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Aberrant activation of hedgehog (Hh) signaling has been observed in a wide variety of tumors and accounts for more than 25% of human cancer deaths. Inhibitors targeting the Hh signal transducer Smoothened (SMO) are widely used and display a good initial efficacy in patients suffering from basal cell carcinoma (BCC); however, a large number of patients relapse. Though SMO mutations may explain acquired therapy resistance, a growing body of evidence suggests that the non-canonical, SMO-independent activation of the Hh pathway in BCC patients can also account for this adverse effect. In this review, we highlight the importance of glioma-associated oncogene (GLI) transcription factors (the main downstream effectors of the canonical and the non-canonical Hh cascade) and their putative role in the regulation of multiple oncogenic signaling pathways. Moreover, we discuss the contribution of the Hh signaling to malignant transformation and propose GLIs as central hubs in tumor signaling networks and thus attractive molecular targets in anti-cancer therapies.

Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer's disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review.

Succinic semialdehyde dehydrogenase deficiency (SSADH-D) is a genetic disorder that results from the aberrant metabolism of the neurotransmitter γ-amino butyric acid (GABA). The disease is caused by impaired activity of the mitochondrial enzyme succinic semialdehyde dehydrogenase. SSADH-D manifests as varying degrees of mental retardation, autism, ataxia, and epileptic seizures, but the clinical picture is highly heterogeneous. So far, there is no approved curative therapy for this disease. In this review, we briefly summarize the molecular genetics of SSADH-D, the past and ongoing clinical trials, and the emerging features of the molecular pathogenesis, including redox imbalance and mitochondrial dysfunction. The main aim of this review is to discuss the potential of further therapy approaches that have so far not been tested in SSADH-D, such as pharmacological chaperones, read-through drugs, and gene therapy. Special attention will also be paid to elucidating the role of patient advocacy organizations in facilitating research and in the communication between researchers and patients.

The disturbance of hemostatic balance, associated with increased tissue factor (TF) expression and activity, occurs in the lungs of patients with idiopathic pulmonary fibrosis (IPF). However, the molecular mechanisms responsible for the regulation of TF expression under profibrotic conditions have not been assessed. We found that transforming growth factor–β1 (TGF-β1) markedly enhanced TF expression in primary human lung fibroblasts (HLFs), whereas platelet-derived growth factor (PDGF)–BB and IGF (insulin-like growth factor)–1 showed only a moderate effect, and PDGB-CC exerted no effect. TGF-β1–induced TF expression correlated with its elevated cell-surface activity, it required de novo gene transcription and protein synthesis, and it was dependent on JNK and Akt activity, because pharmacological inhibition or the knockdown of the previously mentioned kinases prevented TF synthesis. Exposure of HLFs to TGF-β1 activated JNK in a PI3K-dependent manner and induced Akt phosphorylation at threonine 308 and serine 473, but did not change the phosphorylation status of threonine 450. Akt phosphorylation at serine 473 correlated with JNK activity, and co-immunoprecipitation studies revealed a direct interaction between JNK and Akt. Furthermore, TGF-β1–induced TF expression required the recruitment of c-Fos and JunD into a heterodimeric activator protein (AP)-1 complex. Moreover, strong immunoreactivity for phosphorylated Akt and JNK as well as c-Fos and JunD was observed in fibroblasts and myofibroblasts in IPF lungs. In conclusion, PI3K/JNK/Akt and AP-1 synergize to induce TF expression in HLFs after TGF-β1 challenge. Our findings provide new insights into the molecular mechanisms responsible for the regulation of TF expression, and open new perspectives on the treatment of pulmonary fibrosis and other diseases characterized by the inappropriate expression of this cell-surface receptor.

Succinic semialdehyde dehydrogenase (SSADH) is a mitochondrial enzyme involved in the catabolism of the neurotransmitter γ-amino butyric acid. Pathogenic variants in the gene encoding this enzyme cause SSADH deficiency, a developmental disease that manifests as hypotonia, autism, and epilepsy. SSADH deficiency patients usually have family-specific gene variants. Here, we describe a family exhibiting four different SSADH variants: Val90Ala, Cys93Phe, and His180Tyr/Asn255Asp (a double variant). We provide a structural and functional characterization of these variants and show that Cys93Phe and Asn255Asp are pathogenic variants that affect the stability of the SSADH protein. Due to the impairment of the cofactor NAD+ binding, these variants show a highly reduced enzyme activity. However, Val90Ala and His180Tyr exhibit normal activity and expression. The His180Tyr/Asn255Asp variant exhibits a highly reduced activity as a recombinant species, is inactive, and shows a very low expression in eukaryotic cells. A treatment with substances that support protein folding by either increasing chaperone protein expression or by chemical means did not increase the expression of the pathogenic variants of the SSADH deficiency patient. However, stabilization of the folding of pathogenic SSADH variants by other substances may provide a treatment option for this disease.

