (HS) priming (the first HS) enhanced the expression of HaFT-1 during the second HS and subsequent recovery phase. The subcellular localization of YFP-HaFT-1 fusion protein was mainly detected in cytoplasm. HaFT-1 overexpression increased the germination rate of transgenic Arabidopsis seeds, and the survival rate of HaFT-1 overexpression seedlings was higher than that of wild-type (WT) Arabidopsis after priming-and-triggering and non-primed control treatments. Cell death staining showed that HaFT-1 overexpression lines exhibited significantly reduced cell death during HS compared to WT. Transcriptome analysis showed that genes associated with energy generation, protein metabolism, proline metabolism, autophagy, chlorophyll metabolism and reactive oxygen species (ROS) scavenging were important to the thermotolerance of HS-primed HaFT-1 transgenic plants. Growth physiology analysis indicated that priming-and-triggering treatment of Arabidopsis seedlings overexpressing HaFT-1 increased proline content and strengthened ROS scavenging activity. These results demonstrated that overexpression of HaFT-1 increased not only HS priming but also tolerance to the second HS of transgenic Arabidopsis, suggesting that HaFT-1 is a positive regulator in acquired thermotolerance.

Molecular basis and dual ligand regulation of tetrameric estrogen receptor α/14-3-3ζ protein complex.

Therapeutic strategies targeting nuclear receptors (NRs) beyond their endogenous ligand binding pocket have gained significant scientific interest driven by a need to circumvent problems associated with drug resistance and pharmacological profile. The hub protein 14-3-3 is an endogenous regulator of various NRs, providing a novel entry point for small molecule modulation of NR activity. Exemplified, 14-3-3 binding to the C-terminal F-domain of the estrogen receptor alpha (ERα), and small molecule stabilization of the ERα/14-3-3ζ protein complex by the natural product Fusicoccin A (FC-A), was demonstrated to downregulate ERα-mediated breast cancer proliferation. This presents a novel drug discovery approach to target ERα; however, structural and mechanistic insights into ERα/14-3-3 complex formation are lacking. Here, we provide an in-depth molecular understanding of the ERα/14-3-3ζ complex by isolating 14-3-3ζ in complex with an ERα protein construct comprising its ligand-binding domain (LBD) and phosphorylated F-domain. Bacterial co-expression and co-purification of the ERα/14-3-3ζ complex, followed by extensive biophysical and structural characterization, revealed a tetrameric complex between the ERα homodimer and the 14-3-3ζ homodimer. 14-3-3ζ binding to ERα, and ERα/14-3-3ζ complex stabilization by FC-A, appeared to be orthogonal to ERα endogenous agonist (E2) binding, E2-induced conformational changes, and cofactor recruitment. Similarly, the ERα antagonist 4-hydroxytamoxifen inhibited cofactor recruitment to the ERα LBD while ERα was bound to 14-3-3ζ. Furthermore, stabilization of the ERα/14-3-3ζ protein complex by FC-A was not influenced by the disease-associated and 4-hydroxytamoxifen resistant ERα-Y537S mutant. Together, these molecular and mechanistic insights provide direction for targeting ERα via the ERα/14-3-3 complex as an alternative drug discovery approach.

The Cm14-3-3µ protein and CCT transcription factor CmNRRa delay flowering in chrysanthemum.

