Julia M. Flynn

Bio: Julia M. Flynn is an academic researcher from University of Massachusetts Medical School. The author has contributed to research in topics: Biology & Fitness landscape. The author has an hindex of 15, co-authored 24 publications receiving 2134 citations. Previous affiliations of Julia M. Flynn include Howard Hughes Medical Institute & Massachusetts Institute of Technology.

Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis

Abstract: The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9–11 at the C terminus of the tag, whereas ClpA recognizes positions 8–10 in addition to residues 1–2 at the N terminus. SspB interacts with residues 1–4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.

Modulating substrate choice: the SspB adaptor delivers a regulator of the extracytoplasmic-stress response to the AAA+ protease ClpXP for degradation

Abstract: The AAA+ protease ClpXP performs a diverse array of cellular tasks, including degrading incomplete polypeptides, adjusting the activity of metabolic enzymes, and altering the levels of regulatory proteins in response to stress (Gottesman et al. 1998; Wang et al. 1999; Maurizi and Rasulova 2002; Flynn et al. 2003; Gottesman 2003). As a result, many substrates compete for degradation by a relatively small number of ClpXP protease molecules (Ortega et al. 2004). The priority of substrate recognition and degradation can also be controlled by adaptor proteins, which enhance or inhibit interactions between specific substrates and ClpXP or other AAA+ proteases (Dougan et al. 2002a). How widely adaptor proteins are used to control substrate choice is not currently understood. In the ClpXP protease, ClpX—a hexameric-ring ATPase—binds native substrate proteins, denatures these molecules, and translocates the unfolded polypeptides into an internal degradation chamber of the ClpP peptidase (Maurizi et al. 1990, 1994; Wojtkowiak et al. 1993; Wang et al. 1997; Weber-Ban et al. 1999; Kim et al. 2000; Kim and Kim 2003). ClpX binds to short unstructured peptides called recognition signals or degradation tags, usually located near the amino or C terminus of substrates (Levchenko et al. 1997; Gottesman et al. 1998; Gonciarz-Swiatek et al. 1999; Flynn et al. 2003). The ssrA degradation tag is a well-characterized 11-residue peptide (AANDENYALAA), which is added cotranslationally to nascent polypeptides when ribosomes stall (Keiler et al. 1996). SsrA tagging frees these distressed ribosomes for new rounds of translation and targets the incomplete polypeptides for degradation by ClpXP and other proteases (Gottesman et al. 1998; Withey and Friedman 2003). The SspB adaptor was originally identified by its ability to enhance ClpXP degradation of ssrA-tagged proteins (Levchenko et al. 2000) and is one of the best-characterized proteins that functions in substrate delivery (Wah et al. 2002, 2003; Dougan et al. 2003; Levchenko et al. 2003; Song and Eck 2003; Bolon et al. 2004). SspB enhances recognition of ssrA-tagged proteins by mediating the assembly of ternary complexes in which the substrate, adaptor, and protease are tethered by the following three sets of protein-peptide interactions: (1) the AAA+ domain of ClpX binds to the C-terminal LAA sequence of the ssrA tag; (2) the substrate-binding domain of SspB interacts with a sequence spanning the N-terminal seven residues of the ssrA tag; and (3) a short peptide sequence at the end of a flexible SspB tail binds directly to the N-terminal domain of ClpX (Levchenko et al. 2000, 2003; Flynn et al. 2001; Wah et al. 2003; Bolon et al. 2004). Whether SspB delivers any substrates without ssrA tags for ClpXP degradation has not been addressed. Here, we show that SspB directs ClpXP recognition of Escherichia coli proteins, which are not ssrA tagged. One of these substrates, RseA, functions as a master regulator of the extracytoplasmic-stress response by inhibiting the transcription factor (σE) that activates expression of stress genes (De Las Penas et al. 1997b; Missiakas et al. 1997; Dartigalongue et al. 2001; Rezuchova et al. 2003). RseA is a transmembrane protein with an N-terminal cytoplasmic domain, which normally binds to and inhibits σE (De Las Penas et al. 1997b; Missiakas et al. 1997). In response to the stress-induced accumulation of unfolded or unassembled outer-membrane proteins in the periplasm, RseA is processed via multiple cleavage events in a sequential cascade. DegS protease initially cleaves RseA within its periplasmic domain, activating a second cleavage on the cytoplasmic side of the membrane by YaeL protease (Alba et al. 2001, 2002; Kanehara et al. 2002). These cleavage events release the cytoplasmic domain of RseA from the membrane, but this inhibitory domain remains bound to σE, and thus, additional steps are required before σE can activate gene expression (Missiakas et al. 1997; Campbell et al. 2003). Our experiments demonstrate that ClpXP and SspB play a role in the final step of the proteolytic cascade that activates σE. Cleavage of RseA on the cytoplasmic side of the membrane generates a fragment ending in a ClpX recognition signal, similar to the LAA sequence at the end of the ssrA tag. By binding simultaneously to this RseA1-108 fragment and ClpX, SspB brings the σE · RseA1-108 complex and the ClpXP protease together. The RseA fragment is, however, the only component of this complex that is degraded. Surprisingly, the peptide sequences bound by SspB in RseA1-108 and the ssrA tag are not similar, suggesting the SspB has different modes of protein recognition. These results establish that the SspB adaptor recognizes and delivers different classes of cellular proteins for degradation by ClpXP.

Latent ClpX-recognition signals ensure LexA destruction after DNA damage

Abstract: The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these fragments using sequence motifs that flank the auto-cleavage site but are dormant in intact LexA. Furthermore, ClpXP degradation of the LexA-DNA-binding fragment is important to cell survival after DNA damage. These results demonstrate how one protein-processing event can activate latent protease recognition signals, triggering a cascade of protein turnover in response to environmental stress.

AAA+ proteins: have engine, will work.

Abstract: The AAA+ (ATPases associated with various cellular activities) family is a large and functionally diverse group of enzymes that are able to induce conformational changes in a wide range of substrate proteins. The family's defining feature is a structurally conserved ATPase domain that assembles into oligomeric rings and undergoes conformational changes during cycles of nucleotide binding and hydrolysis. Here, we review the structural organization of AAA+ proteins, the conformational changes they undergo, the range of different reactions they catalyse, and the diseases associated with their dysfunction.