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The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli . This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae . Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

ATP synthase, a highly conserved protein complex that has a subunit composition of α3 β3 γδeab2 c8-15 for the bacterial enzyme, is a key player in supplying energy to living organisms. This protein complex consists of a peripheral F1 sector (α3 β3 γδe) and a membrane-integrated Fo sector (ab2 c8-15 ). Structural analyses of the isolated protein components revealed that, remarkably, the C-terminal domain of its e-subunit seems to adopt two dramatically different structures, but the physiological relevance of this conformational change remains largely unknown. In an attempt to decipher this, we developed a high-throughput in vivo protein photo-cross-linking analysis pipeline based on the introduction of the unnatural amino acid into the target protein via the scarless genome-targeted site-directed mutagenesis technique, and probing the cross-linked products via the high-throughput polyacrylamide gel electrophoresis technique. Employing this pipeline, we examined the interactions involving the C-terminal helix of the e-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the bacterial ATP synthase exists as an equilibrium between the 'inserted' and 'noninserted' state in cells, maintaining a moderate but significant level of net ATP synthesis when shifting to the former upon exposing to unfavorable energetically stressful conditions. Such a mechanism allows the bacterial ATP synthases to proportionally and instantly switch between two reversible functional states in responding to changing environmental conditions. Importantly, this high-throughput approach could allow us to decipher the physiological relevance of protein-protein interactions identified under in vitro conditions or to unveil novel physiological context-dependent protein-protein interactions that are unknown before.

BACKGROUND The coronavirus disease 2019 (COVID-19) pandemic is hitting many countries. It is hypothesized the epidemic is differentially progressing in different countries. AIM To investigate how the COVID-19 epidemic is going on in different countries by analyzing representative countries. METHODS The status of COVID-19 epidemic in over 60 most affected countries was characterized. The data of daily new cases of each country were collected from Worldometer. The data of daily tests for the United States, Italy, and South Korea were collected from the Website of One World Data. Levels of daily positive COVID-19 tests in the two most affected states of the United States (New York and New Jersey) were collected from the website of the COVID Tracking Project. Statistics were analyzed using Microcal Origin software with ANOVA algorithm, and significance level was set at a P value of 0.05. RESULTS The COVID-19 epidemic was differentially progressing in different countries. Comparative analyses of daily new cases as of April 19, 2020 revealed that 61 most affected countries can be classified into four types: Downward (22), upward (20), static-phase (12), and uncertain ones (7). In particular, the 12 static-phase countries including the United States were characterized by largely constant numbers of daily new cases in the past over 14 d. Furthermore, these static-phase countries were overall significantly lower in testing density (P = 0.016) but higher in the level of positive COVID-19 tests than downward countries (P = 0.028). These findings suggested that the testing capacity in static-phase countries was lagging behind the spread of the outbreak, i.e., daily new cases (confirmed) were likely less than daily new infections and the remaining undocumented infections were thus still expanding, resulting in unstoppable epidemic. CONCLUSION Increasing the testing capacity and/or reducing the COVID-19 transmission are urgently needed to stop the potentially unstoppable, severing crisis in static-phase countries.

An ongoing outbreak of atypical pneumonia caused by the 2019 novel coronavirus (SARS-CoV-2) is hitting Wuhan City and has spread to other provinces/cities of China and overseas. It very urgent to forecast the future course of the outbreak. Here, we provide an estimate of the potential total number of confirmed cases in mainland China by applying Boltzmann-function based regression analyses. We found that the cumulative number of confirmed cases from Jan 21 to Feb 14, 2020 for mainland China, Hubei Province, Wuhan City and other provinces were all well fitted with the Boltzmann function (R2 being close to 0.999). The potential total number of confirmed cases in the above geographic regions were estimated at 95% confidence interval (CI) as 79589 (71576, 93855), 64817 (58223, 77895), 46562 (40812, 57678) and 13956 (12748, 16092), respectively. Notably, our results suggest that the number of daily new confirmed cases of SARS-CoV-2 in mainland China (including Hubei Province) will become minimal between Feb 28 and Mar 10, 2020, with 95% CI. In addition, we found that the data of cumulative confirmed cases of 2003 SARS-CoV in China and Worldwide were also well fitted to the Boltzmann function. To our knowledge this is the first study revealing that the Boltzmann function is suitable to simulate epidemics. The estimated potential total number of confirmed cases and key dates for the SARS-CoV-2 outbreak may provide certain guidance for governments, organizations and citizens to optimize preparedness and response efforts.