The floral transition from vegetative to reproductive growth is pivotal in the plant life cycle. NUTRITION RESPONSE AND ROOT GROWTH (OsNRRa), as a CONSTANS, CONSTANS-LIKE, TOC1 (CCT) domain protein, delays flowering in rice, and an orthologous protein, CmNRRa, inhibits flowering in chrysanthemum; however, the underlying mechanism remains unknown. In this study, using yeast two-hybrid screening, we identified the 14-3-3 protein family member Cm14-3-3µ as a CmNRRa-interacting protein. A combination of bimolecular fluorescence complementation, pull-down, and co-immunoprecipitation assays was performed to confirm the physical interaction between CmNRRa and Cm14-3-3µ. In addition, expression analysis showed that CmNRRa but not Cm14-3-3µ responded to the diurnal rhythm, whereas both genes were highly expressed in leaves. Moreover, the function of Cm14-3-3µ in flowering time regulation was similar to that of CmNRRa. Furthermore, CmNRRa repressed chrysanthemum FLOWERING LOCUS T-like 3 (CmFTL3) and an APETALA 1 (AP1)/FRUITFULL (FUL)-like gene (CmAFL1) but induced TERMINAL FLOWER1 (CmTFL1) directly by binding to their promoters. Cm14-3-3µ enhanced the ability of CmNRRa to regulate the expression of these genes. These findings suggest that there is a synergistic relationship between CmNRRa and Cm14-3-3µ in flowering repression in chrysanthemum.

The role of 14-3-3 in the progression of vascular inflammation induced by lipopolysaccharide.

OBJECTIVE: To explore the role of 14-3-3 protein and the Hippo and yes-associated protein 1 (YAP) signaling pathway in lipopolysaccharide (LPS)-induced vascular inflammation. METHODS: Human umbilical vein endothelial cells (HUVECs) and C57B6 mice were treated with LPS to establish cell and animal models of vascular inflammation. Lentiviral transfection, Western blot, qPCR, immunofluorescence, immunohistochemistry, co-immunoprecipitation, and enzyme-linked immunosorbent assays were used to measure inflammatory factors and expression of 14-3-3 protein and phosphorylation of YAP at S127. HUVECs and C57B6 mice were pretreated with a YAP inhibitor, Verteporfin, to observe changes in YAP expression and downstream vascular inflammation. RESULTS: LPS induced acute and chronic inflammatory responses in HUVECs and mice and upregulated the expression of several inflammatory factors. LPS also induced expression of 14-3-3 protein and phosphorylation of YAP at S127 in response to acute vascular inflammation and downregulated these markers in response to chronic vascular inflammation. Verteporfin reduced these LPS-induced effects on vascular inflammation. CONCLUSION: In chronic vascular inflammation, 14-3-3 protein is downregulated, which promotes inflammation by increasing Hippo/YAP nuclear translocation.

Clinical Correlates of Cerebrospinal Fluid 14-3-3 Protein in Non-Prion Rapid Progressive Dementia.

BACKGROUND: The 14-3-3 protein in cerebrospinal fluid (CSF) is a suitable biomarker for the diagnosis of Creutzfeldt-Jakob disease (CJD). However, it has also been detected in various non-prion-related rapidly progressive dementia (RPD), which affected its diagnostic performance and clinical utilization. OBJECTIVE: To investigate the general disease distribution with positive 14-3-3 result and to evaluate the association between CSF 14-3-3 protein and the clinical features in patients with non-prion RPD. METHODS: A total of 150 patients with non-prion RPD were enrolled. The clinical data were collected and CSF 14-3-3 test was performed for all patients. The distribution of various diseases with a positive 14-3-3 result was analyzed and the association of CSF 14-3-3 with clinical features was tested. RESULTS: The CSF 14-3-3 protein was detected in 23.3% of non-prion RPD patients, and the most frequent diagnoses were autoimmune encephalitis (22.9%) and neurodegenerative disease (22.9%). CSF 14-3-3 protein was more common in older patients (p = 0.028) and those presenting myoclonus (p = 0.008). In subgroup analysis, the positive 14-3-3 test was more common in neurodegenerative disease with a long time from the symptom onset to CSF 14-3-3 test (p = 0.014). CONCLUSION: CSF 14-3-3 protein could be detected in a broad spectrum of non-prion RPD. In particular, patients with autoimmune encephalitis and rapidly progressive neurodegenerative diseases and those with myoclonus have a greater likelihood of a positive 14-3-3 result. These results could help clinicians interpret the results of CSF 14-3-3 protein more reasonably.

Phytophthora infestans RxLR Effector PITG06478 Hijacks 14-3-3 to Suppress PMA Activity Leading to Necrotrophic Cell Death.

