Clinical outcomes of single blastocyst transfer with machine learning guided noninvasive chromosome screening grading system in infertile patients.

BACKGROUND: Prospective observational studies have demonstrated that the machine learning (ML) -guided noninvasive chromosome screening (NICS) grading system, which we called the noninvasive chromosome screening-artificial intelligence (NICS-AI) grading system, can be used embryo selection. The current prospective interventional clinical study was conducted to investigate whether this NICS-AI grading system can be used as a powerful tool for embryo selection. METHODS: Patients who visited our centre between October 2018 and December 2021 were recruited. Grade A and B embryos with a high probability of euploidy were transferred in the NICS group. The patients in the control group selected the embryos according to the traditional morphological grading. Finally, 90 patients in the NICS group and 161 patients in the control group were compared statistically for their clinical outcomes. RESULTS: In the NICS group, the clinical pregnancy rate (70.0% vs. 54.0%, p < 0.001), the ongoing pregnancy rate (58.9% vs. 44.7%, p = 0.001), and the live birth rate (56.7% vs. 42.9%, p = 0.001) were significantly higher than those of the control group. When the female was ≥ 35 years old, the clinical pregnancy rate (67.7% vs. 32.1%, p < 0.001), ongoing pregnancy rate (56.5% vs. 25.0%, p = 0.001), and live birth rate (54.8% vs. 25.0%, p = 0.001) in the NICS group were significantly higher than those of the control group. Regardless of whether the patients had a previous record of early spontaneous abortion or not, the live birth rate of the NICS group was higher than that of the control group (61.0% vs. 46.9%; 57.9% vs. 34.8%; 33.3% vs. 0%) but the differences were not statistically significant. CONCLUSIONS: NICS-AI was able to improve embryo utilisation rate, and the live birth rate, especially for those ≥ 35 years old, with transfer of Grade A embryos being preferred, followed by Grade B embryos. NICS-AI can be used as an effective tool for embryo selection in the future.

aPC/PAR1 confers endothelial anti-apoptotic activity via a discrete, ß-arrestin-2-mediated SphK1-S1PR1-Akt signaling axis.

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via ß-arrestin-2 (ß-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a ß-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates ß-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on ß-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-ß-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, ß-arr2-driven signaling pathways in caveolae.

CenterADNet: Infrared Video Target Detection Based on Central Point Regression.

Infrared video target detection is a fundamental technology within infrared warning and tracking systems. In long-distance infrared remote sensing images, targets often manifest as circular spots or even single points. Due to the weak and similar characteristics of the target to the background noise, the intelligent detection of these targets is extremely complex. Existing deep learning-based methods are affected by the downsampling of image features by convolutional neural networks, causing the features of small targets to almost disappear. So, we propose a new infrared video weak-target detection network based on central point regression. We focus on suppressing the image background by fusing the different features between consecutive frames with the original image features to eliminate the background's influence. We also employ high-resolution feature preservation and incorporate a spatial-temporal attention module into the network to capture as many target features as possible and improve detection accuracy. Our method achieves superior results on the infrared image weak aircraft target detection dataset proposed by the National University of Defense Technology, as well as on the simulated dataset generated based on real-world observation. This demonstrates the efficiency of our approach for detecting weak point targets in infrared continuous images.

The α-arrestin ARRDC3 suppresses breast carcinoma invasion by regulating G protein-coupled receptor lysosomal sorting and signaling.

Aberrant G protein-coupled receptor (GPCR) expression and activation has been linked to tumor initiation, progression, invasion, and metastasis. However, compared with other cancer drivers, the exploitation of GPCRs as potential therapeutic targets has been largely ignored, despite the fact that GPCRs are highly druggable. Therefore, to advance the potential status of GPCRs as therapeutic targets, it is important to understand how GPCRs function together with other cancer drivers during tumor progression. We now report that the α-arrestin domain-containing protein-3 (ARRDC3) acts as a tumor suppressor in part by controlling signaling and trafficking of the GPCR, protease-activated receptor-1 (PAR1). In a series of highly invasive basal-like breast carcinomas, we found that expression of ARRDC3 is suppressed whereas PAR1 is aberrantly overexpressed because of defective lysosomal sorting that results in persistent signaling. Using a lentiviral doxycycline-inducible system, we demonstrate that re-expression of ARRDC3 in invasive breast carcinoma is sufficient to restore normal PAR1 trafficking through the ALG-interacting protein X (ALIX)-dependent lysosomal degradative pathway. We also show that ARRDC3 re-expression attenuates PAR1-stimulated persistent signaling of c-Jun N-terminal kinase (JNK) in invasive breast cancer. Remarkably, restoration of ARRDC3 expression significantly reduced activated PAR1-induced breast carcinoma invasion, which was also dependent on JNK signaling. These findings are the first to identify a critical link between the tumor suppressor ARRDC3 and regulation of GPCR trafficking and signaling in breast cancer.

