Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamerhexamer interface, with portals of ∼45 Å providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state.ATP-dependent protease | EM structure | IbpB | substrate gating | regulated proteolysis P rotein quality control is vital under stress conditions that promote protein unfolding and aggregation. Escherichia coli Lon degrades many unfolded proteins (1-3) and also degrades folded proteins, including SulA (supressor of Lon protein) and the inclusion-body binding proteins A and B (IbpA and B) (4-6). In E. coli and many other bacteria, Lon is up-regulated under numerous stress conditions (7-10). In mitochondria, Lon helps combat oxidative stress (11)(12)(13)(14), and human mitochondrial Lon was recently identified as a potential antilymphoma target (15). It is widely believed that a major role of Lon in all organisms is to degrade misfolded proteins (2, 10, 16).Lon subunits consist of an N domain, a central ATPase associated with cellular activities (AAA+) ATPase module, and a Cterminal peptidase domain. Although early reports suggested that Lon might be a tetramer (17), it is now clear that six subunits of the E. coli enzyme assemble into a hexamer with an internal degradation chamber accessible via an axial pore in the AAA+ ring (18,19). Lon substrates are recognized, unfolded if necessary by ATP-dependent reactions mediated by the AAA+ ring, and then translocated through the pore and into the peptidase chamber for degradation (20).In many families of ATP-dependent proteases, the AAA+ unfolding/translocation ring and the self-compartmentalized peptidase are encoded by distinct polypeptides, which assemble into independent oligomers before interacting to form the functional protease (21,22). For example, the ClpXP protease consists of AAA+ ClpX hexamers, which dock with the self-compartmentalized ClpP peptidase. This interaction suppresses the ATPase rate of ClpX and enhances the peptidase activity of ClpP (22). Lon activity cannot be controlled in this way because the ATPase and protease domains are always physically attached. Little is currently known about how Lon activity is regulated, although mutational studies show that the AAA+ and peptidase domains influence each other's activities (23-25). In some...

To achieve high-throughput analysis of allele frequencies in human SNPs, we have developed automated methods for designing PCR and DNA sequencing primers. We found we could run the PCR assays at quite stringent, uniform conditions. The design process used freely available databases, including dbSNP, SNPper, and TSC, and publicly available software including RepeatMasker and Primer3. We describe parameters for the software and other considerations that increase experimental success. As anticipated, some assays failed at the design stage due primarily to the genomic locations of repetitive sequences, extreme GC content regions, or lack of sufficient sequence. However, over 23 000 assays, including 96% of those recently analyzed, have been experimentally successful. Similar design methods could be used for PCR assays in any organism with substantial available sequence.

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

Mapping the stability distributions of proteins in their native folded states provides a critical link between structure, thermodynamics, and function. Linear repeat proteins have proven more amenable to this kind of mapping than globular proteins. C-terminal deletion studies of YopM, a large, linear leucine-rich repeat (LRR) protein, show that stability is distributed quite heterogeneously, yet a high level of cooperativity is maintained [1]. Key components of this distribution are three interfaces that strongly stabilize adjacent sequences, thereby maintaining structural integrity and promoting cooperativity. To better understand the distribution of interaction energy around these critical interfaces, we studied internal (rather than terminal) deletions of three LRRs in this region, including one of these stabilizing interfaces. Contrary to our expectation that deletion of structured repeats should be destabilizing, we find that internal deletion of folded repeats can actually stabilize the native state, suggesting that these repeats are destabilizing, although paradoxically, they are folded in the native state. We identified two residues within this destabilizing segment that deviate from the consensus sequence at a position that normally forms a stacked leucine ladder in the hydrophobic core. Replacement of these nonconsensus residues with leucine is stabilizing. This stability enhancement can be reproduced in the context of nonnative interfaces, but it requires an extended hydrophobic core. Our results demonstrate that different LRRs vary widely in their contribution to stability, and that this variation is context-dependent. These two factors are likely to determine the types of rearrangements that lead to folded, functional proteins, and in turn, are likely to restrict the pathways available for the evolution of linear repeat proteins.

The protein quality control network (pQC) plays critical roles in maintaining protein and cellular homeostasis, especially during stress. Lon is a major pQC AAA+ protease, conserved from bacteria to human mitochondria. It is the principal enzyme that degrades most unfolded or damaged proteins. Degradation by Lon also controls cellular levels of several key regulatory proteins. Recently, our group determined that Escherichia coli Lon, previously thought to be an obligate homo-hexamer, also forms a dodecamer. This larger assembly has decreased ATPase activity and displays substrate-specific alterations in degradation compared with the hexamer. Here we experimentally probe the physical hexamer-hexamer interactions and the biological roles of the Lon dodecamer. Using structure prediction methods coupled with mutagenesis, we identified a key interface and specific residues within the Lon N domain that participates in an intermolecular coiled coil unique to the dodecamer. With this knowledge, we made a Lon variant (Lon VQ ) that forms a dodecamer with increased stability, as determined by analytical ultracentrifugation and electron microscopy. Using this altered Lon, we characterize the Lon dodecamer's activities using a panel of substrates. Lon dodecamers are clearly functional, and complement critical lon-phenotypes but also exhibit altered substrate specificity. For example, the small heat shock proteins IbpA and IbpB are only efficiently degraded well by the hexamer. Thus, by elucidating the intermolecular contacts connecting the hexamers, we are starting to illuminate how Abbreviations: AUC, analytical ultracentrifugation; EM, electron microscopy; Ibp, inclusion body binding protein; sHSP, small heat shock protein.Additional Supporting Information may be found in the online version of this article.Summary statement Lon protease degrades several critical regulatory proteins and maintains protein quality control. We characterized a variant of Escherichia coli Lon that preferentially forms a dodecamer; an assembly that has distinct activities and substrate recognition compared with the Lon hexamer. This variant served as a molecular probe to investigate the biological roles of the dodecamer-hexamer equilibrium in vivo and in vitro. This work thus highlights new aspects of the complex molecular processes that control Lon activity.Tejas Kalastavadi's current address is