Improving the efficacy of existing antibiotics is a promising strategy for combating antibiotic-resistant/tolerant bacterial pathogens that have become a severe threat to human health. We previously reported that aminoglycoside antibiotics could be dramatically potentiated against stationary-phase Escherichia coli cells under hypoionic shock conditions (i.e., treatment with ion-free solutions), but the underlying molecular mechanism remains unknown. Here, we show that mechanosensitive (MS) channels, a ubiquitous protein family sensing mechanical forces of cell membrane, mediate such hypoionic shock-induced aminoglycoside potentiation. Two-minute treatment under conditions of hypoionic shock (e.g., in pure water) greatly enhances the bactericidal effects of aminoglycosides against both spontaneous and triggered E. coli persisters, numerous strains of Gram-negative pathogens in vitro, and Pseudomonas aeruginosa in mice. Such potentiation is achieved by hypoionic shock-enhanced bacterial uptake of aminoglycosides and is linked to hypoionic shock-induced destabilization of the cytoplasmic membrane in E. coli. Genetic and biochemical analyses reveal that MscS-family channels directly and redundantly mediate aminoglycoside uptake upon hypoionic shock and thus potentiation, with MscL channel showing reduced effect. Molecular docking and site-directed mutagenesis analyses reveal a putative streptomycin-binding pocket in MscS, critical for streptomycin uptake and potentiation. These results suggest that hypoionic shock treatment destabilizes the cytoplasmic membrane and thus changes the membrane tension, which immediately activates MS channels that are able to effectively transport aminoglycosides into the cytoplasm for downstream killing. Our findings reveal the biological effects of hypoionic shock on bacteria and can help to develop novel adjuvants for aminoglycoside potentiation to combat bacterial pathogens via activating MS channels.

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. This article is protected by copyright. All rights reserved.

The COVID-19 outbreak is ongoing in China. Here, Boltzmann function-based analyses reveal the potential total numbers of COVID-19 deaths: 3,260 (95% confidence interval [CI], 3187–3394) in China; 110 (95% CI, 109–112) in Hubei Province; 3,174 (95% CI, 3095–3270) outside Hubei; 2,550 (95% CI, 2494–2621) in Wuhan City; and 617 (95% CI, 607–632) outside Wuhan.

Human diseases are usually linked to multiloci genetic alterations, including single‐nucleotide polymorphisms (SNPs). Methods to use these SNPs for disease risk prediction (DRP) are of clinical interest. DRP algorithms explored by commercial companies to date have tended to be complex and led to controversial prediction results. Here, we present a general approach for establishing a logistic model‐based DRP algorithm, in which multiple SNP risk factors from different publications are directly used. In particular, the coefficient β of each SNP is set as the natural logarithm of the reported odds ratio, and the constant coefficient β0 is comprehensively determined by the coefficient and frequency of each SNP and the average disease risk in populations. Furthermore, homozygous SNP is considered a dummy variable, and the SNPs are updated (addition, deletion and modification) if necessary. Importantly, we validated this algorithm as a proof of concept: two patients with lung cancer were identified as the maximum risk cases from 57 Chinese individuals. Our logistic model‐based DRP algorithm is apparently more intuitive and self‐evident than the algorithms explored by commercial companies, and it may facilitate DRP commercialization in the era of personalized medicine.

The COVID-19 pandemic has hit many countries, and in some European countries it has been mitigated since April. Here we applied Richards function to simulate and forecast the course of COVID-19 epidemics in Italy, Spain, France, Germany, Turkey, Belgium, Ireland, Netherlands, Portugal and Switzerland. Potential total COVID-19 confirmed cases in these countries were estimated to be 240400±1300, 294100±4000, 178500±800, 176900±700, 155400±1000, 57900±400, 24000±200, 46200±300, 30000±300 and 30700±100 respectively. Most of these countries are predicted to approach ending stage between late May and early June such that daily new cases will become minimal, which may guide societal and economic restorations. In addition, total COVID-19 deaths were estimated to be 33500±300, 28200±200, 27800±200, 8740±80, 4500±30, 9250±70, 1530±20, 6240±50, 1380±10 and 1960±8, respectively. To our best knowledge, this is the first study forecasting the COVID-19 epidemic by applying the Richard function-based regression analysis.

