scholarly journals ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

2020 ◽  
Author(s):  
Sora Kim ◽  
Kristin L Zuromski ◽  
Tristan A Bell ◽  
Robert T Sauer ◽  
Tania A Baker
Keyword(s):  
Protein Degradation ◽  
Protease Activity ◽  
Protein Substrates ◽  
Hand Model ◽  
Clpap Protease ◽  
Optimal Function

AbstractAAA+ proteases, which perform regulated protein degradation in all kingdoms of life, consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX must rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP omplexes unfold, translocate, and degrade protein substrates, albeit modestly slower han uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP therefore is ot required for ClpAP protease activity, although some flexibility in how the AAA+ ring ocks on ClpP may be necessary for optimal function.

scholarly journals ClpAP proteolysis does not require rotation of the ClpA unfoldase relative to ClpP

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sora Kim ◽  
Kristin L Zuromski ◽  
Tristan A Bell ◽  
Robert T Sauer ◽  
Tania A Baker
Keyword(s):  
Protein Degradation ◽  
Protease Activity ◽  
Protein Substrates ◽  
Hand Model ◽  
Clpap Protease ◽  
Optimal Function

AAA+ proteases perform regulated protein degradation in all kingdoms of life and consist of a hexameric AAA+ unfoldase/translocase in complex with a self-compartmentalized peptidase. Based on asymmetric features of cryo-EM structures and a sequential hand-over-hand model of substrate translocation, recent publications have proposed that the AAA+ unfoldases ClpA and ClpX rotate with respect to their partner peptidase ClpP to allow function. Here, we test this model by covalently crosslinking ClpA to ClpP to prevent rotation. We find that crosslinked ClpAP complexes unfold, translocate, and degrade protein substrates in vitro, albeit modestly slower than uncrosslinked enzyme controls. Rotation of ClpA with respect to ClpP is therefore not required for ClpAP protease activity, although some flexibility in how the AAA+ ring docks with ClpP may be necessary for optimal function.

scholarly journals FURTHER STUDIES ON THE ROLE OF PROTEASES IN THE ALLERGIC REACTION

1961 ◽  
Vol 113 (2) ◽  
pp. 359-380 ◽  
Author(s):  
Georges Ungar ◽  
Takuso Yamura ◽  
Jacqueline B. Isola ◽  
Sidney Kobrin
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Proteolytic Activity ◽  
Guinea Pigs ◽  
Protease Activity ◽  
Amino Acid Esters ◽  
Protein Substrates ◽  
Protease Activation ◽  
Acid Esters

Protease activity was measured through the hydrolysis of synthetic amino acid esters in body fluids and tissues of guinea pigs, rats, mice, and humans. Significant in vitro activation was observed in serum and lung slices of sensitized guinea pigs on addition of the specific antigen. Increased proteolytic activity was also seen in reverse anaphylaxis. More marked activation occurred when guinea pig serum was treated with peptone and guinea pig or rat serum was treated with agar. Protease activation was demonstrated in specimens of human skin under the influence of a poison ivy extract or croton oil added in vitro. Urinary protease activity of guinea pigs increased significantly during the first hours of anaphylactic shock and very markedly in peptone shock. Peptone shock, elicited in mice pretreated with H. pertussis, was accompanied by a considerable increase in protease activity in the peritoneal fluid as compared with non-pretreated mice which were insensitive to peptone. Proteolytic activity resulting from the activation procedures was due to a number of proteases. The dominant substrate affinity and inhibition patterns suggest that serum and urine proteases are similar to but not identical with plasmin. Anaphylactic activation exhibited patterns different from those resulting from the action of anaphylactoid agents. Tissue enzymes are either of cathepsin- or chymotrypsin-type or mixtures of both. Some of the activated enzymes, although remarkably effective in hydrolyzing amino acid esters, show no activity on protein substrates. This does not justify, however, their designation as "esterases." They probably belong to the class of specific proteases acting only on a single or a small number of functionally significant protein substrates. There is at present sufficient evidence to prove not only that protease activation does occur in anaphylaxis and anaphylactoid conditions but also that it is an important component of the chain of reactions leading to the allergic response.

Pattern of protein degradation during germination of Streptomyces antibioticus spores

1983 ◽  
Vol 29 (6) ◽  
pp. 637-643 ◽  
Author(s):  
J. A. Guijarro ◽  
J. E. Suarez ◽  
J. A. Salas ◽  
C. Hardisson
Keyword(s):  
Protein Degradation ◽  
Protease Activity ◽  
Germ Tube ◽  
Synthetic Medium ◽  
Distilled Water ◽  
Streptomyces Antibioticus ◽  
Constant Rate ◽  
Degradation Pattern ◽  
Tube Emergence ◽  
Inhibition Studies

The pattern of protein degradation during germination of Streptomyces antibioticus spores was studied by the pulse and chase technique. Two different protein fractions were found. First, a fraction of the proteins synthesized during the darkening process (20–30%) was quickly degraded in the 30 min following the labelling period. This rapid protein degradation was partially inhibited by protease inhibitors: p-chloromercuribenzoic acid, phenylmethylsulphonylfluoride, and o-phenanthroline. Second, the remaining 70–80% and the entire protein population formed during spore swelling and germ tube emergence were degraded with a lower and constant rate (3.3–6.0%/h). A stable mRNA fraction of the dormant spores was translated upon incubation of the spores in a minimal synthetic medium (MSM) or in distilled water. However, the degradation of these proteins did not occur unless the spores were then incubated in the MSM. A strong correlation between the degradation pattern of these proteins and that of those quickly degraded at the beginning of germination was observed. Protease activity in cell-free extracts of dormant spores was detected. Inhibition studies suggest the presence of serine, thiol, and metalloproteases. The protease activity, using casein as substrate, remained constant during the darkening process and started to increase progressively from the beginning of spore swelling.

