Collagen structure and stability Shoulders, Matthew D; Raines, Ronald T
Annual review of biochemistry,
01/2009, Letnik:
78, Številka:
1
Journal Article
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Collagen is the most abundant protein in animals. This fibrous, structural protein comprises a right-handed bundle of three parallel, left-handed polyproline II-type helices. Much progress has been ...made in elucidating the structure of collagen triple helices and the physicochemical basis for their stability. New evidence demonstrates that stereoelectronic effects and preorganization play a key role in that stability. The fibrillar structure of type I collagen-the prototypical collagen fibril-has been revealed in detail. Artificial collagen fibrils that display some properties of natural collagen fibrils are now accessible using chemical synthesis and self-assembly. A rapidly emerging understanding of the mechanical and structural properties of native collagen fibrils will guide further development of artificial collagenous materials for biomedicine and nanotechnology.
Directed evolution experiments are typically carried out using in vitro systems, bacteria, or yeast-even when the goal is to probe or modulate mammalian biology. Performing directed evolution in ...systems that do not match the intended mammalian environment severely constrains the scope and functionality of the targets that can be evolved. We review new platforms that are now making it possible to use the mammalian cell itself as the setting for directed evolution and present an overview of frontier challenges and high-impact targets for this approach.
The unfolded protein response (UPR) maintains endoplasmic reticulum (ER) proteostasis through the activation of transcription factors such as XBP1s and ATF6. The functional consequences of these ...transcription factors for ER proteostasis remain poorly defined. Here, we describe methodology that enables orthogonal, small-molecule-mediated activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the same cell independent of stress. We employ transcriptomics and quantitative proteomics to evaluate ER proteostasis network remodeling owing to the XBP1s and/or ATF6 transcriptional programs. Furthermore, we demonstrate that the three ER proteostasis environments accessible by activating XBP1s and/or ATF6 differentially influence the folding, trafficking, and degradation of destabilized ER client proteins without globally affecting the endogenous proteome. Our data reveal how the ER proteostasis network is remodeled by the XBP1s and/or ATF6 transcriptional programs at the molecular level and demonstrate the potential for selective restoration of aberrant ER proteostasis of pathologic, destabilized proteins through arm-selective UPR activation.
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► Orthogonal, ligand-dependent control of XBP1s and/or ATF6 in a single cell ► Proteomic and transcriptomic characterization of XBP1s and/or ATF6 activation ► XBP1s and/or ATF6 influences pathogenic protein fates, but not the endogenous proteome ► Arm-selective UPR activation reduces secretion of destabilized transthyretin variants
The unfolded protein response adapts endoplasmic reticulum (ER) proteostasis via stress-responsive transcription factors, including XBP1s and ATF6. Here, Wiseman and colleagues implement technology for the orthogonal, ligand-dependent activation of XBP1s and/or ATF6 in a single cell. They characterize how XBP1s and/or ATF6 activation affects ER proteostasis pathway composition and function. Adapted ER environments influence the proteostasis of destabilized protein variants without affecting the endogenous proteome. The work informs the development of proteostasis-environment-adapting therapeutics for protein-misfolding-related diseases.
Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the ...components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.
Cas9-based technologies have transformed genome engineering and the interrogation of genomic functions, but methods to control such technologies across numerous dimensions-including dose, time, ...specificity, and mutually exclusive modulation of multiple genes-are still lacking. We conferred such multidimensional controls to diverse Cas9 systems by leveraging small-molecule-regulated protein degron domains. Application of our strategy to both Cas9-mediated genome editing and transcriptional activities opens new avenues for systematic genome interrogation.
Laboratory time scale evolution in vivo relies on the generation of large, mutationally diverse gene libraries to rapidly explore biomolecule sequence landscapes. Traditional global mutagenesis ...methods are problematic because they introduce many off-target mutations that are often lethal and can engender false positives. We report the development and application of the MutaT7 chimera, a potent and highly targeted in vivo mutagenesis agent. MutaT7 utilizes a DNA-damaging cytidine deaminase fused to a processive RNA polymerase to continuously direct mutations to specific, well-defined DNA regions of any relevant length. MutaT7 thus provides a mechanism for in vivo targeted mutagenesis across multi-kb DNA sequences. MutaT7 should prove useful in diverse organisms, opening the door to new types of in vivo evolution experiments.
