RESUMEN
BACKGROUND: PD-L1, a transmembrane ligand for immune checkpoint receptor PD1, has been successfully targeted to activate an anti-tumor immune response in a variety of solid tumors, including non-small cell lung cancer (NSCLC). Despite the success of targeting PD-L1, only about 20% of patients achieve a durable response. The reasons for the heterogeneity in response are not understood, although some molecular subtypes (e.g., mutant EGF receptor tumors) are generally poor responders. Although PD-L1 is best characterized as a transmembrane PD1 ligand, the emerging view is that PD-L1 has functions independent of activating PD1 signaling. It is not known whether these cell-intrinsic functions of PD-L1 are shared among non-transformed and transformed cells, if they vary among cancer molecular subtypes, or if they are impacted by anti-PD-L1 therapy. METHODS: Here we use quantitative microscopy techniques and APEX2 proximity mapping to describe the behavior of PD-L1 and to identify PD-L1's proximal proteome in human lung epithelial cells. RESULTS: Our data reveal growth factor control of PD-L1 recycling as a mechanism for acute and reversible regulation of PD-L1 density on the plasma membrane. In addition, we describe novel PD-L1 biology restricted to mutant EGFR cells. Anti-PD-L1 antibody treatment of mutant EGFR cells perturbs cell intrinsic PD-L1 functions, leading to reduced cell migration, increased half-life of EGFR and increased extracellular vesicle biogenesis, whereas anti-PD-L1 antibody does not induce these changes in wild type EGFR cells. CONCLUSIONS: Growth factor acute regulation of PD-L1 trafficking, by contributing to the control of plasma membrane density, might contribute to the regulation of PD-L1's immune checkpoint activity, whereas the specific effects of anti-PD-L1 on mutant EGFR cells might contribute to the poor anti-PD-L1 response of mutant EGFR tumors. Video Abstract.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteoma , Ligandos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Pulmón/metabolismo , Antígeno B7-H1/metabolismo , MutaciónRESUMEN
An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.
Asunto(s)
Aneuploidia , Fibroblastos/patología , Trisomía/genética , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Fibroblastos/metabolismo , Dosificación de Gen/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Lípidos/biosíntesis , Sustancias Macromoleculares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Advanced prostate cancer initially responds to hormonal treatment, but ultimately becomes resistant and requires more potent therapies. One mechanism of resistance observed in around 10-20% of these patients is lineage plasticity, which manifests in a partial or complete small cell or neuroendocrine prostate cancer (NEPC) phenotype. Here, we investigate the role of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex in NEPC. Using large patient datasets, patient-derived organoids and cancer cell lines, we identify mSWI/SNF subunits that are deregulated in NEPC and demonstrate that SMARCA4 (BRG1) overexpression is associated with aggressive disease. We also show that SWI/SNF complexes interact with different lineage-specific factors in NEPC compared to prostate adenocarcinoma. These data point to a role for mSWI/SNF complexes in therapy-related lineage plasticity, which may also be relevant for other solid tumors.
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Linaje de la Célula , Plasticidad de la Célula , Proteínas Cromosómicas no Histona/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Línea Celular Tumoral , Estudios de Cohortes , ADN Helicasas/genética , ADN Helicasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , Modelos Biológicos , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/genética , Subunidades de Proteína/metabolismo , Factores de Transcripción/genética , Transcriptoma/genéticaRESUMEN
Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.
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Carcinoma/patología , Cromatina/metabolismo , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Metástasis de la Neoplasia/genética , Animales , Carcinogénesis/genética , Carcinoma/genética , Línea Celular Tumoral , Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/genética , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Progresión de la Enfermedad , Epigénesis Genética , Transición Epitelial-Mesenquimal/genética , Femenino , Histonas/genética , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas/genética , RNA-Seq , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors, as is exemplified by reprogramming somatic cells to pluripotent stem cells through the expression of OCT4, KLF4, SOX2 and cMYC. How transcription factors orchestrate the complex molecular changes around their target gene loci remains incompletely understood. Here, using KLF4 as a paradigm, we provide a transcription-factor-centric view of chromatin reorganization and its association with three-dimensional enhancer rewiring and transcriptional changes during the reprogramming of mouse embryonic fibroblasts to pluripotent stem cells. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in enhancer connectivity, whereas disruption of individual KLF4 binding sites within pluripotent-stem-cell-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce the expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying transcription factor and offers novel insights into the nature of the molecular events that follow transcription factor binding.
