Mechanisms of protein misfolding in conformational lung diseases

Genetic or environmentally-induced alterations in protein structure interfere with the correct folding, assembly and trafficking of proteins. In the lung the expression of misfolded proteins can induce a variety of pathogenetic effects. Cystic fibrosis (CF) and alpha-1 antitrypsin (AAT) deficiency are two major clinically relevant pulmonary disorders associated with protein misfolding. Both are genetic diseases the primary causes of which are expression of mutant alleles of the cystic fibrosis transmembrane conductance regulator (CFTR) and SERPINA1, respectively. The most common and best studied mutant forms of CFTR and AAT are ΔF508 CFTR and the Glu342Lys mutant of AAT called ZAAT, respectively. Non-genetic mechanisms can also damage protein structure and induce protein misfolding in the lung. Cigarette-smoke contains oxidants and other factors that can modify a protein's structure, and is one of the most significant environmental causes of protein damage within the lung. Herein we describe the mechanisms controlling the folding of wild type and mutant versions of CFTR and AAT proteins, and explore the consequences of cigarette-smoke-induced effects on the protein folding machinery in the lung.     

A genetic screening strategy identifies novel regulators of the proteostasis network

A hallmark of diseases of protein conformation and aging is the appearance of protein aggregates associated with cellular toxicity. We posit that the functional properties of the proteostasis network (PN) protect the proteome from misfolding and combat the proteotoxic events leading to cellular pathology. In this study, we have identified new components of the proteostasis network that can suppress aggregation and proteotoxicity, by performing RNA interference (RNAi) genetic screens for multiple unrelated conformationally challenged cytoplasmic proteins expressed in Caenorhabditis elegans. We identified 88 suppressors of polyglutamine (polyQ) aggregation, of which 63 modifiers also suppressed aggregation of mutant SOD1(G93A). Of these, only 23 gene-modifiers suppressed aggregation and restored animal motility, revealing that aggregation and toxicity can be genetically uncoupled. Nine of these modifiers were shown to be effective in restoring the folding and function of multiple endogenous temperature-sensitive (TS) mutant proteins, of which five improved folding in a HSF-1-dependent manner, by inducing cytoplasmic chaperones. This triage screening strategy also identified a novel set of PN regulatory components that, by altering metabolic and RNA processing functions, establish alternate cellular environments not generally dependent on stress response activation and that are broadly protective against misfolded and aggregation-prone proteins.     

Small-molecule catalysts of oxidative protein folding

Oxidative protein folding occurs both in vivo and in vitro and involves the formation and rearrangement of protein disulfide bonds (SS bonds). In vivo these reactions are catalyzed by enzymes, including the eukaryotic enzyme protein disulfide isomerase (PDI). Using the physical properties of PDI as a guide, several small-molecule catalysts of oxidative protein folding have been designed, synthesized, and tested. These small molecules can improve the folding rate of the model substrate ribonuclease A by a factor of over 10 and improve the yield by up to a factor of 3 over traditional conditions. The molecules have also been demonstrated to significantly improve the in vivo folding of proteins as well.     

TRIM family proteins: roles in proteostasis and neurodegenerative diseases

Neurodegenerative diseases (NDs) are a diverse group of disorders characterized by the progressive degeneration of the structure and function of the central or peripheral nervous systems. One of the major features of NDs, such as Alzheimer''s disease (AD), Parkinson''s disease (PD) and Huntington''s disease (HD), is the aggregation of specific misfolded proteins, which induces cellular dysfunction, neuronal death, loss of synaptic connections and eventually brain damage. By far, a great amount of evidence has suggested that TRIM family proteins play crucial roles in the turnover of normal regulatory and misfolded proteins. To maintain cellular protein quality control, cells rely on two major classes of proteostasis: molecular chaperones and the degradative systems, the latter includes the ubiquitin-proteasome system (UPS) and autophagy; and their dysfunction has been established to result in various physiological disorders including NDs. Emerging evidence has shown that TRIM proteins are key players in facilitating the clearance of misfolded protein aggregates associated with neurodegenerative disorders. Understanding the different pathways these TRIM proteins employ during episodes of neurodegenerative disorder represents a promising therapeutic target. In this review, we elucidated and summarized the diverse roles with underlying mechanisms of members of the TRIM family proteins in NDs.     

蛋白质构象病

蛋白质的空间结构又称为三维结构或构象（conformation）,特定的空间构象是蛋白质发挥其各种功能的结构基础。由于蛋白质担负着复杂的生化反应,因此在生物合成以后,蛋白质本身也经历着复杂的生理过程;蛋白质自翻译以后,需进行一系列的翻译后过程,包括跨膜转运、修饰加工、折叠复性、生化反应、生物降解等,这些过程都伴随着蛋白质的结构转换。随着对疯牛病的研究,人们发现：蛋白质分子的氨基酸序列虽不改变,但其空间结构或构象的改变也能引起疾病。同时,越来越多的研究表明,一些遗传性疾病是由于基因突变导致了蛋白质的错误折叠,这些突变并不直接影响蛋白质的功能结构域,但由于蛋白质的错误折叠,干扰了其正确运输,形成对细胞有毒性作用的聚积物。     

Small-molecule FRET probes for protein kinase activity monitoring in living cells

In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Förster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.     

