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- NMR (2)
- UBA5 (2)
- UFM1 (2)
- Bacterial Signal Transduction (1)
- CUE domain (1)
- Capsule Synthesis (1)
- Disulphide Bridges (1)
- ERAD (1)
- Histidine Kinases (1)
- LC3/GABARAP (1)
- LIR (1)
- Lipoprotein Structure (1)
- Metabolic Regulation (1)
- NMR Solution Structure (1)
- Outer Membrane Protein RcsF (1)
- Two-component System (1)
- UFC1 (1)
- UFIM (1)
- complex structure (1)
- isothermal titration calorimetry (ITC) (1)
- nuclear magnetic resonance (NMR) (1)
- protein motif (1)
- protein-protein interactions (1)
- signal transduction (1)
- ubiquitin chain conformation (1)
- ubiquitination (1)
- x-ray crystallography (1)
The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
RcsF, a proposed auxiliary regulator of the regulation of capsule synthesis (rcs) phosphorelay system, is a key element for understanding the RcsC-D-A/B signaling cascade, which is responsible for the regulation of more than 100 genes and is involved in cell division, motility, biofilm formation, and virulence. The RcsC-D-A/B system is one of the most complex bacterial signal transduction pathways, consisting of several membrane-bound and soluble proteins. RcsF is a lipoprotein attached to the outer membrane and plays an important role in activating the RcsC-d-A/B pathway. The exact mechanism of activation of the rcs phosphorelay by RcsF, however, remains unknown. We have analyzed the sequence of RcsF and identified three structural elements: 1) an N-terminal membrane-anchored helix (residues 3-13), 2) a loop (residues 14-48), and 3) a C-terminal folded domain (residues 49-134). We have determined the structure of this C-terminal domain and started to investigate its interaction with potential partners. Important features of its structure are two disulfide bridges between Cys-74 and Cys-118 and between Cys-109 and Cys-124. To evaluate the importance of this RcsF disulfide bridge network in vivo, we have examined the ability of the full-length protein and of specific Cys mutants to initiate the rcs signaling cascade. The results indicate that the Cys-74/Cys-118 and the Cys-109/Cys-124 residues correlate pairwise with the activity of RcsF. Interaction studies showed a weak interaction with an RNA hairpin. However, no interaction could be detected with reagents that are believed to activate the rcs phosphorelay, such as lysozyme, glucose, or Zn(2+) ions.
In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.
Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.