Asunto(s)
Reprogramación Celular/genética , Ensamble y Desensamble de Cromatina/genética , Elementos de Facilitación Genéticos , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Factor 4 Similar a Kruppel , Masculino , Ratones , Células Madre Pluripotentes/metabolismoRESUMEN
Mass-spectrometry-based phosphoproteomics has revolutionized phosphoprotein analysis and enhanced our understanding of diverse and fundamental cellular processes important for human health and disease. Because of their relative scarcity, phosphopeptides must be enriched before analysis. Many different enrichment methods and materials have been described, and many reports have made claims about the advantages of particular materials and methodological variations. We demonstrate an effective and highly reproducible single-step enrichment method using an off-the-shelf preparation of calcium titanate. Using two different cell lines and replicate analysis, we show that our method achieves a purity and depth of analysis comparable or superior to a widely used TiO2-based method at a reduced cost and effort. This method provides a new and immediately available tool for expanding the reach of phosphoproteomic inquiry.
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Fosfopéptidos/química , Fosfoproteínas/química , Proteómica/métodos , Calcio/química , Línea Celular , Humanos , Fosforilación , Espectrometría de Masas en Tándem , Titanio/químicaRESUMEN
In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.
Asunto(s)
Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Holoenzimas/química , Proteínas Recombinantes de Fusión/química , Salmonella typhimurium/enzimología , Factor sigma/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Escherichia coli/enzimología , Escherichia coli/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Cinética , Simulación de Dinámica Molecular , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/genética , Factor sigma/genética , Factor sigma/metabolismo , Solventes , TermodinámicaRESUMEN
Aneuploidy disrupts cellular homeostasis. However, the molecular mechanisms underlying the physiological responses and adaptation to aneuploidy are not well understood. Deciphering these mechanisms is important because aneuploidy is associated with diseases, including intellectual disability and cancer. Although tumors and mammalian aneuploid cells, including several cancer cell lines, show altered levels of sphingolipids, the role of sphingolipids in aneuploidy remains unknown. Here, we show that ceramides and long-chain bases, sphingolipid molecules that slow proliferation and promote survival, are increased by aneuploidy. Sphingolipid levels are tightly linked to serine synthesis, and inhibiting either serine or sphingolipid synthesis can specifically impair the fitness of aneuploid cells. Remarkably, the fitness of aneuploid cells improves or deteriorates upon genetically decreasing or increasing ceramides, respectively. Combined targeting of serine and sphingolipid synthesis could be exploited to specifically target cancer cells, the vast majority of which are aneuploid.
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Aneuploidia , Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Esfingolípidos/biosíntesis , Proliferación Celular , Ceramidas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Serina/biosíntesis , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.
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Regulación de la Expresión Génica , Lipogénesis , Procesamiento Postranscripcional del ARN , Transducción de Señal , Animales , Núcleo Celular/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Femenino , Xenoinjertos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
The bacterial RNA polymerase (RNAP) holoenzyme is a multisubunit core enzyme associated with a σ factor that is required for promoter-specific transcription initiation. Besides a primary σ responsible for most of the gene expression during active growth, bacteria contain alternative σ factors that control adaptive responses. A recurring strategy in the control of σ factor activity is their sequestration by anti-sigma factors that occlude the RNAP binding determinants, reducing their activity. In contrast, the unconventional transcription factor Crl binds specifically to the alternative σ factor σS/RpoS, and favors its association with the core RNAP, thereby increasing its activity. σS is the master regulator of the general stress response that protects many Gram-negative bacteria from several harmful environmental conditions. It is also required for biofilm formation and virulence of Salmonella enterica serovar Typhimurium. In this report, we discuss current knowledge on the regulation and function of Crl in Salmonella and Escherichia coli, two bacterial species in which Crl has been studied. We review recent advances in the structural characterization of the Crl-σS interaction that have led to a better understanding of this unusual mechanism of σ regulation.
