Substrate selection by the ClpXP protease : a tail of destruction

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, September 2004."August 2004."Includes bibliographical references (p. 195-211).(cont.) sites for CIpA and SspB; this spatial arrangement of signals allows for efficient modulation of proteolysis of ssrA-tagged proteins. Finally, additional substrates whose degradation may be regulated by the adaptor protein SspB were determined by identifying substrates captured in ClpXP[trap] in an sspB⁺ strain but not an sspB⁻ strain. This analysis led to the identification of the N-terminal fragment of RseA, the master regulator of the extracytoplasmic stress response, as a protein whose ClpXP-mediated degradation is also enhanced by SspB. Degradation of N-RseA leads to activation of [sigma]E and thus induction of the extra-cytoplasmic stress response. This thesis work has contributed to the understanding of how intracellular proteolysis is regulated to accommodate the selective degradation of a broad range of substrates. ClpXP uses an assortment of recognition strategies, including degradation tags and adaptor proteins, in a combinatorial fashion to regulate protein degradation.Intracellular proteolysis plays a vital role in many regulatory pathways, helping cells survive a battery of stresses including oxidative damage, heat shock, and starvation. The majority of cellular proteins are degraded very slowly; however, certain proteins are extremely unstable. The concentration of unstable regulatory proteins can be adjusted quickly in response to altered cellular conditions by changes in their rate of degradation and synthesis. In addition, proteases can remove proteins from the cell when their activities are no longer required. Proteolysis is thus a powerful mechanism to regulate many cellular pathways. To execute these tasks, it is imperative that intracellular proteases select their substrates swiftly and discerningly. This thesis explores the strategies used by the Escherichia coli energy-dependent protease, ClpXP, to correctly select its substrates for destruction. Prior to our work, only a small group of ClpXP substrates were known. To identify a larger group, we captured intact substrates in vivo inside of a ClpXP[trap]. Sequence analysis of these identified substrates combined with peptide binding experiments revealed five common motifs that are directly recognized by CIpXP, representing the first general description of rules governing substrate recognition by this protease. Direct recognition of these accessible degradation tags can be further modulated by adaptor proteins. SspB is an adaptor protein identified for its ability to enhance the degradation of ssrA-tagged proteins by ClpXP. We dissected the sequence information in the ssrA tag required for recognition by ClpX, SspB, and ClpA, another ClpP partner. The ssrA tag contains contiguous bindings sites for CIpX and SspB, butby Julia M. Flynn.Ph.D

Pervasive contingency and entrenchment in a billion years of Hsp90 evolution [preprint]

Interactions among mutations within a protein have the potential to make molecular evolution contingent and irreversible, but the extent to which epistasis actually shaped historical evolutionary trajectories is unclear. We addressed this question by identifying all amino acid substitutions that occurred during the billion-year evolutionary history of the heat shock protein 90 (Hsp90) ATPase domain beginning from a deep eukaryotic ancestor to modern Saccharomyces cerevisiae and then precisely measuring their fitness effects when introduced into both extant and reconstructed ancestral Hsp90 proteins. We find a pervasive influence of epistasis: of 98 derived states that evolved during history, most were deleterious at times before they happened, and the vast majority also became subsequently entrenched, with the ancestral state becoming deleterious after its substitution. This epistasis was primarily caused by specific interactions among sites rather than a general permissive or restrictive effect on the protein\u27s tolerance to mutation. Our results show that epistasis continually opens and closes windows of mutational opportunity over evolutionary timescales, producing histories and biological states that reflect the transient internal constraints imposed by a protein\u27s fleeting sequence states

The adaptive potential of the M-domain of yeast Hsp90 [preprint]

Comparing the distribution of fitness effects (DFE) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in other environments. So far, results regarding the cost of adaptation across environments have been mixed, and there were no sufficiently large data sets to map its variation along the genome. Here, we study the DFEs of ≈2500 amino-acid changing mutations obtained from deep mutational scanning of the 118 amino-acid-long middle domain of the heat-shock protein Hsp90 in five environments and at two expression levels. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-M and M-C interdomains and regulation of ATPase-chaperone activity. Despite the diverse and stressful environments, we find that fitness correlates well across environments, with the exception of one environment, diamide. Consistent with these results, we find very little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client binding interfaces or residues that are involved in ATPase chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information

A New 13 Million Year Old Gavialoid Crocodylian from Proto-Amazonian Mega-Wetlands Reveals Parallel Evolutionary Trends in Skull Shape Linked to Longirostry

Gavialoid crocodylians are the archetypal longirostrine archosaurs and, as such, understanding their patterns of evolution is fundamental to recognizing cranial rearrangements and reconstructing adaptive pathways associated with elongation of the rostrum (longirostry). The living Indian gharial Gavialis gangeticus is the sole survivor of the group, thus providing unique evidence on the distinctive biology of its fossil kin. Yet phylogenetic relationships and evolutionary ecology spanning ~70 million-years of longirostrine crocodylian diversification remain unclear. Analysis of cranial anatomy of a new proto-Amazonian gavialoid, Gryposuchus pachakamue sp. nov., from the Miocene lakes and swamps of the Pebas Mega-Wetland System reveals that acquisition of both widely separated and protruding eyes (telescoped orbits) and riverine ecology within South American and Indian gavialoids is the result of parallel evolution. Phylogenetic and morphometric analyses show that, in association with longirostry, circumorbital bone configuration can evolve rapidly for coping with trends in environmental conditions and may reflect shifts in feeding strategy. Our results support a long-term radiation of the South American forms, with taxa occupying either extreme of the gavialoid morphospace showing preferences for coastal marine versus fluvial environments. The early biogeographic history of South American gavialoids was strongly linked to the northward drainage system connecting proto-Amazonian wetlands to the Caribbean region