ClpA and ClpP Remain Associated during Multiple Rounds of ATP-Dependent Protein Degradation by ClpAP Protease

Biochemistry ◽  
1999 ◽  
Vol 38 (45) ◽  
pp. 14906-14915 ◽  
Author(s):  
Satyendra K. Singh ◽  
Fusheng Guo ◽  
Michael R. Maurizi
Keyword(s):  
Protein Degradation ◽  
Clpap Protease ◽  
Dependent Protein

scholarly journals Methods to Discover and Evaluate Proteasome Small Molecule Stimulators

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2341 ◽  
Author(s):  
Rachel A. Coleman ◽  
Darci J. Trader
Keyword(s):  
Neurodegenerative Diseases ◽  
Protein Degradation ◽  
Small Molecule ◽  
Protease Activity ◽  
Ubiquitin Proteasome System ◽  
Proteasome Activity ◽  
Protein Accumulation ◽  
Toxic Protein ◽  
Disease States ◽  
Ubiquitin Proteasome

Protein accumulation has been identified as a characteristic of many degenerative conditions, such as neurodegenerative diseases and aging. In most cases, these conditions also present with diminished protein degradation. The ubiquitin-proteasome system (UPS) is responsible for the degradation of the majority of proteins in cells; however, the activity of the proteasome is reduced in these disease states, contributing to the accumulation of toxic protein. It has been hypothesized that proteasome activity, both ubiquitin-dependent and -independent, can be chemically stimulated to reduce the load of protein in diseased cells. Several methods exist to identify and characterize stimulators of proteasome activity. In this review, we detail the ways in which protease activity can be enhanced and analyze the biochemical and cellular methods of identifying stimulators of both the ubiquitin-dependent and -independent proteasome activities.

scholarly journals The non-dominant AAA+ ring in the ClpAP protease functions as an anti-stalling motor to accelerate protein unfolding and translocation

2019 ◽  
Author(s):  
Hema Chandra Kotamarthi ◽  
Robert. T. Sauer ◽  
Tania. A. Baker
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Protein Unfolding ◽  
Protein Homeostasis ◽  
Protein Substrates ◽  
Clpap Protease

AbstractATP-powered unfoldases containing D1 and D2 AAA+ rings play important roles in protein homeostasis, but uncertainty about the function of each ring remains. Here we use single-molecule optical-tweezers to assay mechanical unfolding and translocation by a variant of the ClpAP protease containing an ATPase-inactive D1 ring. This variant displays substantial mechanical defects both in unfolding and translocation of protein substrates. Notably, when D1 is hydrolytically inactive, ClpAP often stalls for times as long as minutes, and the substrate can “back-slip” through the enzyme when ATP concentrations are low. The inactive D1 variant also has substantially more difficulty traveling in the N-to-C direction on a polypeptide track than moving C-to-N. These results indicate that D1 normally functions as an auxiliary/regulatory motor to promote uninterrupted enzyme advancement that is fueled largely by the D2 ring.

scholarly journals Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability

2015 ◽  
Vol 82 (5) ◽  
pp. 1391-1400 ◽  
Author(s):  
Francois Rineau ◽  
Jelle Stas ◽  
Nhu H. Nguyen ◽  
Thomas W. Kuyper ◽  
Robert Carleer ◽  
...  
Keyword(s):  
Organic Matter ◽  
Protein Degradation ◽  
Protease Activity ◽  
Nitrogen Availability ◽  
N Uptake ◽  
Organic N ◽  
Content Type ◽  
N Source ◽  
Ecm Fungi

ABSTRACTIn temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH4+and NO3−) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13Suillusspecies with different ecological preferences for theirin vitroprotein degradation abilities.Suillusspecies preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability.

The Multiple Levels of Mitonuclear Coregulation

2018 ◽  
Vol 52 (1) ◽  
pp. 511-533 ◽  
Author(s):  
R. Stefan Isaac ◽  
Erik McShane ◽  
L. Stirling Churchman
Keyword(s):  
Gene Expression ◽  
Oxidative Phosphorylation ◽  
Protein Degradation ◽  
Current Understanding ◽  
Mitochondrial Genomes ◽  
Expression Systems ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Optimal Function ◽  
Multiple Levels

Together, the nuclear and mitochondrial genomes encode the oxidative phosphorylation (OXPHOS) complexes that reside in the mitochondrial inner membrane and enable aerobic life. Mitochondria maintain their own genome that is expressed and regulated by factors distinct from their nuclear counterparts. For optimal function, the cell must ensure proper stoichiometric production of OXPHOS subunits by coordinating two physically separated and evolutionarily distinct gene expression systems. Here, we review our current understanding of mitonuclear coregulation primarily at the levels of transcription and translation. Additionally, we discuss other levels of coregulation that may exist but remain largely unexplored, including mRNA modification and stability and posttranslational protein degradation.

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