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Proteínas Bacterianas/agonistas , Proteínas Bacterianas/metabolismo , Factor sigma/agonistas , Factor sigma/metabolismo , Proteínas Bacterianas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Unión Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Factor sigma/química , Estrés Fisiológico , Activación TranscripcionalRESUMEN
In many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (S. Typhimurium), the sigma factor RpoS/σ(S) accumulates during stationary phase of growth, and associates with the core RNA polymerase enzyme (E) to promote transcription initiation of genes involved in general stress resistance and starvation survival. Whereas σ factors are usually inactivated upon interaction with anti-σ proteins, σ(S) binding to the Crl protein increases σ(S) activity by favouring its association to E. Taking advantage of evolution of the σ(S) sequence in bacterial species that do not contain a crl gene, like Pseudomonas aeruginosa, we identified and assigned a critical arginine residue in σ(S) to the S. Typhimurium σ(S)-Crl binding interface. We solved the solution structure of S. Typhimurium Crl by NMR and used it for NMR binding assays with σ(S) and to generate in silico models of the σ(S)-Crl complex constrained by mutational analysis. The σ(S)-Crl models suggest that the identified arginine in σ(S) interacts with an aspartate of Crl that is required for σ(S) binding and is located inside a cavity enclosed by flexible loops, which also contribute to the interface. This study provides the basis for further structural investigation of the σ(S)-Crl complex.
Asunto(s)
Proteínas Bacterianas/química , ARN Polimerasas Dirigidas por ADN/química , Pseudomonas aeruginosa/metabolismo , Salmonella/metabolismo , Factor sigma/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Sitios de Unión , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/ultraestructura , Modelos Químicos , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Subunidades de Proteína , Factor sigma/metabolismo , Factor sigma/ultraestructura , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
The general stress response in Enterobacteria, like Escherichia coli or Salmonella, is controlled by the transcription factor σ(S), encoded by the rpoS gene, which accumulates during stationary phase growth and associates with the core RNA polymerase enzyme (E) to promote transcription of genes involved in cell survival. Tight regulation of σ(S) is essential to preserve the balance between self-preservation under stress conditions and nutritional competence in the absence of stress. Whereas σ factors are generally inactivated upon interaction with anti-sigma proteins, σ(S) binding by the Crl protein facilitates the formation of the holoenzyme Eσ(S), and therefore σ(S)-controlled transcription. Previously, critical residues in both Crl and σ(S) were identified and assigned to the binding interface in the Crl-σ(S) complex. However, high-resolution structural data are missing to fully understand the molecular mechanisms underlying σ(S) activation by Crl, in particular the possible role of Crl in triggering domain rearrangements in the multi-domain protein σ(S). Here we provide the (1)H, (13)C and (15)N resonance assignments of Salmonella enterica serovar Typhimurium Crl, as a starting point for CrlSTM structure determination and further structural investigation of the CrlSTM-σ STM (S) complex.
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Proteínas Bacterianas/química , Espectroscopía de Resonancia Magnética con Carbono-13 , Espectroscopía de Protones por Resonancia Magnética , Salmonella typhimurium/metabolismo , Factor sigma/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Proteus mirabilis/metabolismo , Alineación de SecuenciaRESUMEN
In many γ-proteobacteria, the RpoS/σS sigma factor associates with the core RNAP (RNA polymerase) to modify global gene transcription in stationary phase and under stress conditions. The small regulatory protein Crl stimulates the association of σS with the core RNAP in Escherichia coli and Salmonella enterica serovar Typhimurium, through direct and specific interaction with σS. The structural determinants of Crl involved in σS binding are unknown. In the present paper we report the X-ray crystal structure of the Proteus mirabilis Crl protein (CrlPM) and a structural model for Salmonella Typhimurium Crl (CrlSTM). Using a combination of in vivo and in vitro assays, we demonstrated that CrlSTM and CrlPM are structurally similar and perform the same biological function. In the Crl structure, a cavity enclosed by flexible arms contains two patches of conserved and exposed residues required for σS binding. Among these, charged residues that are likely to be involved in electrostatic interactions driving Crl-σS complex formation were identified. CrlSTM and CrlPM interact with domain 2 of σS with the same binding properties as with full-length σS. These results suggest that Crl family members share a common mechanism of σS binding in which the flexible arms of Crl might play a dynamic role.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteus mirabilis/metabolismo , Salmonella typhimurium/metabolismo , Factor sigma/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteus mirabilis/química , Proteus mirabilis/enzimología , Proteus mirabilis/genética , Salmonella typhimurium/química , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Factor sigma/química , Factor sigma/genéticaRESUMEN
Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.