Pathogens often induce cell death for their successful proliferation in the host plant. Plasma membrane H+-ATPases (PMAs) are targeted by either pathogens or plant immune receptors in immune response regulation. Although PMAs play pivotal roles in host cell death, the molecular mechanism of effector-mediated regulation of PMA activity has not been described. Here, we report that the Phytophthora infestans RxLR effector PITG06478 can induce cell death in Nicotiana benthamiana but the induced cell death is inhibited by fusicoccin (FC), an irreversible PMA activator. PITG06478, which is localized at the plasma membrane, is not directly associated with the PMA but is associated with Nb14-3-3s, a PMA activator. Immunoblot analyses revealed that the interaction between PITG06478 and Nb14-3-3s was disrupted by FC. PMA activity in PITG06478-expressing plants was eventually inhibited, and cell death likely occurred because the 14-3-3 protein was hijacked. Our results further confirm the significance of PMA activity in host cell death and provide new insight into how pathogens utilize essential host components to sustain their life cycle. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The 14-3-3γ isoform binds to and regulates the localization of endoplasmic reticulum (ER) membrane protein TMCC3 for the reticular network of the ER.

The reticular network of the endoplasmic reticulum (ER) is formed by connecting ER tubules through three-way junctions and undergoes constant remodeling through formation and loss of the three-way junctions. Transmembrane and coiled-coil domain family 3 (TMCC3), an ER membrane protein localizing at three-way junctions, has been shown to positively regulate formation of the reticular ER network. However, elements that negatively regulate TMCC3 localization have not been characterized. In this study, we report that 14-3-3γ, a phospho-serine/phospho-threonine-binding protein involved in various signal transduction pathways, is a negative regulator of TMCC3. We demonstrate that overexpression of 14-3-3γ reduced localization of TMCC3 to three-way junctions and decreased the number of three-way junctions. TMCC3 bound to 14-3-3γ through the N terminus and had deduced 14-3-3 binding motifs. Additionally, we determined that a TMCC3 mutant substituting alanine for serine to be phosphorylated in the binding motif reduced binding to 14-3-3γ. The TMCC3 mutant was more prone than wildtype TMCC3 to localize at three-way junctions in the cells overexpressing 14-3-3γ. Furthermore, the TMCC3 mutant rescued the ER sheet expansion caused by TMCC3 knockdown less than wild-type TMCC3. Taken together, these results indicate that 14-3-3γ binding negatively regulates localization of TMCC3 to the three-way junctions for the proper reticular ER network, implying that the negative regulation of TMCC3 by 14-3-3γ would underlie remodeling of the reticular network of the ER.

Human 14-3-3 Proteins Site-selectively Bind the Mutational Hotspot Region of SARS-CoV-2 Nucleoprotein Modulating its Phosphoregulation.

Phosphorylation of SARS-CoV-2 nucleoprotein recruits human cytosolic 14-3-3 proteins playing a well-recognized role in replication of many viruses. Here we use genetic code expansion to demonstrate that 14-3-3 binding is triggered by phosphorylation of SARS-CoV-2 nucleoprotein at either of two pseudo-repeats centered at Ser197 and Thr205. According to fluorescence anisotropy measurements, the pT205-motif,presentin SARS-CoV-2 but not in SARS-CoV, is preferred over the pS197-motif by all seven human 14-3-3 isoforms, which collectively display an unforeseen pT205/pS197 peptide binding selectivity hierarchy. Crystal structures demonstrate that pS197 and pT205 are mutually exclusive 14-3-3-binding sites, whereas SAXS and biochemical data obtained on the full protein-protein complex indicate that 14-3-3 binding occludes the Ser/Arg-rich region of the nucleoprotein, inhibiting its dephosphorylation. This Ser/Arg-rich region is highly prone to mutations, as exemplified by the Omicron and Delta variants, with our data suggesting that the strength of 14-3-3/nucleoprotein interaction can be linked with the replicative fitness of the virus.

Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF's E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ's ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ's interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF's E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.