Amyloid cores in prion domains: Key regulators for prion conformational conversion

Despite the significant efforts devoted to decipher the particular protein features that encode for a prion or prion-like behavior, they are still poorly understood. The well-characterized yeast prions constitute an ideal model system to address this question, because, in these proteins, the prion activity can be univocally assigned to a specific region of their sequence, known as the prion forming domain (PFD). These PFDs are intrinsically disordered, relatively long and, in many cases, of low complexity, being enriched in glutamine/asparagine residues. Computational analyses have identified a significant number of proteins having similar domains in the human proteome. The compositional bias of these regions plays an important role in the transition of the prions to the amyloid state. However, it is difficult to explain how composition alone can account for the formation of specific contacts that position correctly PFDs and provide the enthalpic force to compensate for the large entropic cost of immobilizing these domains in the initial assemblies. We have hypothesized that short, sequence-specific, amyloid cores embedded in PFDs can perform these functions and, accordingly, act as preferential nucleation centers in both spontaneous and seeded aggregation. We have shown that the implementation of this concept in a prediction algorithm allows to score the prion propensities of putative PFDs with high accuracy. Recently, we have provided experimental evidence for the existence of such amyloid cores in the PFDs of Sup35, Ure2, Swi1, and Mot3 yeast prions. The fibrils formed by these short stretches may recognize and promote the aggregation of the complete proteins inside cells, being thus a promising tool for targeted protein inactivation.     

The unfolded protein response and proteostasis in Alzheimer disease

Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore proteostasis in the ER. Previously, we demonstrated that UPR activation is an early event in Alzheimer disease (AD) brain. In our recent work we investigated whether activation of the UPR is employed to enhance the capacity of the ubiquitin proteasome system or autophagy in neuronal cells. We showed that the levels, composition and activity of the proteasome are not regulated by the UPR. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.     

Use of yeast as a model system to investigate protein conformational diseases

Protein conformational diseases arise when a cellular protein adopts an aberrant shape that either directly or indirectly alters the physiology of its host cell. Notable conformational diseases include cystic fibrosis, Huntington’s disease, the prion-related diseases, Alzheimer’s disease, and antitrypsin deficiency. In principle, the severity and progression of conformational diseases can be altered by cellular factors that recognize and attempt to ameliorate the harmful effects of the disease-causing, misshapen protein. To better define the mechanistic underpinnings of cellular factors that mediate quality control, and to understand why a single misfolded protein can impact cell viability, specific proteins that cause each of the diseases listed above have been expressed in a model eukaryote, the yeast Saccharomyces cerevisiae. In this review, we describe what has been learned from these studies, and speculate on future uses of yeast expression systems.     

Feature space resampling for protein conformational search

De novo protein structure prediction requires location of the lowest energy state of the polypeptide chain among a vast set of possible conformations. Powerful approaches include conformational space annealing, in which search progressively focuses on the most promising regions of conformational space, and genetic algorithms, in which features of the best conformations thus far identified are recombined. We describe a new approach that combines the strengths of these two approaches. Protein conformations are projected onto a discrete feature space which includes backbone torsion angles, secondary structure, and beta pairings. For each of these there is one “native” value: the one found in the native structure. We begin with a large number of conformations generated in independent Monte Carlo structure prediction trajectories from Rosetta. Native values for each feature are predicted from the frequencies of feature value occurrences and the energy distribution in conformations containing them. A second round of structure prediction trajectories are then guided by the predicted native feature distributions. We show that native features can be predicted at much higher than background rates, and that using the predicted feature distributions improves structure prediction in a benchmark of 28 proteins. The advantages of our approach are that features from many different input structures can be combined simultaneously without producing atomic clashes or otherwise physically inviable models, and that the features being recombined have a relatively high chance of being correct. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Computational strategies for protein conformational ensemble detection

Protein function is constrained by the three-dimensional structure but is delineated by its dynamics. This framework must satisfy specificity of function along with adaptability to changing environments and evolvability under external constraints. The accessibility of the available regions of the energy landscape for a set of conditions and shifts in the populations upon their modulation have effects propagating across scales, from biomolecular interactions, to organisms, to populations. Developing the ability to detect and juggle protein conformations supplemented by a physics-based understanding has implications for not only in vivo problems but also for resistance impeding drug discovery and bionano-sensor design.     

Tryptophan-based fluorophores for studying protein conformational changes

With the continuing interest in deciphering the interplay between protein function and conformational changes, small fluorescence probes will be especially useful for tracking changes in the crowded protein interior space. Presently, we describe the potential utility of six unnatural amino acid fluorescence donors structurally related to tryptophan and show how they can be efficiently incorporated into a protein as fluorescence probes. We also examine the various photophysical properties of the new Trp analogues, which are significantly redshifted in their fluorescence spectra relative to tryptophan. In general, the Trp analogues were well tolerated when inserted into Escherichia coli DHFR, and did not perturb enzyme activity, although substitution for Trp22 did result in a diminution in DHFR activity. Further, it was demonstrated that D and E at position 37 formed efficient FRET pairs with acridon-2-ylalanine (Acd) at position 17. The same was also true for a DHFR construct containing E at position 79 and Acd at position 17. Together, these findings demonstrate that these tryptophan analogues can be introduced into DHFR with minimal disruption of function, and that they can be employed for the selective study of targeted conformational changes in proteins, even in the presence of unmodified tryptophans.     

microRNAs:心血管疾病重要的调控因子

微RNA（microRNA,miRNA）是一类内源性19—25个核苷酸大小的非编码RNA分子,在进化中具有高度保守性,并且能够通过碱基匹配原则识别靶基因3’非翻译区的靶位点,从而抑制编码蛋白靶基因的翻译或（和）降解靶基因。目前的研究表明,miRNA在生物体发育、心血管疾病以及肿瘤发生等过程中起重要作用。本文对miRNA在心血管系统生理病理中的作用做一综述。