64 research outputs found
Structures Involved in the Oligomerization of Prestin
No full textHearing is an essential part of daily life for most people, yet little is known its molecular constituents. Cochlear amplification is the mechanism by which the hearing process is tuned and boosted in the inner ear. Somatic motility, a unique property of the outer hair cells in the inner ear, is a major component of cochlear amplification. Outer hair cell somatic motility is driven by the motor protein prestin, but little is known about the structure-function relationship of the motor protein prestin. This has lead to disputes over its role in cochlear amplification. This work seeks to clarify the structure-function relationship of prestin by testing the hypothesis that the prestin protein family’s function is dependent on homo-oligomerization through the STAS domain. Förster resonance energy transfer demonstrated that homo-oligomerization occurs in several prestin homologous sequences. Subsequent sequence analysis of prestin homologous sequences revealed a model of the STAS domain, a putative protein-protein motif in the STAS domain, and two putative pore regions in the transmembrane region. Scanning cysteine mutagenesis suggested that one cysteine (C415) affects both structure and function and may have a role in disulfide bond formation. Mutation of the protein-protein motif in the STAS domain also significantly altered both structure and function, but it is unclear the role this motif plays in homo-oligomerization. These results, along with recently published structural data, were used to generate a refined model of prestin. This model postulates that the STAS domain acts as an ‘ATP-gate’ regulating prestin function. If correct, this model may help further our understanding of the structure-function relationship of prestin and its role in human hearing.ProQuest Traditional Publishing Optionvii, 213 page
A New Approach for Sequence Analysis
No full textUnderstanding the structure-function relationship of proteins offers the key to biological processes, and can offer knowledge for better investigation of matters with widespread impact, such as pathological disease and drug intervention. This relationship is dictated at the simplest level by the primary protein sequence. Since useful structures and functions are conserved within biology, a sequence with known structure-function relationship can be compared to related sequences to aid in novel structure-function prediction. Sequence analysis provides a means for suggesting evolutionary relationships, and inferring structural or functional similarity. It is crucial to consider these parameters while comparing sequences as they influence both the algorithms used and the implications of the results. For example, proteins that are closely related on an evolutionary time scale may have very similar structure, but entirely different functions. In contrast, proteins which have undergone convergent evolution may have dissimilar primary structure, but perform similar functions. This chapter details how the aspects of evolution, structure, and function can be taken into account when performing sequence analysis, and proposes an expansion on traditional approaches resulting in direct improvement of said analysis. This model is applied to a case study in the prestin protein and shows that the proposed approach provides a better understanding of input and output and can improve the performance of sequence analysis by means of motif detection software.</jats:p
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Abstract 1151: Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment
No full textAbstract Glioblastoma multiforme (GBM) is the most aggressive form of primary brain tumor, with no curative treatment options. Multiple studies have characterized at single cell resolution the GBM as being composed of transcriptional cell states interconnected with components in the tumor immune microenvironment (TME). Our group proposed and validated the first single cell guided functional classification of GBM in four tumor-intrinsic cell states which informed clinical outcome and delivered therapeutic options. However, single cell technologies are unable to unravel the spatial relationships among the cell states of GBM and between GBM cell states and TME. Spatially resolved transcriptomic technologies are emerging as powerful tools to reconstruct the spatial architecture of a tissue. We performed spatial transcriptomics of multicellular regions of interest (ROI) in 6 primary IDH wild-type GBM and 2 recurrent GBM with both CosMx Spatial Molecular Imager, which analyzes 1,000 RNA probes and 64 proteins at single cell resolution, and GeoMx Digital Spatial Profiler which profiles the whole transcriptome (~18,000 genes) at ROI resolution. The development of computational tools aimed to integrate spatial proximity and CosMx derived single-cell transcriptomics revealed spatial segregation of the tumor cell clones and cellular states and highlighted recurrent patterns of cell states, distinct TME cell types associated with coherent histopathological features across multiple samples. The development of a spatial informed intercellular communication algorithm and the reconstruction of ligand-receptor-target networks will allow the discovery of tumor cell states-TME cross-talks and the biological signaling regulated by these interactions that are driving the heterogeneity of GBM and therefore potentially therapeutically targetable. Analysis of matched regions of interest profiled by GeoMx and spatial proteomics with CosMx further cross-validated the spatial ecosystem of glioblastoma as reconstructed at single-cell resolution. Our studies established a scalable approach to resolve the transcriptional heterogeneity of GBM and reconstruct the architecture of GBM cell states and tumor microenvironment. Citation Format: Bruno Adabbo, Simona Migliozzi, Luciano Garofano, Fulvio D'Angelo, Pedro Davila, Sakir H. Gultekin, Daniel Bilbao Cortes, Benjamin Currall, Sion L. Williams, Marc Sanson, Franck Bielle, Anna Luisa Di Stefano, Michele Ceccarelli, Anna Lasorella, Antonio Iavarone. Reconstruction of the spatial ecosystem of glioblastoma reveals relationships between tumor cell states and microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1151
The roles of conserved and nonconserved cysteinyl residues in the oligomerization and function of mammalian prestin
No full text The creation of several prestin knockout and knockin mouse lines has demonstrated the importance of the intrinsic outer hair cell membrane protein prestin to mammalian hearing. However, the structure of prestin remains largely unknown, with even its major features in dispute. Several studies have suggested that prestin forms homo-oligomers that may be stabilized by disulfide bonds. Our phylogenetic analysis of prestin sequences across chordate classes suggested that the cysteinyl residues could be divided into three groups, depending on the extent of their conservation between prestin orthologs and paralogs or homologs. An alanine scan functional analysis was performed of all nine cysteinyl positions in mammalian prestin. Prestin function was assayed by measurement of prestin-associated nonlinear capacitance. Of the nine cysteine-alanine substitution mutations, all were properly membrane targeted and all demonstrated nonlinear capacitance. Four mutations (C124A, C192A, C260A, and C415A), all in nonconserved cysteinyl residues, significantly differed in their nonlinear capacitance properties compared with wild-type prestin. In the two most severely disrupted mutations, substitution of the polar residue seryl for cysteinyl restored normal function in one (C415S) but not the other (C124S). We assessed the relationship of prestin oligomerization to cysteine position using fluorescence resonance energy transfer. With one exception, cysteine-alanine substitutions did not significantly alter prestin-prestin interactions. The exception was C415A, one of the two nonconserved cysteinyl residues whose mutation to alanine caused the most disruption in function. We suggest that no disulfide bond is essential for prestin function. However, C415 likely participates by hydrogen bonding in both nonlinear capacitance and oligomerization. </jats:p
An intelligent data-centric approach toward identification of conserved motifs in protein sequences
No full textThe continued integration of the computational and biological sciences has revolutionized genomic and proteomic studies. However, efficient collaboration between these fields requires the creation of shared standards. A common problem arises when biological input does not properly fit the expectations of the algorithm, which can result in misinterpretation of the output. This potential confounding of input/output is a drawback especially when regarding motif finding software. Here we propose a method for improving output by selecting input based upon evolutionary distance, domain architecture, and known function. This method improved detection of both known and unknown motifs in two separate case studies. By standardizing input considerations, both biologists and bioinformaticians can better interpret and design the evolving sophistication of bioinformatic software
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