The COVID-19 epidemics are differentially progressing in different countries. Here, comparative analyses of daily new cases reveal that 61 most affected countries can be classified into four types: downward (22), upward (20), static-phase (12) and uncertain ones (7). In particular, the 12 static-phase countries including US and UK are characterized by largely constant numbers of daily new cases in the past over 14 days. Furthermore, these static-phase countries are overall significantly lower in testing density but higher in the level of positive COVID-19 tests than downward countries. These findings suggest that the testing capacity in static-phase countries is lagging behind the spread of the outbreak, i.e., daily new cases (confirmed) are likely less than daily new infections and the remaining undocumented infections are thus still expanding, resulting in unstoppable epidemics. As such, increasing the testing capacity and/or reducing the COVID-19 transmission are urgently needed to stop the severing crisis in static-phase countries.Highlights61 most affected countries can be classified into four types: downward, upward, static-phase and uncertain ones.The 12 static-phase countries including US and UK are characterized by largely constant numbers of daily new cases in the past over 14 days.Static-phase countries show lower testing density but higher level of positive COVID-19 tests than downward countries.In static-phase countries, daily new cases (confirmed) are likely less than daily new infections and the remaining undocumented infections are thus still expanding.

ObjectiveSchool reopening has not yet started in China where the COVID-19 outbreak has reached ending stage, largely due to a great concern about COVID-19 infections on students. We attempted to quantitatively evaluate the risk of COVID-19 infections on students caused by school reopening.Study designWe collected the data of the numbers of teachers, population size and newly confirmed COVID-19 cases in the past 14 days in typical provinces/cities of China, and then analyzed the risk of COVID-19 infections in schools with respect to each province/city.MethodsA step-by-step procedure was explored to calculate the probability of COVID-19 infections on students as transmitted from infected teachers. Two critical assumptions for analysis were proposed: (i) only locally generated cases were counted while imported cases were omitted; (ii) the secondary attack rate of the COVID-19 virus in schools is similar to that in households in China, ranging from 3-10%.ResultsThe probability of COVID-19 resurgence within one week on students of primary, middle and high schools in China (outside Hubei) is extremely low (ConclusionsOur work provides guidance for local governments to make risk level-based policies for school reopening. Currently, the risk of COVID-19 infections on students is extremely low in China (outside Hubei) and therefore school reopening can be initiated without the endanger of infections on students.

The COVID-19 outbreak in China appears to reach the late stage since late March 2020, and a stepwise restoration of economic operations is implemented. Risk assessment for such economic restoration is of significance. Here, we estimated the probability of COVID-19 resurgence caused by work resuming in typical provinces/cities and found that such probability is very limited (

We report that COVID-19 mortality and recovery rates in Hubei Province, China exponentially decays (R2>0.93) and grows (R2>0.95), respectively. A great number of newly supplied medical resources, including more than 42000 aided health workers, over 26000 makeshift beds and 23000 acute care beds, enabled overwhelming patients to be treated effectively in hospitals. This may inform other countries to deal with the coming COVID-19 pandemic when patients are overwhelming the local health care system.

The COVID-19 outbreak is on-going in China. Here we estimated the potential total numbers of COVID-19 deaths in China, outside Hubei (in China), Hubei Province, Wuhan City and outside Wuhan (in Hubei) by Boltzmann function-based analyses, which are 3342 (95% CI, 3214, 3527), 111 (109, 114), 3245 (3100, 3423), 2613 (2498, 2767) and 627 (603, 654), respectively. The results may help to evaluate the severity of COVID-19 outbreaks and facilitate timely mental service for the families of passed patients.

