Degp degrades a wide range of substrate proteins in Escherichia coli under stress conditions.

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 ß-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 ß-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 ß-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 ß-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 ß-barrel OMPs under stress conditions and likely also by participating in the folding of chimeric ß-barrel OMPs. A working model is proposed to illustrate the finely tuned functions of DegP with respect to different substrate proteins.

Subunit interactions as mediated by "non-interface" residues in living cells for multiple homo-oligomeric proteins.

Protein-protein interaction, including protein homo-oligomerization, is commonly believed to occur through a specific interface made of a limited number of amino acid residues. Here our systematic in vivo photo-crosslinking analysis via genetically incorporated unnatural amino acids unexpectedly shows that the dimerization of HdeA, an acid stress chaperone, is mediated by the residues along its whole polypeptide. These include those "forbidden" residues that are far away from the dimerization interface as judged according to the reported 3-D structure. We demonstrate that such dimerization, though intriguing, is neither a result of protein over-expression nor of any structural disturbance caused by the residue replacement. Similar unexpected dimerization also occurs for two other oligomeric proteins, IbpB (a molecular chaperone existing as polydispersed oligomers in vitro) and DegP (a protease existing as hexamers in vitro). In contrast to these three proteins, dimerization of a few other oligomeric proteins (e.g., OmpF, LamB, SurA, FtsZ and FkpA) that we similarly examined in living cells seems to be mediated only by specific residues. Together, our unexpected observations suggest that, for some oligomeric proteins such as HdeA, IbpB and DegP, their subunit interactions in living cells can also be mediated by residues other than those located at the interfaces as revealed by in vitro structure determination. Our observations might be partially explained by the formation of "encounter complex" or by protein conformational dynamics. Our findings provide new insights on understanding protein-protein interactions and encounter complex formation in living cells.

A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of ß-Barrel Outer Membrane Proteins in Bacteria.

ß-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent ß-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent ß-barrel OMPs largely via its N-domain but with ß-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent ß-barrel OMPs interact with SurA via their N- and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving ß-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for ß-barrel OMP biogenesis.

The function of the DegP (HtrA) protein: Protease versus chaperone.

The DegP (or HtrA) is a highly conserved family of proteins functioning in all living organisms. It was initially identified as a protease functioning in the periplasmic space of the Gram-negative bacterial cells. It was later reported to also exhibit chaperone activity and thus has been designated as a bifunctional protein. However, recent studies demonstrated that in living cells it more likely functions only as a protease with hardly detectable chaperone activities. In this review, I will summarize the evidences clarifying that DegP more likely only functions as a protease rather than as a chaperone in cells. © 2016 IUBMB Life, 68(11):904-907, 2016.

An oxidative fluctuation hypothesis of aging generated by imaging H2O2 levels in live Caenorhabditis elegans with altered lifespans.

Reactive oxygen species (ROS) are important factors mediating aging according to the free radical theory of aging. Few studies have systematically measured ROS levels in relationship to aging, partly due to the lack of tools for detection of specific ROS in live animals. By using the H2O2-specific fluorescence probe Peroxy Orange 1, we assayed the H2O2 levels of live Caenorhabditis elegans with 41 aging-related genes being individually knocked down by RNAi. Knockdown of 14 genes extends the lifespan but increases H2O2 level or shortens the lifespan but decreases H2O2 level, contradicting the free radical theory of aging. Strikingly, a significant inverse correlation between lifespan and the normalized standard deviation of H2O2 levels was observed (p < 0.0001). Such inverse correlation was also observed in worms cultured under heat shock conditions. An oxidative fluctuation hypothesis of aging is thus proposed and suggests that the ability of animals to homeostatically maintain the ROS levels within a narrow range is more important for lifespan extension than just minimizing the ROS levels though the latter still being crucial.

Identification of FkpA as a key quality control factor for the biogenesis of outer membrane proteins under heat shock conditions.