Insulin Receptor Antibody-α-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts.

Mucopolysaccharidosis Type IIIB (MPSIIIB) is caused by mutations in the gene encoding the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU). MPSIIIB presents with severe disease of the central nervous system, but intravenous NAGLU enzyme replacement therapy has not been developed because the NAGLU enzyme does not cross the blood-brain barrier (BBB). A BBB-penetrating form of the enzyme was produced by re-engineering NAGLU as an IgG-enzyme fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb traverses the BBB via transport on the endogenous insulin receptor and acts as a molecular Trojan horse to ferry the fused NAGLU across the BBB from blood. The NAGLU was fused to the carboxyl terminus of each heavy chain of the HIRMAb via an extended 31-amino acid linker, and the fusion protein is designated HIRMAb-LL-NAGLU. The fusion protein retains high affinity binding to the HIR, and on a molar basis has an enzyme activity equal to that of recombinant human NAGLU. Treatment of MPSIIIB fibroblasts with the fusion protein normalizes intracellular NAGLU enzyme activity and reduces sulfate incorporation into intracellular glycosoaminoglycan. The fusion protein is targeted to the lysosomal compartment of the cells as shown by confocal microscopy. The fusion protein was radiolabeled with the [(125)I]-Bolton-Hunter reagent and injected intravenously in the adult Rhesus monkey. The fusion protein was rapidly cleared from plasma by all major peripheral organs. The high brain uptake of the fusion protein, 1% injected dose/brain, enables normalization of brain NAGLU enzyme activity with a therapeutic dose of 1 mg/kg. The HIRMAb-LL-NAGLU fusion protein is a new treatment of the brain in MPSIIIB, which can be administered by noninvasive intravenous infusion.

Cofactoring and dimerization of proteinase-activated receptors.

Proteinase-activated receptors (PARs) are G protein-coupled receptors that transmit cellular responses to extracellular proteases and have important functions in vascular physiology, development, inflammation, and cancer progression. The established paradigm for PAR activation involves proteolytic cleavage of the extracellular N terminus, which reveals a new N terminus that functions as a tethered ligand by binding intramolecularly to the receptor to trigger transmembrane signaling. Most cells express more than one PAR, which can influence the mode of PAR activation and signaling. Clear examples include murine PAR3 cofactoring of PAR4 and transactivation of PAR2 by PAR1. Thrombin binds to and cleaves murine PAR3, which facilitates PAR4 cleavage and activation. This process is essential for thrombin signaling and platelet activation, since murine PAR3 cannot signal alone. Although PAR1 and PAR4 are both competent to signal, PAR1 is able to act as a cofactor for PAR4, facilitating more rapid cleavage and activation by thrombin. PAR1 can also facilitate PAR2 activation through a different mechanism. Cleavage of the PAR1 N terminus by thrombin generates a tethered ligand domain that can bind intermolecularly to PAR2 to activate signaling. Thus, PARs can regulate each other's activity by localizing thrombin when in complex with PAR3 and PAR4 or by cleaved PAR1, providing its tethered ligand domain for PAR2 activation. The ability of PARs to cofactor or transactivate other PARs would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer. Here, we discuss the cofactoring and dimerization of PARs and the functional consequences on signaling.

Transactivation of the PAR1-PAR2 heterodimer by thrombin elicits ß-arrestin-mediated endosomal signaling.

Thrombin cleaves the N terminus of PAR1, generating a new N-terminal domain that functions as a tethered ligand that binds intermolecularly to activate PAR2 in trans. The mechanisms that regulate PAR1-PAR2 heterodimer signaling and trafficking are not known. We now report that PAR1 and PAR2 form a heterodimer that exhibits unique trafficking and signaling behaviors compared with receptor protomers. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, co-immunoprecipitation, and cells expressing receptors exogenously and endogenously, we show that PAR1 and PAR2 specifically interact and form stable dimers. Intriguingly, the PAR1-PAR2 heterodimer displays constitutive internalization that is driven by PAR1 C-terminal tail sorting motifs and is a process that enhances dimer formation. Upon thrombin activation, PAR1-PAR2 dimers co-internalize and recruit ß-arrestins to endosomes. Remarkably, PAR1-PAR2 heterodimers appear to utilize a distinct interface for ß-arrestin interaction compared with receptor protomers. Moreover, thrombin-activated PAR1-PAR2 heterodimers enhance ß-arrestin-mediated ERK1/2 activation in the cytoplasm, whereas activated ERK1/2 induced by the thrombin-activated PAR1 protomer redistributes to the nucleus. Thus, the formation of PAR1-PAR2 heterodimers provides additional modes of thrombin-stimulated signaling responses that appear to be distinctly regulated compared with the receptor protomer.

