Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature.

The 5'-end region of cspA mRNA contains a Cold Box sequence conserved among several cold-shock mRNAs. This region forms a stable stem-loop structure followed by an AU-rich sequence. Here we show that the Cold Box region is essential for the normal scale of cspA mRNA induction after cold shock because a deletion of the stem-loop significantly destabilizes the mRNA and reduces the cold shock-induced cspA mRNA amount by approximately 50%. The AU-rich track, however, slightly destabilizes the mRNA. The integrity of the stem is essential for the stabilizing function, whereas that of the loop sequence is less important. Overexpression of a mutant cspA mRNA devoid of both the AUG initiation codon and the coding sequence results in a severe growth inhibition at low temperature along with a derepression of the chromosomal cspA expression. Furthermore, the overexpressed RNA is stably associated with the 30 S and 70 S ribosomes. Our results demonstrate that the AUG initiation codon and the coding region containing the downstream box are not required for cspA mRNA to bind ribosomes and that the 5'-untranslated region by itself has a remarkable affinity to ribosomes at low temperature.     

Multiomic Metabolic Enrichment Network Analysis Reveals Metabolite–Protein Physical Interaction Subnetworks Altered in Cancer

Metabolism is recognized as an important driver of cancer progression and other complex diseases, but global metabolite profiling remains a challenge. Protein expression profiling is often a poor proxy since existing pathway enrichment models provide an incomplete mapping between the proteome and metabolism. To overcome these gaps, we introduce multiomic metabolic enrichment network analysis (MOMENTA), an integrative multiomic data analysis framework for more accurately deducing metabolic pathway changes from proteomics data alone in a gene set analysis context by leveraging protein interaction networks to extend annotated metabolic models. We apply MOMENTA to proteomic data from diverse cancer cell lines and human tumors to demonstrate its utility at revealing variation in metabolic pathway activity across cancer types, which we verify using independent metabolomics measurements. The novel metabolic networks we uncover in breast cancer and other tumors are linked to clinical outcomes, underscoring the pathophysiological relevance of the findings.     

Evolution of green lacewings (Neuroptera: Chrysopidae): a molecular supermatrix approach

We present a time‐calibrated phylogeny of the charismatic green lacewings (Neuroptera: Chrysopidae). Previous phylogenetic studies on the family using DNA sequences have suffered from sparse taxon sampling and/or limited amounts of data. Here we combine all available previously published DNA sequence data and add to it new DNA sequences generated for this study. We analysed these data in a supermatrix using Bayesian and maximum likelihood methods and provide a phylogenetic hypothesis for the family that recovers strong support for the monophyly of all subfamilies and resolves relationships among a large proportion of chrysopine genera. Chrysopinae tribes Leucochrysini and Belonopterygini were recovered as monophyletic sister clades, while the species‐rich tribe Chrysopini was rendered paraphyletic by Ankylopterygini. Relationships among the subfamilies were resolved, although with relatively low statistical support, and the topology varied based on the method of analysis. Greatest support was found for Apochrysinae as sister to Nothochrysinae and Chrysopinae, which is in contrast to traditional concepts that place Nothochrysinae as sister to the rest of the family. Divergence estimates suggest that the stem groups to the various subfamilies diverged during the Triassic‐Jurassic, and that stem groups of the chrysopine tribes diverged during the Cretaceous.