The role of the cysteine-rich region of the β2 integrin subunit in the leukocyte function-associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) heterodimer formation and ligand binding

The cysteine-rich region (CRR) of the β2 integrin subunit was replaced by that of β1 to give the chimera β2NV1. β2NV1 can combine with αL to form a variant leukocyte-function-associated antigen (LFA)-1 on COS cell surface, suggesting that the specificity of the β2 interaction with αL does not lie in the CRR. Unlike those expressing wild-type LFA-1, COS cells expressing αLβ2NV1 are constitutively active in intercellular adhesion molecule (ICAM)-1 adhesion. These results suggest that activation of LFA-1 involves the release of an intramolecular constraint, which is maintained, in part, by the authentic β2 CRR.     

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.     

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.     

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

DC‐UbP/UBTD2 is a ubiquitin (Ub) domain‐containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C‐terminal Ub‐like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC‐UbP, we then solved the solution structure of the N‐terminal domain of DC‐UbP (DC‐UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC‐UbP_N holds a novel structural fold and acts as a Ub‐binding domain (UBD) but with low affinity. This implies that the DC‐UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells.     

An “orientation sphere” visualization for examining animal head movements

1. Animal behavior is elicited, in part, in response to external conditions, but understanding how animals perceive the environment and make the decisions that bring about these behavioral responses is challenging.
2. Animal heads often move during specific behaviors and, additionally, typically have sensory systems (notably vision, smell, and hearing) sampling in defined arcs (normally to the front of their heads). As such, head‐mounted electronic sensors consisting of accelerometers and magnetometers, which can be used to determine the movement and directionality of animal heads (where head “movement” is defined here as changes in heading [azimuth] and/or pitch [elevation angle]), can potentially provide information both on behaviors in general and also clarify which parts of the environment the animals might be prioritizing (“environmental framing”).
3. We propose a new approach to visualize the data of such head‐mounted tags that combines the instantaneous outputs of head heading and pitch in a single intuitive spherical plot. This sphere has magnetic heading denoted by “longitude” position and head pitch by “latitude” on this “orientation sphere” (O‐sphere).
4. We construct the O‐sphere for the head rotations of a number of vertebrates with contrasting body shape and ecology (oryx, sheep, tortoises, and turtles), illustrating various behaviors, including foraging, walking, and environmental scanning. We also propose correcting head orientations for body orientations to highlight specific heading‐independent head rotation, and propose the derivation of O‐sphere‐metrics, such as angular speed across the sphere. This should help identify the functions of various head behaviors.
5. Visualizations of the O‐sphere provide an intuitive representation of animal behavior manifest via head orientation and rotation. This has ramifications for quantifying and understanding behaviors ranging from navigation through vigilance to feeding and, when used in tandem with body movement, should provide an important link between perception of the environment and response to it in free‐ranging animals.

     

The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.     

Repeated pre-training sleep restriction in adolescent rats impaired spatial performance

Sleep and Biological Rhythms - Chronic sleep deprivation (SD) is an overwhelming problem in young students. Firstly, we investigated whether different levels of pre-training SD had effects on...