Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.     

Proteasome inhibitors improve the function of mutant lysosomal α-glucosidase in fibroblasts from Pompe disease patient carrying c.546G>T mutation

Pompe disease (glycogen storage disease type II) is an autosomal recessive myopathic disorder arising from the deficiency of lysosomal acid α-glucosidase (GAA). Recently, we found that mutant GAA in patient fibroblasts carrying c.546G>T mutation is stabilized by treatment with proteasome inhibitor as well as pharmacological chaperon N-butyl-deoxynojirimycin. In this study, we characterized the effect of two proteasome inhibitors, bortezomib and MG132, on maturation, subcellular localization and residual activity of mutant GAA in the patient fibroblasts carrying c.546G>T mutation. Each proteasome inhibitor promoted the stabilization of patient GAA and processing of them to mature forms without cytotoxic effect. Immunocytochemical analysis showed increased colocalization of GAA with the lysosomal marker LAMP2 in patient fibroblasts treated with proteasome inhibitors. Furthermore, bortezomib and MG132 also increased enzyme activity in the patient fibroblasts (about 4-fold and 2-fold, respectively). These findings indicate that proteasome inhibitor may be a novel drug as potential pharmacological chaperone therapy for Pompe disease patient carrying chaperon-responsive mutation.     

A pathogenic mutation in cytochrome c oxidase results in impaired proton pumping while retaining O2-reduction activity

In this work we have investigated the effect of a pathogenic mitochondrial DNA mutation found in human colon cells, at a functional-molecular level. The mutation results in the amino-acid substitution Tyr19His in subunit I of the human CytcO and it is associated with respiratory deficiency. It was introduced into Rhodobacter sphaeroides, which carries a cytochrome c oxidase (cytochrome aa₃) that serves as a model of the mitochondrial counterpart. The residue is situated in the middle of a pathway that is used to transfer substrate protons as well as protons that are pumped across the membrane. The Tyr33His (equivalent residue in the bacterial CytcO) structural variant of the enzyme was purified and its function was investigated. The results show that in the structurally altered CytcO the activity decreased due to slowed proton transfer; proton transfer from an internal proton donor, the highly-conserved Glu286, to the catalytic site was slowed by a factor of ∼ 5, while reprotonation of the Glu from solution was slowed by a factor of ∼ 40. In addition, in the structural variant proton pumping was completely impaired. These results are explained in terms of introduction of a barrier for proton transfer through the D pathway and changes in the coordination of water molecules surrounding the Glu286 residue. The study offers an explanation, at the molecular level, to the link between a specific amino-acid substitution and a pathogenic phenotype identified in human colon cells.     

The Application of the Tissue Microarray (TMA) Technology to Analyze Cerebral Organoids

“Multi-Omics” technologies have contributed greatly to the understanding of various diseases by enabling researchers to accurately and rapidly investigate the molecular circuitry that connects cellular systems. The tissue-engineered, three-dimensional (3D), in vitro disease model “organoid” integrates the “omics” results in a model system, elucidating the complex links between genotype and phenotype. These 3D structures have been used to model cancer, infectious disease, toxicity, and neurological disorders. Here, we describe the advantage of using the tissue microarray (TMA) technology to analyze human-induced pluripotent stem cell–derived cerebral organoids. Compared with the conventional processing of individual samples, sectioning and staining of TMA slides are faster and can be automated, decreasing labor and reagent costs. The TMA technology faithfully captures cell morphology variations and detects specific biomarkers. The use of this technology can scale up organoid research results in at least two ways: (1) in the number of specimens that can be analyzed simultaneously and (2) in the number of consecutive sections that can be produced for analysis with different probes and antibodies.     

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom,HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.

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Infants prefer the faces of strangers or mothers to morphed faces: an uncanny valley between social novelty and familiarity

The ‘uncanny valley’ response is a phenomenon involving the elicitation of a negative feeling and subsequent avoidant behaviour in human adults and infants as a result of viewing very realistic human-like robots or computer avatars. It is hypothesized that this uncanny feeling occurs because the realistic synthetic characters elicit the concept of ‘human’ but fail to satisfy it. Such violations of our normal expectations regarding social signals generate a feeling of unease. This conflict-induced uncanny valley between mutually exclusive categories (human and synthetic agent) raises a new question: could an uncanny feeling be elicited by other mutually exclusive categories, such as familiarity and novelty? Given that infants prefer both familiarity and novelty in social objects, we address this question as well as the associated developmental profile. Using the morphing technique and a preferential-looking paradigm, we demonstrated uncanny valley responses of infants to faces of mothers (i.e. familiarity) and strangers (i.e. novelty). Furthermore, this effect strengthened with the infant''s age. We excluded the possibility that infants detect and avoid traces of morphing. This conclusion follows from our finding that the infants equally preferred strangers’ faces and the morphed faces of two strangers. These results indicate that an uncanny valley between familiarity and novelty may accentuate the categorical perception of familiar and novel objects.     

Comparative analysis detects dependencies among the 5' splice-site positions

Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5' splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5' splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, -2 and +5, and -1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5' splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5' splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5' splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5' splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5' splice site.     

In vivo hyperglycemia and its effect on Glut-1 expression in the embryonic heart

BACKGROUND: Maternal diabetes exposes embryos to periods of hyperglycemia. Glucose is important for normal cardiogenesis, and Glut-1 is the predominant glucose transporter in the embryo. METHODS: Pregnant mice were exposed to 6 or 12 hr hyperglycemia during organogenesis using intraperitoneal (IP) injections of D-glucose on gestational day (GD) 9.5 (plug = GD 0.5). Embryos were examined for morphology and total cardiac protein, and embryonic hearts were evaluated for Glut-1 protein and mRNA expression immediately after treatment (GD 9.75, GD 10.0), as well as on GD 10.5 and GD 12.5. RESULTS: IP glucose injections were effective in producing sustained maternal hyperglycemia. Maternal hyperglycemia for 6 or 12 hr on GD 9.5, followed by normoglycemia, produced a decrease in overall size and total cardiac protein in embryos evaluated on GD 10.5 but no difference on GD 12.5. Cardiac Glut-1 expression was immediately upregulated in embryos exposed to 6 or 12 hr maternal hyperglycemia. On GD 10.5, cardiac Glut-1 expression was not different in embryos exposed to maternal hyperglycemia for 6 hr but was downregulated in embryos exposed for 12 hr. On GD 12.5, cardiac Glut-1 expression in embryos exposed to maternal hyperglycemia on GD 9.5 for 6 or 12 hr, followed by normoglycemia, was not different from controls. The temporal pattern was the same for Glut-1 protein and mRNA expression. CONCLUSIONS: Hyperglycemia-induced alterations in Glut-1 expression likely interfere with balance of glucose available to the embryonic heart that may affect cardiac morphogenesis.