Search

Intrinsic protein properties that may not be apparent by only examining three-dimensional structures can be revealed by careful analysis of mutant protein variants. Deep mutational scanning is a technique that allows the functional analysis of millions of protein variants in a single experiment. To enable this high-throughput technique, the mutant genotype of protein variants must be coupled to a selectable function. This chapter outlines how artificial genetic circuits in the yeast Saccharomyces cerevisiae can maintain the genotype-phenotype link, thus enabling the general application of this approach. To do this, we describe how to engineer genetic selections in yeast, methods to construct mutant libraries, and how to analyze sequencing data. We investigate the structure-function relationships of the antimicrobial resistance protein TetX to illustrate this process. In doing so, we demonstrate that deep mutational scanning is a powerful method to dissect the importance of individual residues for the inactivation of antibiotic analogues, with consequences for the rational design of new drugs to combat antimicrobial resistance., (© 2020 Elsevier Inc. All rights reserved.)

The remarkable growth of multi-platform genomic profiles has led to the challenge of multiomics data integration. In this study, we present a novel network-based multiomics clustering founded on the Wasserstein distance from optimal mass transport. This distance has many important geometric properties making it a suitable choice for application in machine learning and clustering. Our proposed method of aggregating multiomics and Wasserstein distance clustering (aWCluster) is applied to breast carcinoma as well as bladder carcinoma, colorectal adenocarcinoma, renal carcinoma, lung non-small cell adenocarcinoma, and endometrial carcinoma from The Cancer Genome Atlas project. Subtypes were characterized by the concordant effect of mRNA expression, DNA copy number alteration, and DNA methylation of genes and their neighbors in the interaction network. aWCluster successfully clusters all cancer types into classes with significantly different survival rates. Also, a gene ontology enrichment analysis of significant genes in the low survival subgroup of breast cancer leads to the well-known phenomenon of tumor hypoxia and the transcription factor ETS1 whose expression is induced by hypoxia. We believe aWCluster has the potential to discover novel subtypes and biomarkers by accentuating the genes that have concordant multiomics measurements in their interaction network, which are challenging to find without the network inference or with single omics analysis. [ABSTRACT FROM AUTHOR]

Although the basic molecular components that promote regulated neurotransmitter release are well established, the contribution of these proteins as regulators of the plasticity of neurotransmission and refinement of synaptic connectivity during development is elaborated less fully. For example, during the period of synaptic growth and maturation in brain, the expression of synaptosomal protein 25 kDa (SNAP-25), a neuronal t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) essential for action potential-dependent neuroexocytosis, is altered through alternative splicing of pre-mRNA transcripts. We addressed the role of the two splice-variant isoforms of SNAP-25 with a targeted mouse mutation that impairs the shift from SNAP-25a to SNAP-25b. Most of these mutant mice die between 3 and 5 weeks of age, which coincides with the time when SNAP-25b expression normally reaches mature levels in brain and synapse formation is essentially completed. The altered expression of these SNAP-25 isoforms influences short-term synaptic function by affecting facilitation but not the initial probability of release. This suggests that mechanisms controlling alternative splicing between SNAP-25 isoforms contribute to a molecular switch important for survival that helps to guide the transition from immature to mature synaptic connections, as well as synapse regrowth and remodeling after neural injury. [ABSTRACT FROM AUTHOR]

Axon outgrowth during development and neurotransmitter release depends on exocytotic mechanisms, although what protein machinery is common to or differentiates these processes remains unclear. Here we show that the neural t-SNARE (target-membrane-associated–soluble N-ethylmaleimide fusion protein attachment protein (SNAP) receptor) SNAP-25 is not required for nerve growth or stimulus-independent neurotransmitter release, but is essential for evoked synaptic transmission at neuromuscular junctions and central synapses. These results demonstrate that the development of neurotransmission requires the recruitment of a specialized SNARE core complex to meet the demands of regulated exocytosis. [ABSTRACT FROM AUTHOR]