The outer membrane proteins (OMPs) of Gram-negative bacterial cells, as well as the mitochondrion and chloroplast organelles, possess unique and highly stable ß-barrel structures. Biogenesis of OMPs in Escherichia coli involves such periplasmic chaperones as SurA and Skp. In this study, we found that the ΔsurA Δskp double-deletion strain of E. coli, although lethal and defective in the biogenesis of OMPs at the normal growth temperature, is viable and effective at the heat shock temperature. We identified FkpA as the multicopy suppressor for the lethal phenotype of the ΔsurA Δskp strain. We also demonstrated that the deletion of fkpA from the ΔsurA cells resulted in only a mild decrease in the levels of folded OMPs at the normal temperature but a severe decrease as well as lethality at the heat shock temperature, whereas the deletion of fkpA from the Δskp cells had no detectable effect on OMP biogenesis at either temperature. These results strongly suggest a functional redundancy between FkpA and SurA for OMP biogenesis under heat shock stress conditions. Mechanistically, we found that FkpA becomes a more efficient chaperone for OMPs under the heat shock condition, with increases in both binding rate and affinity. In light of these observations and earlier reports, we propose a temperature-responsive OMP biogenesis mechanism in which the degrees of functional importance of the three chaperones are such that SurA > Skp > FkpA at the normal temperature but FkpA ≥ SurA > Skp at the heat shock temperature.

A small heat shock protein enables Escherichia coli to grow at a lethal temperature of 50°C conceivably by maintaining cell envelope integrity.

It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.

In vivo substrate diversity and preference of small heat shock protein IbpB as revealed by using a genetically incorporated photo-cross-linker.

Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones found in all forms of life, are known to be able to protect cells against stresses and suppress the aggregation of a variety of model substrate proteins under in vitro conditions. Nevertheless, it is poorly understood what natural substrate proteins are protected by sHSPs in living cells. Here, by using a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we identified a total of 95 and 54 natural substrate proteins of IbpB (an sHSP from Escherichia coli) in living cells with and without heat shock, respectively. Functional profiling of these proteins (110 in total) suggests that IbpB, although binding to a wide range of cellular proteins, has a remarkable substrate preference for translation-related proteins (e.g. ribosomal proteins and amino-acyl tRNA synthetases) and moderate preference for metabolic enzymes. Furthermore, these two classes of proteins were found to be more prone to aggregation and/or inactivation in cells lacking IbpB under stress conditions (e.g. heat shock). Together, our in vivo data offer novel insights into the chaperone function of IbpB, or sHSPs in general, and suggest that the preferential protection on the protein synthesis machine and metabolic enzymes may dominantly contribute to the well known protective effect of sHSPs on cell survival against stresses.

Small heat shock protein IbpB acts as a robust chaperone in living cells by hierarchically activating its multi-type substrate-binding residues.

As ubiquitous molecular chaperones, small heat shock proteins (sHSPs) are crucial for protein homeostasis. It is not clear why sHSPs are able to bind a wide spectrum of non-native substrate proteins and how such binding is enhanced by heat shock. Here, by utilizing a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we systematically characterized the substrate-binding residues in IbpB (a sHSP from Escherichia coli) in living cells over a wide spectrum of temperatures (from 20 to 50 °C). A total of 20 and 48 residues were identified at normal and heat shock temperatures, respectively. They are not necessarily hydrophobic and can be classified into three types: types I and II were activated at low and normal temperatures, respectively, and type III mediated oligomerization at low temperature but switched to substrate binding at heat shock temperature. In addition, substrate binding of IbpB in living cells began at temperatures as low as 25 °C and was further enhanced upon temperature elevation. Together, these in vivo data provide novel structural insights into the wide substrate spectrum of sHSPs and suggest that sHSP is able to hierarchically activate its multi-type substrate-binding residues and thus act as a robust chaperone in cells under fluctuating growth conditions.

Differential degradation for small heat shock proteins IbpA and IbpB is synchronized in Escherichia coli: implications for their functional cooperation in substrate refolding.

Small heat shock proteins (sHSPs), as a conserved family of ATP-independent molecular chaperones, are known to bind non-native substrate proteins and facilitate the substrate refolding in cooperation with ATP-dependent chaperones (e.g., DnaK and ClpB). However, how different sHSPs function in coordination is poorly understood. Here we report that IbpA and IbpB, the two sHSPs of Escherichia coli, are coordinated by synchronizing their differential in vivo degradation. Whereas the individually expressed IbpA and IbpB are respectively degraded slowly and rapidly in cells cultured under both heat shock and normal conditions, their simultaneous expression leads to a synchronized degradation at a moderate rate. Apparently, such synchronization is linked to their hetero-oligomerization and cooperation in binding substrate proteins. In addition, truncation of the flexible N- and C-terminal tails dramatically suppresses the IbpB degradation, and somehow accelerates the IbpA degradation. In view of these in vivo data, we propose that the synchronized degradation for IbpA and IbpB are crucial for their synergistic promoting effect on DnaK/ClpB-mediated substrate refolding, conceivably via the formation of IbpA-IbpB-substrate complexes. This scenario may be common for different sHSPs that interact with each other in cells.

A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance.