Genetic and biochemical analysis of high iron toxicity in yeast: iron toxicity is due to the accumulation of cytosolic iron and occurs under both aerobic and anaerobic conditions.

Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity.

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity.

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865-33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal- binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains.

Bi-functional IgG-lysosomal enzyme fusion proteins for brain drug delivery.

Most lysosomal storage disorders affect the central nervous system. However, lysosomal enzymes do not cross the blood-brain barrier (BBB), and intravenous enzyme infusion is not effective for the brain. Lysosomal enzymes can be re-engineered for BBB transport as IgG-enzyme fusion proteins, where the IgG domain is a monoclonal antibody (MAb) against an endogenous BBB receptor/transporter, and which acts as a molecular Trojan horse to deliver the enzyme to brain. However, the problem is retention of high enzyme activity following enzyme fusion to the IgG. The present investigation shows this is possible with a versatile approach that employs fusion of the enzyme to either the IgG heavy chain or light chain using a long flexible linker. The model IgG is a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The enzyme activity of the HIRMAb-enzyme fusion protein is preserved for hexosaminidase A, which is mutated in Tay Sachs disease, for protein palmitoylthioesterase-1, which is mutated in Batten disease type 1, acid sphingomyelinase, which is mutated in Niemann Pick disease type A, and beta galactosidase-1, which is mutated in GM1 gangliosidosis.

Association Between Antipsychotic Agents and Risk of Acute Respiratory Failure in Patients With Chronic Obstructive Pulmonary Disease.

Importance: Acute respiratory failure (ARF) is a life-threatening event that has been linked in case reports to antipsychotic use, but this association lacks population-based evidence. Particular attention should be focused on patients with chronic obstructive pulmonary disease (COPD) regarding this drug safety concern because these patients are prone to ARF and are commonly treated with antipsychotics. Objective: To determine whether the use of antipsychotics is associated with an increased risk of ARF in patients with COPD. Design, Setting, and Participants: A population-based case-crossover study analyzing the Taiwan National Health Insurance Research Database was conducted of all patients with COPD, who were newly diagnosed with ARF in hospital or emergency care settings necessitating intubation or mechanical ventilation from January 1, 2000, to December 31, 2011. Patients with prior ARF, lung cancer, and cardiogenic, traumatic, or septic ARF were excluded to analyze idiopathic ARF. The pilot study was conducted from November 1 to December 31, 2013, and full data analysis was performed from October 15, 2015, to November 8, 2016. Exposures: The use of antipsychotics was self-compared during days 1 to 14 (the risk period according to previous case reports) and days 75 to 88 (control period) preceding the ARF event or index date. The antipsychotic class, route of administration, and dose were also examined. Main Outcomes and Measures: Risk of ARF. Results: There were 5032 patients with ARF (mean [SD] age, 74.4 [9.9] years; 3533 males [70.2%]) among the 61 620 patients with COPD. Five hundred ninety patients with ARF (11.7%) filled at least 1 antipsychotic prescription during the case period compared with 443 (8.8%) during the control period, corresponding to a 1.66-fold (95% CI, 1.34-2.05; P < .001) adjusted increased risk of ARF regardless of antipsychotic class and administration route. A dose-dependent risk of ARF associated with antipsychotics was identified (test for trend, adjusted odds ratio, 1.35; 95% CI, 1.19-1.52; P < .001), which increased from a 1.52-fold risk for a low daily dose (95% CI, 1.20-1.92; P < .001) to a 3.74-fold risk for a high dose (95% CI, 1.68-8.36; P = .001). The increased risk persisted under a case-time-control analysis (adjusted odds ratio, 1.62; 95% CI, 1.16-2.27; P = .005) and nested case-control study (adjusted odds ratio, 2.16; 95% CI, 1.91-2.15; P < .001). Conclusions and Relevance: Antipsychotic use is associated with an acute and dose-dependent increased risk of ARF in patients with COPD. Clinicians should exercise caution when prescribing antipsychotics to patients with COPD and avoid high doses if possible.

The α-arrestin ARRDC3 mediates ALIX ubiquitination and G protein-coupled receptor lysosomal sorting.