Asunto(s)
Azul de Metileno/química , Priones/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Priones/genética , Priones/metabolismo , Conformación Proteica , OvinosRESUMEN
Prion protein (PrP) is involved in lethal neurodegenerative diseases, and many issues remain unclear about its physio-pathological role. Quadruplex-forming nucleic acids (NAs) have been found to specifically bind to both PrP cellular and pathological isoforms. To clarify the relevance of these interactions, thermodynamic, kinetic and structural studies have been performed, using isothermal titration calorimetry, surface plasmon resonance and circular dichroism methodologies. Three quadruplex-forming sequences, d(TGGGGT), r(GGAGGAGGAGGA), d(GGAGGAGGAGGA), and various forms of PrP were selected for this study. Our results showed that these quadruplexes exhibit a high affinity and specificity toward PrP, with K(D) values within the range 62÷630 nM, and a weaker affinity toward a PrP-ß oligomer, which mimics the pathological isoform. We demonstrated that the NA quadruplex architecture is the structural determinant for the recognition by both PrP isoforms. Furthermore, we spotted both PrP N-terminal and C-terminal domains as the binding regions involved in the interaction with DNA/RNAs, using several PrP truncated forms. Interestingly, a reciprocally induced structure loss was observed upon PrP-NA interaction. Our results allowed to surmise a quadruplex unwinding-activity of PrP, that may have a feedback in vivo.
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G-Cuádruplex , Priones/química , Sitios de Unión , Calorimetría , Dicroismo Circular , ADN/química , Cinética , Priones/metabolismo , Unión Proteica , ARN/química , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
Hyperphenylalaninemias are genetic diseases prevalently caused by mutations in the phenylalanine hydroxylase (PAH) gene. The wild-type PAH enzyme is a homotetramer regulated by its substrate, cofactor and phosphorylation. We reproduced a full-length wild-type protein and seven natural full-length PAH variants, p.I65M, p.N223Y, p.R297L, p.F382L, p.K398N, p.A403V, and p.Q419R, and analyzed their biochemical and biophysical behavior. All mutants exhibited reduced enzymatic activity, namely from 38% to 69% of wild-type activity. Biophysical characterization was performed by size-exclusion chromatography, light scattering and circular dichroism. In the purified wild-type PAH, we identified the monomer in equilibrium with the dimer and tetramer. In most mutants, the equilibrium shifted toward the dimer and most tended to form aggregates. All PAH variants displayed different biophysical behaviors due to loss of secondary structure and thermal destabilization. Specifically, p.F382L was highly unstable at physiological temperature. Moreover, using confocal microscopy with the number and brightness technique, we studied the effect of BH4 addition directly in living human cells expressing wild-type PAH or p.A403V, a mild mutant associated with BH4 responsiveness in vivo. Our results demonstrate that BH4 addition promotes re-establishment of the oligomerization equilibrium, thus indicating that the dimer-to-tetramer shift in pA403V plays a key role in BH4 responsiveness. In conclusion, we show that the oligomerization process and conformational stability are altered by mutations that could affect the physiological behavior of the enzyme. This endorses the hypothesis that oligomerization and folding defects of PAH variants are the most common causes of HPAs, particularly as regards mild human phenotypes.