The collagenopathies are a diverse group of diseases caused primarily by mutations in collagen genes. The resulting disruptions in collagen biogenesis can impair development, cause cellular ...dysfunction, and severely impact connective tissues. Most existing treatment options only address patient symptoms. Yet, while the disease-causing genes and proteins themselves are difficult to target, increasing evidence suggests that resculpting the intracellular proteostasis network, meaning the machineries responsible for producing and ensuring the integrity of collagen, could provide substantial benefit. We present a proteostasis-focused perspective on the collagenopathies, emphasizing progress toward understanding how mechanisms of collagen proteostasis are disrupted in disease. In parallel, we highlight recent advances in small molecule approaches to tune endoplasmic reticulum proteostasis that may prove useful in these disorders.
The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at ...least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The discovery and optimization of biomolecules that reliably function in metazoan cells is imperative for both the study of basic biology and the treatment of disease. We describe the development, ...characterization, and proof-of-concept application of a platform for directed evolution of diverse biomolecules of interest (BOIs) directly in human cells. The platform relies on a custom-designed adenovirus variant lacking multiple genes, including the essential DNA polymerase and protease genes, features that allow us to evolve BOIs encoded by genes as large as 7 kb while attaining the mutation rates and enforcing the selection pressure required for successful directed evolution. High mutagenesis rates are continuously attained by trans-complementation of a newly engineered, highly error-prone form of the adenoviral polymerase. Selection pressure that couples desired BOI functions to adenoviral propagation is achieved by linking the functionality of the encoded BOI to the production of adenoviral protease activity by the human cell. The dynamic range for directed evolution can be enhanced to several orders of magnitude via application of a small-molecule adenoviral protease inhibitor to modulate selection pressure during directed evolution experiments. This platform makes it possible, in principle, to evolve any biomolecule activity that can be coupled to adenoviral protease expression or activation by simply serially passaging adenoviral populations carrying the BOI. As proof-of-concept, we use the platform to evolve, directly in the human cell environment, several transcription factor variants that maintain high levels of function while gaining resistance to a small-molecule inhibitor. We anticipate that this platform will substantially expand the repertoire of biomolecules that can be reliably and robustly engineered for both research and therapeutic applications in metazoan systems.
Protein disulfide isomerase A1 (PDIA1) is an endoplasmic reticulum (ER)-localized thiol-disulfide oxidoreductase that is an important folding catalyst for secretory pathway proteins. PDIA1 contains ...two active-site domains (a and a′), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. The two active-site domains share 37% sequence identity and function independently to perform disulfide-bond reduction, oxidation, and isomerization. Numerous inhibitors for PDIA1 have been reported, yet the selectivity of these inhibitors toward the a and a′ sites is poorly characterized. Here, we identify a potent and selective PDIA1 inhibitor, KSC-34, with 30-fold selectivity for the a site over the a′ site. KSC-34 displays time-dependent inhibition of PDIA1 reductase activity in vitro with a k inact/K I of 9.66 × 103 M–1 s–1 and is selective for PDIA1 over other members of the PDI family, and other cellular cysteine-containing proteins. We provide the first cellular characterization of an a-site selective PDIA1 inhibitor and demonstrate that KSC-34 has minimal sustained effects on the cellular unfolded protein response, indicating that a-site inhibition does not induce global protein folding-associated ER stress. KSC-34 treatment significantly decreases the rate of secretion of a destabilized, amyloidogenic antibody light chain, thereby minimizing pathogenic amyloidogenic extracellular proteins that rely on high PDIA1 activity for proper folding and secretion. Given the poor understanding of the contribution of each PDIA1 active site to the (patho)physiological functions of PDIA1, site selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1.
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