DegP, a periplasmic dual-functional protease and chaperone in Gram-negative bacteria, is critical for bacterial stress resistance, but the precise underlying mechanisms are not fully understood. Here, we show that the protease function of DegP is critical for Escherichia coli cells to maintain membrane integrity, particularly under heat shock conditions (42°C). Site-directed photo-cross-linking, mass spectrometry and immunoblotting analyses reveal that both periplasmic proteins (e.g. OppA and MalE) and β-barrel outer membrane proteins (OMPs) are DegP-interacting proteins and that OppA is degraded by DegP in vitro and in vivo at 42°C. In addition, OmpA and BamA, chimeric β-barrel OMPs containing a soluble periplasmic domain, are bound to DegP in both unfolded and folded forms, whereas only the unfolded forms are degradable by DegP. The presence of folded OmpA as a substrate of DegP is attributed to its periplasmic domain, which is resistant to DegP degradation and even generally protects pure β-barrel OMPs from degradation in an intra-molecular way. Furthermore, a pair of residues (R262 and V328) in the PDZ domain-1 of DegP play important roles for binding unfolded and folded β-barrel OMPs, with R262 being critical. Our study, together with earlier reports, indicates that DegP plays a critical role in protein quality control in the bacterial periplasm by degrading both periplasmic proteins and β-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric β-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.
E 2 (17β-oestradiol), a female sex hormone, has important biological functions in a woman's body. The pancreas, often considered a non-classical E 2 -targeting organ, is known to be functionally regulated by E 2 , but little is known about how oestrogen actions are regulated in this organ. In the present study we report that PDIp (pancreas-specific protein disulfide isomerase), a protein-folding catalyst, can act as a major intracellular E 2 storage protein in a rat model to modulate the pancreatic tissue level, metabolism and action of E 2 . The purified endogenous PDIp from both rat and human pancreatic tissues can bind E 2 with a K d value of approximately 150 nM. The endogenous PDIp-bound E 2 accounts for over 80% of the total protein-bound E 2 present in rat and human pancreatic tissues, and this binding protects E 2 from metabolic disposition and prolongs its duration of action. Importantly, we showed in ovariectomized female rats that the E 2 level in the pancreas reaches its highest level (9-fold increase over its basal level) at 24–48 h after a single injection of E 2 , and even at 96 h its level is still approximately 5-fold higher. In contrast, the E 2 level in the uterus quickly returns to its basal level at 48 h after reaching its maximal level (approximately 2-fold increase) at 24 h. Taken together, these results show for the first time that PDIp is a predominant intracellular oestrogen storage protein in the pancreas, which offers novel mechanistic insights into the accumulation and action of oestrogen inside pancreatic cells.
Conditions are reported under which the reassembled intermediates of the heat-shock protein Hsp16.3 after being denatured in 8M urea were detected by mainly using urea-gradient PAGE (with modifications) and urea-denaturing pore-gradient PAGE. Hsp16.3 is the small heat-shock protein from Mycobacterium tuberculosis , which exists as a specific nonamer and was proposed to form a trimer-of-trimers structure. The refolding and reassembling of this protein was achieved rapidly by dilution or dialysis, suggesting an effectively spontaneous recovery of quaternary structure. Data presented in this report demonstrate that the in vitro reassembling process of Hsp16.3 protein occurs through a spontaneous and effective stepwise mechanism. Modified urea-gradient PAGE may provide a general method for studying the reassembling processes of other oligomeric proteins.