Teorizando a dobradiça cartonera: pesquisa trans-formal para uma prática transformadora

Trata-se de uma tradução do capítulo “Methods: Trans-Formal Research for Transformational Practice”, parte do livro Taking Form, Making Worlds: Cartonera Publishers in Latin America (2022). Ao longo do artigo, os autores acompanham as atividades das cartoneras mexicanas La Rueda Cartonera e Viento Cartonero para teorizar sobre o fazer cartonero e, ao mesmo tempo, elaborar uma reflexão sobre a metodologia para fazê-lo, partindo de uma visão interdisciplinar. Nesse processo, Bell, Flynn e O’Hare defendem o conceito de “dobradiça” para nomear um duplo caminho desse tipo de projeto social e editorial: a presença da estrutura sociopolítica nas práticas cartoneras e, por sua vez, o impacto das cartoneras nas práticas sociopolíticas

Teorizando a dobradiça cartonera: pesquisa trans-formal para uma prática transformadora

Trata-se de uma tradução do capítulo “Methods: Trans-Formal Research for Transformational Practice”, parte do livro Taking Form, Making Worlds: Cartonera Publishers in Latin America (2022). Ao longo do artigo, os autores acompanham as atividades das cartoneras mexicanas La Rueda Cartonera e Viento Cartonero para teorizar sobre o fazer cartonero e, ao mesmo tempo, elaborar uma reflexão sobre a metodologia para fazê-lo, partindo de uma visão interdisciplinar. Nesse processo, Bell, Flynn e O’Hare defendem o conceito de “dobradiça” para nomear um duplo caminho desse tipo de projeto social e editorial: a presença da estrutura sociopolítica nas práticas cartoneras e, por sua vez, o impacto das cartoneras nas práticas sociopolíticas.This is a translation of the chapter “Methods: Trans-Formal Research for Transformational Practice”, part of the book Taking Form, Making Worlds: Cartonera Publishers in Latin America (2022). Throughout the article, the authors follow the activities of the Mexican cartoneras La Rueda Cartonera and Viento Cartonero to elaborate a theory of cartonera practice and, at the same time, to offer a reflection on the methodology they have developed to work on and with cartonera, based on an interdisciplinary vision. In this process, Bell, Flynn and O’Hare propose the concept of the “double fold” to name a double path of this type of social and artistic project: the presence of the sociopolitical structures in cartonera practices and, in turn, the impact of cartoneras in sociopolitical practices

The Adaptive Potential of the Middle Domain of Yeast Hsp90

The distribution of fitness effects (DFEs) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the distribution of fitness effects of the same approximately 2,300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase-chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client-binding interfaces, or residues that are involved in ATPase-chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information

Investigating the influence of environment on the evolution of Hsp90 using comprehensive fitness maps [preprint]

Gene-environment interactions have long been theorized to influence molecular evolution. However, the environmental dependence of most mutations remains unknown. Using deep mutational scanning, we engineered budding yeast with all 44,604 single codon changes encoding 14,160 amino acid variants in Hsp90 and quantified growth effects under standard laboratory conditions and under five stress conditions (elevated temperature, nitrogen starvation, elevated salinity, high ethanol concentration, and oxidative stress caused by diamide). To our knowledge these are the largest comprehensive fitness maps of point mutant growth effects that have been determined. The growth effects of many variants differed between each of the conditions, indicating that environmental conditions can have a large impact on the evolution of Hsp90. Multiple variants provided growth advantages relative to wildtype Hsp90 under individual conditions, however these variants tended to exhibit growth defects in other environments. The diversity of Hsp90 sequences observed in extant eukaryotes preferentially contain amino acid variants that supported robust growth under all tested conditions. Thus, rather than favoring substitutions in individual conditions, the long-term selective pressure on Hsp90 may have been that of fluctuating environments, leading to robustness under a variety of conditions

Lipoprotein Profiles in Class III Obese Caucasian and African American Women with Nonalcoholic Fatty Liver Disease

Triglyceride content in the liver is regulated by the uptake, production and elimination of lipoproteins, and derangements in these processes contribute to nonalcoholic fatty liver disease (NAFLD). Previous studies show a direct relationship between intrahepatic fat and production of apolipoprotein B100 (apoB100) containing particles, VLDL and LDL, but little consensus exists regarding changes in lipoprotein production in the development of simple steatosis (SS) versus nonalcoholic steatohepatitis (NASH). Further, ethnic variations in lipoproteins among SS and NASH are unknown as is how such variations might contribute to the differential prevalence of disease among Caucasians versus African Americans. In this study, we assessed plasma lipoprotein profiles by nuclear magnetic resonance (NMR) spectroscopy in 70 non-diabetic class III obese females recruited from the surgical weight loss clinic. Of these, 51 females were stratified by biopsy-staged NAFLD severity (histologically normal, SS, or NASH). NASH females displayed increased circulating triglycerides and increased VLDL particle number and size relative to those with histologically normal livers, while total and large LDL concentration decreased in SS versus NASH and correlated with increased insulin resistance (via HOMA2-IR). When Caucasian women were examined alone (n = 41), VLDL and triglycerides increased between normal and SS, while total LDL and apoB100 decreased between SS and NASH along with increased insulin resistance. Compared to Caucasians with SS, African American women with SS displayed reduced triglycerides, VLDL, and small LDL and a more favorable small to large HDL ratio despite having increased BMI and HOMA2-IR. These findings suggest that ApoB100 and lipoprotein subclass particle number and size can delineate steatosis from NASH in obese Caucasian females, but should be interpreted with caution in other ethnicities as African Americans with SS display relatively improved lipoprotein profiles. This may reflect variation in the relationship between dyslipidemia and NAFLD progression across gender and ethnicity