Antibiotics have long been used to successfully kill bacterial pathogens, but antibiotic resistance/tolerance usually has led to the failure of antibiotic therapy, and it has become a severe threat to human health. How to improve the efficacy of existing antibiotics is of importance for combating antibiotic-resistant/tolerant pathogens. Here, we report that 10-s rapid freezing with liquid nitrogen dramatically enhanced the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many bacterial pathogens in vitro and also in a mouse skin wound model. In particular, such combined treatment was able to effectively kill persister cells of Escherichia coli and Pseudomonas aeruginosa, which are per se tolerant of conventional treatment with bactericidal antibiotics for several hours. We also demonstrated that freezing-induced aminoglycoside potentiation was apparently linked to freezing-induced cell membrane damage that may have activated the mechanosensitive ion channel MscL, which, in turn, was able to effectively uptake aminoglycoside antibiotics in a proton motive force-independent manner. Our report sheds light on the development of a new strategy against bacterial pathogens by combining existing antibiotics with a conventional physical treatment or with MscL agonists., Bacterial persisters exhibit noninherited antibiotic tolerance and are linked to the recalcitrance of bacterial infections. It is very urgent but also challenging to develop antipersister strategies. Here, we report that 10-s freezing with liquid nitrogen dramatically enhances the bactericidal action of aminoglycoside antibiotics by 2 to 6 orders of magnitude against many Gram-negative pathogens, with weaker potentiation effects on Gram-positive bacteria. In particular, antibiotic-tolerant Escherichia coli and Pseudomonas aeruginosa persisters—which were prepared by treating exponential-phase cells with ampicillin, ofloxacin, the protonophore cyanide m-chlorophenyl hydrazone (CCCP), or bacteriostatic antibiotics—can be effectively killed. We demonstrated, as a proof of concept, that freezing potentiated the aminoglycosides' killing of P. aeruginosa persisters in a mouse acute skin wound model. Mechanistically, freezing dramatically increased the bacterial uptake of aminoglycosides regardless of the presence of CCCP, indicating that the effects are independent of the proton motive force (PMF). In line with these results, we found that the effects were linked to freezing-induced cell membrane damage and were attributable, at least partly, to the mechanosensitive ion channel MscL, which was able to directly mediate such freezing-enhanced aminoglycoside uptake. In view of these results, we propose that the freezing-induced aminoglycoside potentiation is achieved by freezing-induced cell membrane destabilization, which, in turn, activates the MscL channel, which is able to effectively take up aminoglycosides in a PMF-independent manner. Our work may pave the way for the development of antipersister strategies that utilize the same mechanism as freezing but do so without causing any injury to animal cells.

Comparators play a significant role in the semiconductor industry and have become indispensable in the design of ADC. The delay and energy consumption are two important indicators of the comparator. Many designs have been made to reduce the delay and energy consumption, such as separated gata-biasing cross-coupled transistors for a new latching stage, and pMOS is used to replace nMOS in comparators. This paper analyzes the working principle of the proposed comparators designed for different needs reported on different papers. It compares their simulation results about key data such as delay and energy consumption.

Highlights • CFR in Iran in the early stage of the outbreak is highest among all the countries. • CFRs in the USA and Italy are similar to Hubei Province in the early stage. • CFRs in South Korea are similar to outside Hubei, indicating less severity. • Our findings highlight the severity of outbreaks globally, particular in the USA., Graphical Abstract Image, graphical abstract

ATP synthase, a highly conserved multi-subunit enzyme complex having a common stoichiometry of α3β3γδεab2c8-15, functions to supply ATP as the universal energy currency for cells. It comprises of the peripheral F1 sector (α3β3γδε) and the membrane-integrated Fo sector (ab2c8-15). In vitro structural analyses revealed that the C-terminal domain of the ε-subunit could adopt either an “inserted” or “non-inserted” state (with or without interacting with the α/β-subunits), with the former being viewed as inhibitory for the ATP hydrolysis activity of ATP synthase. Nevertheless, as common in current protein researches, the physiological relevance of such an “inserted” state for ATP synthase functioning is hardly known. To decipher this, designed an unnatural amino acid-mediated living-cell protein photocrosslinking analysis pipeline by developing the scarless genome-targeted site-directed mutagenesis and the high-throughput gel polyacrylamide gel electrophoresis (HT-PAGE) techniques. Employing this powerful approach, we systematically examined the interactions involving the C-terminal helix of the ε-subunit in cells living under a variety of experimental conditions. These studies enabled us to uncover that the “inserted” and “non-inserted” states of the ε-subunit exist as an equilibrium in cells cultured under common experimental conditions, shifting to the former upon the appearance of unfavorable conditions, acting as a low-gear state to strengthen the ATP synthesis function. Such a fine-tuning mechanism allows the ATP synthase to reversibly and instantly switch between two functional states. Further, the two powerful techniques that we developed here might be applied to many aspects of protein researches.