The sorting of G protein-coupled receptors (GPCRs) to lysosomes is critical for proper signaling and cellular responses. We previously showed that the adaptor protein ALIX regulates lysosomal degradation of protease-activated receptor-1 (PAR1), a GPCR for thrombin, independent of ubiquitin-binding ESCRTs and receptor ubiquitination. However, the mechanisms that regulate ALIX function during PAR1 lysosomal sorting are not known. Here we show that the mammalian α-arrestin arrestin domain-containing protein-3 (ARRDC3) regulates ALIX function in GPCR sorting via ubiquitination. ARRDC3 colocalizes with ALIX and is required for PAR1 sorting at late endosomes and degradation. Depletion of ARRDC3 by small interfering RNA disrupts ALIX interaction with activated PAR1 and the CHMP4B ESCRT-III subunit, suggesting that ARRDC3 regulates ALIX activity. We found that ARRDC3 is required for ALIX ubiquitination induced by activation of PAR1. A screen of nine mammalian NEDD4-family E3 ubiquitin ligases revealed a critical role for WWP2. WWP2 interacts with ARRDC3 and not ALIX. Depletion of WWP2 inhibited ALIX ubiquitination and blocked ALIX interaction with activated PAR1 and CHMP4B. These findings demonstrate a new role for the α-arrestin ARRDC3 and the E3 ubiquitin ligase WWP2 in regulation of ALIX ubiquitination and lysosomal sorting of GPCRs.

Endosomal signaling by protease-activated receptors.

Protease-activated receptors (PARs) are a family of G protein-coupled receptors (GPCRs) that are uniquely activated by proteolysis. There are four members of the PAR family including: PAR1, PAR2, PAR3, and PAR4. PARs are expressed primarily in the cells of the vasculature and elicit cellular responses to coagulant and anticoagulant proteases. PAR1 exemplifies the unusual proteolytic mechanism of receptor activation. Thrombin binds to and cleaves the N-terminal exodomain of PAR1, generating a new N-terminus that functions as a tethered ligand. The N-terminal tethered ligand domain of PAR1 binds intramolecularly to the receptor to trigger transmembrane signaling and cannot diffuse away. Similar to other GPCRs, activation of PARs promotes coupling to heterotrimeric G proteins at the plasma membrane. After activation, PARs are rapidly internalized to endosomes and then sorted to lysosomes and degraded. Internalization functions to uncouple PARs from heterotrimeric G proteins at the cell surface. However, recent studies indicate that activated internalized PARs signal from endosomes through the recruitment of ß-arrestins and potentially other pathways. Here, we provide an overview of methods and strategies used to examine endosomal signaling by PARs.

AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway.

The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail-localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.

ALIX binds a YPX(3)L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting.

The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.

A single amino acid change in the yeast vacuolar metal transporters ZRC1 and COT1 alters their substrate specificity.

Iron is an essential nutrient but in excess may damage cells by generating reactive oxygen species due to Fenton reaction or by substituting for other transition metals in essential proteins. The budding yeast Saccharomyces cerevisiae detoxifies cytosolic iron by storage in the vacuole. Deletion of CCC1, which encodes the vacuolar iron importer, results in high iron sensitivity due to increased cytosolic iron. We selected mutants that permitted Deltaccc1 cells to grow under high iron conditions by UV mutagenesis. We identified a mutation (N44I) in the vacuolar zinc transporter ZRC1 that changed the substrate specificity of the transporter from zinc to iron. COT1, a vacuolar zinc and cobalt transporter, is a homologue of ZRC1 and both are members of the cation diffusion facilitator family. Mutation of the homologous amino acid (N45I) in COT1 results in an increased ability to transport iron and decreased ability to transport cobalt. These mutations are within the second hydrophobic domain of the transporters and show the essential nature of this domain in the specificity of metal transport.

Identification of FRA1 and FRA2 as genes involved in regulating the yeast iron regulon in response to decreased mitochondrial iron-sulfur cluster synthesis.

The nature of the connection between mitochondrial Fe-S cluster synthesis and the iron-sensitive transcription factor Aft1 in regulating the expression of the iron transport system in Saccharomyces cerevisiae is not known. Using a genetic screen, we identified two novel cytosolic proteins, Fra1 and Fra2, that are part of a complex that interprets the signal derived from mitochondrial Fe-S synthesis. We found that mutations in FRA1 (YLL029W) and FRA2 (YGL220W) led to an increase in transcription of the iron regulon. In cells incubated in high iron medium, deletion of either FRA gene results in the translocation of the low iron-sensing transcription factor Aft1 into the nucleus, where it occupies the FET3 promoter. Deletion of either FRA gene has the same effect on transcription as deletion of both genes and is not additive with activation of the iron regulon due to loss of mitochondrial Fe-S cluster synthesis. These observations suggest that the FRA proteins are in the same signal transduction pathway as Fe-S cluster synthesis. We show that Fra1 and Fra2 interact in the cytosol in an iron-independent fashion. The Fra1-Fra2 complex binds to Grx3 and Grx4, two cytosolic monothiol glutaredoxins, in an iron-independent fashion. These results show that the Fra-Grx complex is an intermediate between the production of mitochondrial Fe-S clusters and transcription of the iron regulon.