Succinic semialdehyde dehydrogenase is a key enzyme involved in the catabolism of g-aminobutyric acid. Biallelic variants in the ALDH5A1 gene cause a rare monogenic neurometabolic disorder, SSADH deficiency, characterized by a total or partial enzyme loss-of-function. Patients present symptoms from infancy on and mainly show developmental delay and intellectual disabilities, with seizures and/or autistic spectrum disorders as the main distinctive traits associated with the severe forms of the disorder. A valuable instrument to interpret variant function is provided by bioinformatic tools that are based on evolutionary and structural data. However, the results need to be complemented by other experimental approaches to dissect the molecular basis for the defect and be supportive of the clinical phenotype of patients. Here, we validate a bioinformatic prediction of loss-of-function using two recombinant enzyme variants and/or transient transfection in a ALDH5A1 knockout HEK-293T cell model. These SSADH protein variants are synthesized by two functionally hemizygous SSADH deficiency patients. The known loss-of-function p.Gly176Arg variant was predicted by bioinformatic analyses to affect oligomerization. Indeed, we demonstrate that the variant acquires a tetrameric altered structure, possibly responsible for the loss-of-function. The pathogenicity of the new p.Pro234Ser variant is mainly due to its failure to reach the mature form, possibly due to its intrinsic instability rather than a defect in the maturation apparatus, thus preventing a correct folding process. Altogether, we provide evidence that combining recombinant protein studies with structural predictions can offer valuable insights into loss-of-function, which is essential for precision therapy.

Abstract Rationale Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. Objectives To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. Methods The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. Measurements and Main Results Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. Conclusions Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare, monogenic disorder affecting the degradation of the main inhibitory neurotransmitter γ-amino butyric acid (GABA). Pathogenic variants in the ALDH5A1 gene that cause an enzymatic dysfunction of succinic semialdehyde dehydrogenase (SSADH) lead to an accumulation of potentially toxic metabolites, including γ-hydroxybutyrate (GHB). Here, we present a patient with a severe phenotype of SSADHD caused by a novel genetic variant c.728T > C that leads to an exchange of leucine to proline at residue 243, located within the highly conserved nicotinamide adenine dinucleotide (NAD)+ binding domain of SSADH. Proline harbors a pyrrolidine within its side chain known for its conformational rigidity and disruption of protein secondary structures. We investigate the effect of this novel variant in vivo, in vitro, and in silico. We furthermore examine the mutational spectrum of all previously described disease-causing variants and computationally assess all biologically possible missense variants of ALDH5A1 to identify mutational hotspots.

Streptococcus pneumoniae is the most frequent cause of community-acquired pneumonia. The infection process involves bacterial cell surface receptors, which interact with host extracellular matrix components to facilitate colonization and dissemination of bacteria. Here, we investigated the role of host-derived extracellular RNA (eRNA) in the process of pneumococcal alveolar epithelial cell infection. Our study demonstrates that eRNA dose-dependently increased S. pneumoniae invasion of alveolar epithelial cells. Extracellular enolase (Eno), a plasminogen (Plg) receptor, was identified as a novel eRNA-binding protein on S. pneumoniae surface, and six Eno eRNA-binding sites including a C-terminal 15 amino acid motif containing lysine residue 434 were characterized. Although the substitution of lysine 434 for glycine (K434G) markedly diminished the binding of eRNA to Eno, the adherence to and internalization into alveolar epithelial cells of S. pneumoniae strain carrying the C-terminal lysine deletion and the mutation of internal Plg-binding motif were only marginally impaired. Accordingly, using a mass spectrometric approach, we identified seven novel eRNA-binding proteins in pneumococcal cell wall. Given the high number of eRNA-interacting proteins on pneumococci, treatment with RNase1 completely inhibited eRNA-mediated pneumococcal alveolar epithelial cell infection. Our data support further efforts to employ RNAse1 as an antimicrobial agent to combat pneumococcal infectious diseases.