Volume 393, Issue 6

Chronic neurodegenerative disorders and acute injuries of the central nervous system exert a prohibitive economic burden, which is aggravated by an unmet medical need for the development of effective neurotherapeutics. The evolutionarily conserved neuropeptide, adrenomedullin (AM), and its binding protein, AMBP-1, also known as complement factor H, play important roles in brain physiology, and their expression is altered in brain pathology. In this review, we discuss the molecular regulation of AM and AMBP-1 and the pivotal roles they play in neuroprotection following brain injury. We assess the reciprocal synergistic effects of AM and AMBP-1 and make suggestions for the design of a novel combination neurotherapy devoid of the potential hypotensive effects of AM while optimizing its neuroprotective property.

Protein degradation occurs through several distinct proteolytic pathways for membrane and cytosolic proteins. There is evidence that these processes are linked and that crosstalk among these major protein degradation pathways occurs. Ubiquilins, a family of ubiquitin-binding proteins, are involved in all protein degradation pathways. This minireview provides an overview of ubiquilin function in protein degradation and contrasts it with sequestosome-1 (p62), a protein that also has been implicated in multiple proteolytic pathways.

Ribotoxins are a family of toxic proteins that exert a highly specific cleavage at the universally conserved sarcin/ricin loop (SRL) of the larger rRNA molecule. Before this ribonucleolytic action, passage through the cell membrane is a necessary step for ribotoxin internalization and the limiting factor for cytotoxicity. Although extensive knowledge of their ribonucleolytic activity and substrate recognition has been accumulated, little is known about the mechanisms of cell entry of ribotoxins. Hirsutellin A (HtA) is a recently described member of this family, which accommodates the main abilities of previously characterized ribotoxins into a shorter sequence, but exhibits some differences regarding membrane interaction properties. This work investigates the contribution of tryptophan (Trp) residues 71 and 78 to both endoribonucleolytic activity and cellular toxicity of this ribotoxin. Substitution mutants W71F and W78F, as well as the double mutant W71/78F, were obtained and assayed against isolated ribosomes, synthetic SRL, and human tumor cells. The results provide evidence that cell membrane passage and internalization, as well as substrate-specific recognition, require the participation of the region involving both Trp 71 and Trp 78. Additionally, the mutant W71/78F is the first non-cytotoxic but specific ribosome-cleaving ribotoxin mutant obtained to date.

Ecdysteroids coordinate essential biological processes in Drosophila through a complex of two nuclear receptors, the ecdysteroid receptor (EcR) and the ultraspiracle protein (Usp). Biochemical experiments have shown that, in contrast to Usp, the EcR molecule is characterized by high intramolecular plasticity. To investigate whether this plasticity is sufficient to form EcR complexes with nuclear receptors other than Usp, we studied the interaction of EcR with the DHR38 nuclear receptor. Previous in vitro experiments suggested that DHR38 can form complexes with Usp and thus disrupt Usp-EcR interaction with the specific hsp27pal response element. This article provides the experimental evidence that EcR is able to form complexes with DHR38 as well. The recombinant DNA-binding domains (DBDs) of EcR and DHR38 interact specifically on hsp27pal. However, the interaction between the receptors is not restricted to their isolated DBDs. We pre\xadsent data that indicate that the full-length EcR and DHR38 can also form specific complexes within the nuclei of living cells. This interaction is mediated by the hinge region of EcR, which was recently classified as an intrinsically disordered region. Our results indicate that DHR38 might modulate the activity of the Usp-EcR heterodimer by forming complexes with both of its components.

The pancreatic β-cell surface protein Tmem27 is promotes the preservation of functional β-cell mass. It is a selective substrate of the protease Bace2, yet the intramolecular features of Tmem27 that regulate its processing by this sheddase have not been characterized. In particular, the importance of homodimerization, glycosylation, trafficking to the plasma membrane (PM), the existence of multiple cleavage sites, and the amino acid residues that govern these features are currently unknown. Using Tmem27 mutational analysis and multiple biochemical approaches, we here show that Tmem27 dimerization is a dynamic process mediated by its intracellular cysteine residue and that prevents Tmem27 cleavage, that extracellular asparagine glycosylation is essential for Tmem27 trafficking to the PM and its processing by Bace2, that the amount of Tmem27 at the PM is proportional to its total cell levels upon glucose stimulation and Bace2 inhibition, and that the double phenylalanine motif in the Tmem27 cleavage site is an intramolecular Bace2 inhibitor. These findings define structural properties of Tmem27 that affect the susceptibility to its protease Bace2 and have implications for the efficiency with which Tmem27 and other Bace2 substrates are cleaved in normal and disease states.

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase containing two homologous domains. While the C-domain plays a major role in blood pressure regulation, the N-domain hydrolyzes the antifibrotic agent N -acetyl-Ser-Asp-Lys-Pro. Thus, N-domain selective (N-selective) inhibitors could be useful in the treatment of conditions relating to excessive tissue fibrosis. New keto-ACE analogues were designed that contained functionalities considered important for N-selective inhibitor RXP407 binding, namely, a P 2 Asp, N -acetyl group, and C-terminal amide. Such functionalities were incorporated to assess the structural determinants for N-selective binding in a novel inhibitor template. Inhibitors containing a C-terminal amide and modified P 2 ′ group were poor inhibitors of the N-domain, with several of these displaying improved inhibition of the C-domain. Molecules with both a C-terminal amide and P 2 Asp were also poor inhibitors and not N-selective. Compounds containing a free C-terminus, a P 2 Asp and protecting group displayed a change of more than 1000-fold N-selectivity compared with the parent molecule. Molecular docking models revealed interaction of these P 2 groups with S 2 residues Tyr369 and Arg381. This study emphasizes the importance of P 2 functionalities in allowing for improved N-selective binding and provides further rationale for the design of N-selective inhibitors, which could be useful in treating tissue fibrosis.

