Disruption of the sumoylation/desumoylation equilibrium is associated with several disease states such as cancer and infections, however the mechanisms regulating the global SUMO balance remain poorly defined. Here, we show that infection by Shigella flexneri, the causative agent of human bacillary dysentery, switches off host sumoylation during epithelial cell infection in vitro and in vivo and that this effect is mainly mediated by a calcium/calpain-induced cleavage of the SUMO E1 enzyme SAE2, thus leading to sumoylation inhibition. Furthermore, we describe a mechanism by which Shigella promotes its own invasion by altering the sumoylation state of RhoGDIa, a master negative regulator of RhoGTPase activity and actin polymerization. Together, our data suggest that SUMO modification is essential to restrain pathogenic bacterial entry by limiting cytoskeletal rearrangement induced by bacterial effectors. Moreover, these findings identify calcium-activated calpains as powerful modulators of cellular sumoylation levels with potentially broad implications in several physiological and pathological situations.
- Pierre Lapaquette
- Anne Dejean
- Philippe Sansonetti
- Anne Dejean
- Anne Dejean
- Anne Dejean
- Sabrina Fritah
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Animal experiments were performed accordingly to the guidelines of the Institut Pasteur's ethical committee for animal use in research (CETEA number 2013-0028).
- Dominique Soldati-Favre, University of Geneva, Switzerland
© 2017, Lapaquette et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Profilin-1 (PFN1) is a cytoskeletal protein that regulates the dynamics of actin and microtubule assembly. Thus, PFN1 is essential for the normal division, motility, and morphology of cells. Unfortunately, conventional fusion and direct labeling strategies compromise different facets of PFN1 function. As a consequence, the only methods used to determine known PFN1 functions have been indirect and often deduced in cell-free biochemical assays. We engineered and characterized two genetically encoded versions of tagged PFN1 that behave identical to each other and the tag-free protein. In biochemical assays purified proteins bind to phosphoinositide lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and bound tubulin dimers (kD = 1.89 µM) to impact microtubule dynamics. In PFN1-deficient mammalian cells, Halo-PFN1 or mApple-PFN1 (mAp-PEN1) restored morphological and cytoskeletal functions. Titrations of self-labeling Halo-ligands were used to visualize molecules of PFN1. This approach combined with specific function-disrupting point-mutants (Y6D and R88E) revealed PFN1 bound to microtubules in live cells. Cells expressing the ALS-associated G118V disease variant did not associate with actin filaments or microtubules. Thus, these tagged PFN1s are reliable tools for studying the dynamic interactions of PFN1 with actin or microtubules in vitro as well as in important cell processes or disease-states.
Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63 linked di-Ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63 linked di-Ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.