An endoplasmic reticulum (ER) retention sequence (ERS) is a characteristic short sequence that mediates protein retention in the ER of eukaryotic cells. However, little is known about the detailed molecular mechanism involved in ERS-mediated protein ER retention. Using a new surface display-based fluorescence technique that effectively quantifies ERS-promoted protein ER retention within cells, we performed comprehensive ERS analyses. We found that the length, type of amino acid residue, and additional residues at positions -5 and -6 of the C-terminal HDEL motif all determined the retention of ERS in the yeast ER. Moreover, the biochemical results guided by structure simulation revealed that aromatic residues (Phe-54, Trp-56, and other aromatic residues facing the ER lumen) in both the ERS (at positions -6 and -4) and its receptor, Erd2, jointly determined their interaction with each other. Our studies also revealed that this aromatic residue interaction might lead to the discriminative recognition of HDEL or KDEL as ERS in yeast or human cells, respectively. Our findings expand the understanding of ERS-mediated residence of proteins in the ER and may guide future research into protein folding, modification, and translocation affected by ER retention.
Human
rhinovirus 3C protease (HRV 3C-P) is a high-value commercial
cysteine protease that could specifically recognize the short peptide
sequence of LEVLFQ↓GP. In here, a strategy based on our previous
Yeast Endoplasmic Reticulum Sequestration Screening (YESS) approach
was developed in Saccharomyces cerevisiae, a model
microorganism, to fully characterize the substrate specificity of
a typical human virus protease, HRV 3C-P, in a quantitative and fast
manner. Our results demonstrated that HRV 3C-P had very high specificity
at P1 and P1′ positions, only recognizing Gln/Glu at the P1
position and Gly/Ala/Cys/Ser at the P1′ position, respectively.
Comparably, it exhibited efficient recognition of most residues at
the P2′ position, except Trp. Further biochemical characterization
through site mutagenesis, enzyme structural modeling, and comparison
with other 3C proteases indicated that the S1 pocket of HRV 3C-P was
constituted by neutral and basic amino acids, in which His160 and
Thr141 specifically interacted with Gln or Glu residues at the substrate
P1 position. Additionally, the stringent S1′ pocket determined
its unique property of only accommodating residues without or with
short side chains. Based on our characterization, LEVLFQ↓GM
was identified as a more favorable substrate than the original LEVLFQ↓GP
at high temperature, which might be caused by the conversion of random
coils to β-turns in HRV 3C-P along with the temperature increase.
Our studies prompted a further understanding of the substrate specificity
and recognition mechanism of HRV 3C-P. Besides, the YESS-PSSC combined
with the enzyme modeling strategy in this study provides a general
strategy for deciphering the substrate specificities of proteases.
Yersinia species are bacterial pathogens that can cause plague and intestinal diseases after invading into human cells through the Three Secretion System (TTSS). The effect of pathogenesis is mediated by Yersinia outer proteins (Yop) and manifested as down-regulation of the cytokine genes expression by inhibiting nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. In addition, its pathogenesis can also manipulate the disorder of host innate immune system and cell death such as apoptosis, pyroptosis, and autophagy. Among the Yersinia effector proteins, YopB and YopD assist the injection of other virulence effectors into the host cytoplasm, while YopE, YopH, YopJ, YopO, and YopT target on disrupting host cell signaling pathways in the host cytosols. Many efforts have been applied to reveal that intracellular proteins such as Rho-GTPase, and transmembrane receptors such as Toll-like receptors (TLRs) both play critical roles in Yersinia pathogenesis, establishing a connection between the pathogenic process and the signaling response. This review will mainly focus on how the effector proteins of Yersinia modulate the intrinsic signals in host cells and disturb the innate immunity of hosts through TTSS.
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