Cadherin-related protein 24 (CDHR24) is a potential tumor suppressor located apically as well as laterally in polarized cells. Here, the role of CDHR24 in contributing to cell morphology and polarity is examined. CDHR24 was predominantly localized at the nonattached part of nonpolarizing cells while another apically sorted protein, aminopeptidase N, was equally distributed over the plasma membrane. Furthermore, CDHR24 expression induced cell aggregation capacity, indicating direct cell-cell interaction. The transepithelial resistance, however, was elevated in polarized MDCK cells, but not in nonpolarizing CHO cells. Our data propose a model in which CDHR24 is directly involved in cell and tissue morphogenesis.

Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer worldwide. In recent decades, the 5-year mortality rate is approximately 50% around the world. As reliable biomarkers of oral cancer are still lacking, it is necessary to identify new target molecules for early diagnosis, effective therapy, and monitoring of the disease. In the present work, we focused on the expression of the enzyme nicotinamide N -methyltransferase (NNMT). We analyzed enzyme activity in 37 paired tumor and non-tumor tissues and found that activity levels are significantly higher in tumor compared with adjacent normal oral mucosa. Interestingly, oral epithelium surrounding tumor of unfavorable cases (N+) seems to display higher activity levels compared with that of favorable ones (N0). Western blot analyses were performed to evaluate protein levels in saliva samples from patients with OSCC and healthy subjects. Preliminary results indicated an up-regulation of salivary NNMT in tumor. This study shows a marked increase in enzyme activity in oral cancer and suggests that adjacent normal tissue of unfavorable cases seems to change toward cancer. Moreover, it is conceivable to hypothesize that NNMT could represent a potential biomarker for early and non-invasive diagnosis of oral cancer.

Mitochondrial protein import (MPI) is essential for the biogenesis of mitochondria in all eukaryotes. Current models of MPI are predominantly based on experiments with one group of eukaryotes, the opisthokonts. Although fascinating genome database-driven hypotheses on the evolution of the MPI machineries have been published, previous experimental research on non-opisthokonts usually focused on the analysis of single pathways or components in, for example, plants and parasites. In this study, we have established the kinetoplastid parasite Leishmania tarentolae as a model organism for the comprehensive analysis of non-opisthokont MPI into all four mitochondrial compartments. We found that opisthokont marker proteins are efficiently imported into isolated L. tarentolae mitochondria. Vice versa , L. tarentolae marker proteins of all compartments are also imported into mitochondria from yeast. The results are remarkable because only a few of the more than 25 classical components of the opisthokont MPI machineries are found in parasite genome databases. Our results demonstrate that different MPI pathways are functionally conserved among eukaryotes despite significant compositional differences of the MPI machineries. Moreover, our model system could lead to the identification of significantly altered or even novel MPI components in non-opisthokonts. Such differences might serve as starting points for drug development against parasitic protists.

C-Jun N-terminal kinases (JNK) are activated in course of many viral infections. Here we analyzed the activity of JNK inhibitors on influenza A virus (IAV) amplification. Human lung epithelial cells were infected with either the highly pathogenic avian virus strain A/FPV/Bratislava/79 (H7N7) or the pandemic swine-origin influenza virus A/Hamburg/4/09 (H1N1v). The application of the JNK inhibitors SP600125 and AS601245 reduced IAV amplification by suppressing viral protein and RNA synthesis. Although AS601245 appeared to generally block the transcription of newly introduced genes, SP600125 specifically affected viral RNA synthesis. Overexpression of a dominant negative mutant of SEK/MKK4 and siRNA-mediated suppression of JNK2 expression confirmed that specific manipulation of the JNK pathway attenuates virus propagation. An IAV minigenome replication assay revealed that SP600125 did not directly affect the activity of the viral RNA polymerase complex but seems to suppress an anti-influenza nonstructural protein 1-mediated virus supportive function. Finally, when H7N7- or H1N1v-infected mice were treated with SP600125, the viral load is reduced in lungs of treated compared with untreated mice. Our data suggest that this class of ATP competitive inhibitors once optimized for antiviral action potentially represent novel drugs for antiviral intervention.

The type II transmembrane serine protease matriptase-2 (TMPRSS6) down-regulates the expression of hepcidin, the main regulator of systemic iron homeostasis, and increases in this way iron plasma levels. Matriptase-2 is up-regulated under hypoxic conditions, providing a new link between hypoxia signaling and iron homeostasis. In this study, we have characterized the TMPRSS6 promoter region and identified a functional hypoxia-responsive element (HRE). Mutations of the hypoxia inducible factor (HIF)-binding site located within the HRE abrogate HIF-1α-dependent induction of TMPRSS6 expression. The action of HIF-1α on TMPRSS6 promoter activity reveals a new regulative element for the suppression of hepcidin synthesis.

Very little is understood about the trafficking of G protein-coupled receptors (GPCRs) from the endoplasmic reticulum (ER) to the plasma membrane. Rab guanosine triphosphatases (GTPases) are known to participate in the trafficking of various GPCRs via a direct interaction during the endocytic pathway, but whether this occurs in the anterograde pathway is unknown. We evaluated the potential interaction of Rab1, a GTPase known to regulate β2-adrenergic receptor (β2AR) trafficking, and its effect on export from the ER. Our results show that GTP-bound Rab1 interacts with the F(x) 6 LL motif of β2AR. Receptors lacking the interaction motif fail to traffic properly, suggesting that a direct interaction with Rab1 is required for β2AR anterograde trafficking.

No abstract available.