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.

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.

Protein biogenesis and quality control are essential to maintaining a functional pool of proteins and involve numerous protein factors that dynamically and transiently interact with each other and with the substrate proteins in living cells. Conventional methods are hardly effective for studying dynamic, transient, and weak protein-protein interactions that occur in cells. Herein, we review how the site-directed photocrosslinking approach, which relies on the genetic incorporation of a photoreactive unnatural amino acid into a protein of interest at selected individual amino acid residue positions and the covalent trapping of the interacting proteins upon ultraviolent irradiation, has become a highly efficient way to explore the aspects of protein contacts in living cells. For example, in the past decade, this approach has allowed the profiling of the in vivo substrate proteins of chaperones or proteases under both physiologically optimal and stressful (e.g., acidic) conditions, mapping residues located at protein interfaces, identifying new protein factors involved in the biogenesis of membrane proteins, trapping transiently formed protein complexes, and snapshotting different structural states of a protein. We anticipate that the site-directed photocrosslinking approach will play a fundamental role in dissecting the detailed mechanisms of protein biogenesis, quality control, and dynamics in the future.

β-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 β-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 β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-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 β-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 β-barrel OMP biogenesis.

DegP (also designated as HtrA) and its homologs are found in prokaryotic cells and such eukaryotic organelles as mitochondria and chloroplasts. DegP has been found to be essential for the growth of Gram-negative bacteria under heat shock conditions and arguably considered to possess both protease and chaperone activities. The function of DegP has not been clearly defined. Using genetically incorporated non-natural amino acids as photo-crosslinkers, here we identified the β-barrel outer membrane proteins (OMPs) as the major natural substrates of DegP in Escherichia coli cells. We also demonstrated that DegP primarily functions as a protease, at both low and high temperatures, to eliminate unfolded OMPs, with hardly any appreciable chaperone activity in cells. We also found that the toxic and cell membrane-damaging misfolded OMPs would accumulate in DegP-lacking cells cultured under heat shock conditions. Together, our study defines the primary function of DegP in OMP biogenesis and offers a mechanistic insight into the essentiality of DegP for cell growth under heat shock conditions. Structured digital abstract DegP physically interacts with ompA by pull down (1, 2) DegP physically interacts with ompX, ompA, ompW, ompF, nmpC and ompC by pull down (View interaction) DegP physically interacts with ompC and ompF by pull down (View interaction) DegP physically interacts with ompC and ompA by pull down (View interaction) DegP physically interacts with ompC, malE, fkpA, ompW, ompA, ompF, nmpC and ompX by cross-linking study (View interaction)

Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones that prevent the aggregation of various non-native proteins and play crucial roles for protein quality control in cells. It is poorly understood what natural substrate proteins, with respect to structural characteristics, are preferentially bound by sHSPs in cells. Here we compared the structural characteristics for the natural substrate proteins of Escherichia coli IbpB and Deinococcus radiodurans Hsp20.2 with the respective bacterial proteome at multiple levels, mainly by using bioinformatics analysis. Data indicate that both IbpB and Hsp20.2 preferentially bind to substrates of high molecular weight or moderate acidity. Surprisingly, their substrates contain abundant charged residues but not abundant hydrophobic residues, thus strongly indicating that ionic interactions other than hydrophobic interactions also play crucial roles for the substrate recognition and binding of sHSPs. Further, secondary structure prediction analysis indicates that the substrates of low percentage of β-sheets or coils but high percentage of α-helices are un-favored by both IbpB and Hsp20.2. In addition, IbpB preferentially interacts with multi-domain proteins but unfavorably with α + β proteins as revealed by SCOP analysis. Together, our data suggest that bacterial sHSPs, though having broad substrate spectrums, selectively bind to substrates of certain structural features. These structural characteristic elements may substantially participate in the sHSP-substrate interaction and/or increase the aggregation tendency of the substrates, thus making the substrates more preferentially bound by sHSPs.