Acid chaperones are essential factors in preserving the protein homeostasis for enteric pathogens to survive in the extremely acidic mammalian stomach (pH 1-3). The client proteins of these chaperones remain largely unknown, primarily because of the exceeding difficulty of determining protein-protein interactions under low-pH conditions. We developed a genetically encoded, highly efficient protein photocrosslinking probe, which enabled us to profile the in vivo substrates of a major acid-protection chaperone, HdeA, in Escherichia coli periplasm. Among the identified HdeA client proteins, the periplasmic chaperones DegP and SurA were initially found to be protected by HdeA at a low pH, but they subsequently facilitated the HdeA-mediated acid recovery of other client proteins. This unique, ATP-independent chaperone cooperation in the ATP-deprived E. coli periplasm may support the acid resistance of enteric bacteria. The crosslinker would be valuable in unveiling the physiological interaction partners of any given protein and thus their functions under normal and stress conditions.

Uncovering the membrane-integrated SecAN protein that plays a key role in translocating nascent outer membrane proteins.

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecAN protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts.

Interaction between bacterial outer membrane proteins and periplasmic quality control factors: a kinetic partitioning mechanism.

The OMPs (outer membrane proteins) of Gram-negative bacteria have to be translocated through the periplasmic space before reaching their final destination. The aqueous environment of the periplasmic space and high permeability of the outer membrane engender such a translocation process inevitably challenging. In Escherichia coli, although SurA, Skp and DegP have been identified to function in translocating OMPs across the periplasm, their precise roles and their relationship remain to be elucidated. In the present paper, by using fluorescence resonance energy transfer and single-molecule detection, we have studied the interaction between the OMP OmpC and these periplasmic quality control factors. The results of the present study reveal that the binding rate of OmpC to SurA or Skp is much faster than that to DegP, which may lead to sequential interaction between OMPs and different quality control factors. Such a kinetic partitioning mechanism for the chaperone-substrate interaction may be essential for the quality control of the biogenesis of OMPs.

The 2011 Joint Sino-U.K. Protein Symposium.

As part of the celebrations marking its Centenary, the Biochemical Society held a joint meeting in Shanghai with the Chinese Protein Society to commemorate the many long-standing collaborations between biochemists from the U.K. and China. Under the overall theme of structural biology of proteins, presentations covering both historical and current research were given by a number of leading biochemists from both countries. Papers based on these talks have been prepared for this issue of Biochemical Society Transactions.

Activation of DegP chaperone-protease via formation of large cage-like oligomers upon binding to substrate proteins.

Cells use molecular chaperones and proteases to implement the essential quality control mechanism of proteins. The DegP (HtrA) protein, essential for the survival of Escherichia coli cells at elevated temperatures with homologues found in almost all organisms uniquely has both functions. Here we report a mechanism for DegP to activate both functions via formation of large cage-like 12- and 24-mers after binding to substrate proteins. Cryo-electron microscopic and biochemical studies revealed that both oligomers are consistently assembled by blocks of DegP trimers, via pairwise PDZ1-PDZ2 interactions between neighboring trimers. Such interactions simultaneously eliminate the inhibitory effects of the PDZ2 domain. Additionally, both DegP oligomers were also observed in extracts of E. coli cells, strongly implicating their physiological importance.

The Caenorhabditis elegans 12-kDa small heat shock proteins with little in vitro chaperone activity play crucial roles for its dauer formation, longevity, and reproduction.

Small heat shock proteins (sHSPs) are known to exhibit in vitro chaperone activity by suppressing the aggregation of misfolded proteins. The 12-kDa sHSPs (Hsp12s) subfamily members from Caenorhabditis elegans, including Hsp12.2, Hsp12.3, and Hsp12.6, however, are devoid of such chaperone activity, and their in vivo functions are poorly understood. Here we verified that Hsp12.1, similar to its homologs Hsp12.2, Hsp12.3, and Hsp12.6, hardly exhibited any chaperone activity. Strikingly, we demonstrated that these Hsp12s seem to play crucial physiological roles in C. elegans, for suppressing dauer formation and promoting both longevity and reproduction. A unique sHSP gene from Filarial nematode worm Brugia malayi was identified such that it encodes two products, one as a full-length Hsp12.6 protein and the other one having an N-terminal arm of normal length but lacks the C-terminal extension. This gene may represent an intermediate form in evolution from a common sHSP to a Hsp12. Together, our study offers insights on what biological functions the chaperone-defective sHSPs may exhibit and also implicates an evolutionary scenario for the unique Hsp12s subfamily.