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The ER is the hub of protein synthesis in a cell. It has robust systems to quality control protein folding and also for degradation of terminally misfolded proteins. Under normal conditions, a small proportion of misfolded proteins that cannot be salvaged need to be transported to the cytoplasm by the ER-associated degradation or ERAD pathways. However, if the ERAD cannot handle the misfolded proteins, the cell activates the unfolded protein response or UPR to adjust the protein folding...
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Structural basis for Ufm1 processing by UfSP1.

Byung Hak Ha1, Hee-Chul Ahn, Sung Hwan Kang

  • 1Life Sciences Division, Korea Institute of Science and Technology, Sungbuk-Gu, Seoul, Korea.

The Journal of Biological Chemistry
|March 7, 2008
PubMed
Summary
This summary is machine-generated.

Ubiquitin-fold modifier 1 (Ufm1) processing requires novel cysteine proteases UfSP1 and UfSP2. The crystal structure of UfSP1 reveals a unique papain-like fold and catalytic triad, defining a new protease subfamily.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein crucial for cellular processes.
  • Ufm1 requires processing of its precursor and removal from conjugated proteins by specific proteases.
  • Two novel proteases, UfSP1 and UfSP2, have been identified for Ufm1 maturation and deconjugation.

Purpose of the Study:

  • To elucidate the structural basis of Ufm1 processing by UfSP1.
  • To characterize the catalytic mechanism and substrate interaction of UfSP1.
  • To determine the potential classification of UfSP1 within protease superfamilies.

Main Methods:

  • X-ray crystallography to determine the 1.7Å resolution structure of mouse UfSP1.
  • Site-directed mutagenesis to investigate the role of active site residues.
  • Isothermal titration calorimetry (ITC) to quantify UfSP1-Ufm1 binding affinity.
  • Nuclear Magnetic Resonance (NMR) spectroscopy to identify Ufm1 binding regions.

Main Results:

  • The crystal structure revealed UfSP1 as a novel cysteine protease with a papain-like fold.
  • A unique catalytic triad (Cys53, Asp175, His177) and an unusual Asp-Pro-His motif were identified.
  • Mutagenesis confirmed the catalytic role of active site residues.
  • ITC analysis showed a high affinity interaction between UfSP1 and Ufm1 (K(D) = 1.6 μM).
  • NMR data indicated that specific regions of Ufm1 are involved in binding to UfSP1.

Conclusions:

  • UfSP1 represents a novel cysteine protease, distinct from canonical papain-like proteases due to its catalytic triad configuration.
  • UfSP1 and related proteases (Atg4B, M48(USP)) may constitute a new subfamily within the cysteine protease superfamily.
  • The structural and biochemical data provide a foundation for understanding Ufm1 conjugation and deconjugation pathways.