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.

As a class of molecular chaperones, small heat shock proteins (sHsps) usually exist as multi-subunit spherical oligomers. In this study, we report that AgsA, a sHsp of Salmonella enterica serovar Typhimurium, spontaneously forms fibrils in vitro. These fibrils tend to be formed at elevated temperature and do not share the characteristics of amyloid. Interestingly, the fibril-forming AgsA is able to suppress the dithiothreitol-induced aggregation of insulin efficiently within a certain range of temperature. During this process, AgsA fibrils disappear and spherical complexes form between AgsA and insulin molecules. These data suggest that AgsA fibrils may represent a distinctive type of structural and functional form of sHsp from spherical oligomers. Our study provides new insights into sHsp structures and chaperone functions.Structured summary of protein interactionsAgsA and AgsA bind by electron microscopy (View interaction).Insulin and Insulin bind by molecular sieving (View interaction).Insulin and Insulin bind by electron microscopy (View interaction).AgsA and AgsA bind by molecular sieving (View interaction).AgsA and AgsA bind by fluorescence technology (View interaction).AgsA and AgsA bind by circular dichroism (View interaction).Insulin and Insulin bind by fluorescence technology (View interaction).AgsA and Insulin bind by molecular sieving (View interaction).AgsA and AgsA bind by electron tomography (View interaction).

Members of the PDI (protein disulfide-isomerase) family are critical for the correct folding of secretory proteins by catalysing disulfide bond formation as well as by serving as molecular chaperones to prevent protein aggregation. In the present paper, we report that the chaperone activity of the human pancreas-specific PDI homologue (PDIp) is independent of its enzymatic activity on the basis of the following lines of evidence. First, alkylation of PDIp by iodoacetamide fully abolishes its enzymatic activity, whereas it still retains most of its chaperone activity in preventing the aggregation of reduced insulin B chain and denatured GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Secondly, mutation of the cysteine residues in PDIp's active sites completely abolishes its enzymatic activity, but does not affect its chaperone activity. Thirdly, the b-b′ fragment of PDIp, which does not contain the active sites and is devoid of enzymatic activity, still has chaperone activity. Mechanistically, we found that both the recombinant PDIp expressed in Escherichia coli and the natural PDIp present in human or monkey pancreas can form stable complexes with thermal-denatured substrate proteins independently of their enzymatic activity. The high-molecular-mass soluble complexes between PDIp and GAPDH are formed in a stoichiometric manner (subunit ratio of 1:3.5–4.5), and can dissociate after storage for a certain time. As a proof-of-concept for the biological significance of PDIp in intact cells, we demonstrated that its selective expression in E. coli confers strong protection of these cells against heat shock and oxidative-stress-induced death independently of its enzymatic activity.

Members of the protein disulfide isomerase (PDI) family play a critical role in catalyzing the formation of disulfide bonds in secretory proteins, and most of these enzymes have a wide tissue distribution. However, the pancreas-specific PDI homolog was previously suggested to be exclusively expressed in the pancreas (thus commonly referred to as PDIp). In the present study, we found that PDIp was also highly expressed in several other tissues in mice, including the stomach, cecum, ileum, adrenal glands, epididymis, and prostate. Notably, in the digestive organs, such as the stomach and pancreas, very high levels of PDIp were selectively expressed in the digestive enzyme-secreting cells (e.g., gastric chief cells and pancreatic acinar cells). This observation suggests that PDIp may function as a protein-folding catalyst for secretory digestive enzymes. In ileum, PDIp was exclusively expressed in Paneth cells. In addition, high levels of PDIp expression were also detected in normal human pancreas, but its expression was mostly absent in human pancreatic duct adenocarcinoma and pancreatic cancer cell lines. The absence of PDIp expression in pancreatic adenocarcinoma may serve as an additional biomarker for pancreatic cancer.

Protein disulfide isomerase (PDI) and its homologs are catalysts of the formation of disulfide bonds in secretory proteins, and they also serve as molecular chaperones. In the present study, we investigated the redox-mediated regulation of the structures and functions of human pancreas-specific PDI homolog (PDIp). We found that formation of an inter-subunit disulfide bond in the recombinant human PDIp can alter not only its structure, but also its functions. PDIp exists predominantly as monomer under reducing conditions, but the dimeric form is significantly increased following the removal of the reducing agent, due to the formation of an inter-subunit disulfide bond. The oxidized PDIp (with an inter-subunit disulfide bond) appears to expose more hydrophobic patches and is more sensitive to protease digestion compared to the reduced form. Along with these structural changes, the oxidized PDIp also exhibits an enhanced chaperone activity. The formation of the inter-subunit disulfide bond in PDIp is mainly contributed by its non-active cysteine residue (cysteine-4), which is only present in human and primate PDIp, but not in rodent PDIp. In addition, we observed that the formation of the inter-subunit disulfide bond in PDIp is redox-dependent and is favored under oxidizing conditions, and that PDIp can function as a chaperone to form stable complexes with various non-native cellular proteins, particularly under oxidizing conditions. In light of these observations, it is concluded that the structures and functions of human PDIp are redox-regulated through formation of an inter-subunit disulfide bond between two cysteine-4 residues.

High-affinity biotinylated derivatives of 17β-estradiol (E2) are of value for isolation of various estrogen-binding proteins (including estrogen receptors) and also for studying protein–protein interactions involving these proteins. In this study, we developed a simplified route for the chemical synthesis of a biotinylated derivative of E2 (compound 7) with a side chain attached to its C-7α position. Compound 7, i.e., 7α-{7-[8-(biotinamido)-octanamido]-heptyl}-estradiol, could be readily synthesized from 6-keto-estradiol-3,17β-di-tetrahydropyranyl ether (compound 2, which can be prepared from E2), with a final yield of 36%. In vitro receptor-binding assay confirmed that the synthesized affinity ligand has a high binding affinity for both human estrogen receptor α and β. When the affinity ligand (compound 7) was immobilized with avidin on an affinity column, it effectively bound human estrogen receptor α, and the receptor protein could be selectively eluted with a biotin-containing buffer. Using the same affinity ligand, prolyl 4-hydroxylase β-subunit (also known as protein disulfide isomerase) was identified as one of the high-affinity E2-binding proteins in the whole cytosolic protein mixture prepared from MCF-7 human breast cancer cells. Computational molecular modeling analysis showed that compound 7 can bind to human estrogen receptor α in a similar manner as ICI-182,780 and raloxifene, and their binding energy values are also similar.

The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.

The ATP synthase (having a typical subunit composition of α3β3γδeab2c8-15) employs an intriguing rotary mechanism for the generation of ATP from ADP and Pi, using energy stored in a transmembrane proton gradient. The conventional rotary model, although being generally accepted, remains difficult to explain certain experimental observations. Here we propose an alternative rotary model for the ATP synthase such that what rotates is the catalytic α3β3 cylinder rather than the central stalk and the membrane-embedded c-ring. Specifically, the membrane translocation of protons would induce a cycled conformational change in the c-ring, leading to a reciprocating motion of the attached central stalk, which in turn drives the unidirectional rotation of the α3β3 cylinder. Such a reciprocating motion-driven rotation mechanism is somehow analogous to the working mechanism of a retractable click ballpoint pen. Our new model not only explains the experimental observations that have been difficult to reconcile with the conventional model but also avoids its theoretical illogicality.

Bacterial persisters, usually being considered as dormant cells that are tolerant to antibiotics, are an important source for recurrent infection and emergence of antibiotic resistant pathogens. Clinical eradication of pathogenic persisters is highly desired but greatly difficult mainly due to the substantial reduction in antibiotics uptake as well as the non-active state of the drug targets. Here we report that bacterial persisters (normal growing cells as well) can be effectively eradicated by aminoglycoside antibiotics upon hypoionic shock (e.g. pure water treatment) even for less than one minute. Such hypoionic shock potentiation effect on aminoglycosides is proton motive force-independent and is apparently achieved by promoting the entrance of aminoglycosides, speculatively through the mechanosensitive ion channels. Our revelations may provide a simple and powerful strategy to eradicate pathogen persisters.