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Hsp70 molecular chaperones play central roles in maintaining a healthy cellular proteome. Hsp70s function by binding to short peptide sequences in incompletely folded client proteins, thus preventing them from misfolding and/or aggregating, and in many cases holding them in a state that is competent for subsequent processes like translocation across membranes. There is considerable interest in predicting the sites where Hsp70s may bind their clients, as the ability to do so sheds light on the cellular functions of the chaperone. In addition, the capacity of the Hsp70 chaperone family to bind to a broad array of clients and to identify accessible sequences that enable discrimination of those that are folded from those that are not fully folded, which is essential to their cellular roles, is a fascinating puzzle in molecular recognition. In this article we discuss efforts to harness computational modeling with input from experimental data to develop a predictive understanding of the promiscuous yet selective binding of Hsp70 molecular chaperones to accessible sequences within their client proteins. We trace how an increasing understanding of the complexities of Hsp70-client interactions has led computational modeling to new underlying assumptions and design features. We describe the trend from purely data-driven analysis toward increased reliance on physics-based modeling that deeply integrates structural information and sequence-based functional data with physics-based binding energies. Notably, new experimental insights are adding to our understanding of the molecular origins of "selective promiscuity" in substrate binding by Hsp70 chaperones and challenging the underlying assumptions and design used in earlier predictive models. Taking the new experimental findings together with exciting progress in computational modeling of protein structures leads us to foresee a bright future for a predictive understanding of selective-yet-promiscuous binding exploited by Hsp70 molecular chaperones; the resulting new insights will also apply to substrate binding by other chaperones and by signaling proteins.

Background: High-sensitivity (hs) assays allow to measure cardiac troponin T and I (cTnT/I) even in healthy individuals. The higher hs-cTn values, the higher the ongoing cardiomyocyte damage, and then reasonably the risk of developing symptomatic cardiac disease. Methods: We retrieved all studies evaluating the prognostic value of hs-cTnT or I in the general population. We calculated pooled hazard ratio (HR) values for all-cause and cardiovascular death, cardiovascular events and heart failure (HF) hospitalization. Results: We included 24 studies for a total of 203,202 subjects; 11 studies assessed hs-cTnT and 14 hs-cTnI. One standard deviation (SD) increase in baseline hs-cTn was associated with a 23% higher risk of all-cause death (HR 1.226, 95% CI 1.083-1.388, p

Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Escherichia coli Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to E. coli alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA. Analysis of SBD complexes with proPhoA peptides by a combination of X-ray crystallography, methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), and paramagnetic relaxation enhancement (PRE) NMR and chemical cross-linking experiments provided detailed descriptions of their binding modes. Importantly, many sequences populate multiple SBD binding modes, including both the canonical N to C orientation and a C to N orientation. The favored peptide binding mode optimizes substrate residue side-chain compatibility with the SBD binding pockets independent of backbone orientation. Relating these results to the binding of the SBD to full-length proPhoA, we observe that multiple chaperones may bind to the protein substrate, and the binding sites, well separated in the proPhoA sequence, behave independently. The hierarchy of chaperone binding to sites on the protein was generally consistent with the apparent binding affinities observed for the peptides corresponding to these sites. Functionally, these results reveal that Hsp70s "read" sequences without regard to the backbone direction and that both binding orientations must be considered in current predictive algorithms.

Objectives Most research addressing needs and concerns of young patients with breast cancer (≤40 years) is retrospective. The HOHO European protocol is a prospective multicenter cohort study of young women with newly diagnosed breast cancer, about fertility, psychosocial and quality of life concerns. Here we report the baseline data and focus on predictors of fertility concerns. Materials and methods Patient surveys and medical record review were used. The baseline survey included sociodemographic, medical and treatment data as well as questions on fertility concerns and preservation strategies. Subscales from the CAncer Rehabilitation Evaluation System-Short Form (CARES-SF) were administered to measure specific quality of life aspects. Uni- and multivariable modeling were used to investigate predictors of greater fertility concern. Results Among 297 eligible respondents, 67% discussed fertility issues before starting therapy, 64% were concerned about becoming infertile after treatment, and 15% decided not to follow prescribed therapies. Fifty-four percent of women wished future children before diagnosis; of these, 71% still desired biologic children afterwards. In multivariable analysis, not having children was the only patient characteristic significantly associated with fertility concerns at diagnosis. Twenty-seven percent used fertility preservation strategies. Women who received chemotherapy reported greater physical (p = 0.021) and sexual difficulties (p = 0.039) than women who did not. Women who were married or had a partner reported less psychosocial problems than single women (p = 0.039). Conclusions Young women with newly diagnosed breast cancer have several concerns, including, but not limited to, fertility. The HOHO European study provides valuable information to develop targeted interventions.

The evidence available suggests that cannabinoid-based products improve spasticity, chronic pain, and bladder dysfunction, which are common symptoms in more than 50% of multiple sclerosis (MS) patients. In this paper we present three cases of patients with progressive or relapsing forms of MS, with different severity, duration, and with various degrees of hypertonia and or bladder dysfuction, treated with nabiximols (Sativex®). Nabiximols was administered as an oral spray to reduce MS-associated hypertonia. The results show the efficacy of the therapy in MS patients with moderate to severe spasticity and resistant or intolerant to common myorelaxants drugs. In conclusion, nabiximols proves to be an effective treatment, easy to administer, even in patients with motor disabilities affecting the hands, and without important side effects.

Introduction Surgical site infection (SSI) is one of the most common healthcare-associated infections (HAIs). However, there is a lack of data available about SSI in children worldwide, especially from low-income and middle-income countries. This study aimed to estimate the incidence of SSI in children and associations between SSI and morbidity across human development settings. Methods A multicentre, international, prospective, validated cohort study of children aged under 16 years undergoing clean-contaminated, contaminated or dirty gastrointestinal surgery. Any hospital in the world providing paediatric surgery was eligible to contribute data between January and July 2016. The primary outcome was the incidence of SSI by 30 days. Relationships between explanatory variables and SSI were examined using multilevel logistic regression. Countries were stratified into high development, middle development and low development groups using the United Nations Human Development Index (HDI). Results Of 1159 children across 181 hospitals in 51 countries, 523 (45·1%) children were from high HDI, 397 (34·2%) from middle HDI and 239 (20·6%) from low HDI countries. The 30-day SSI rate was 6.3% (33/523) in high HDI, 12·8% (51/397) in middle HDI and 24·7% (59/239) in low HDI countries. SSI was associated with higher incidence of 30-day mortality, intervention, organ-space infection and other HAIs, with the highest rates seen in low HDI countries. Median length of stay in patients who had an SSI was longer (7.0 days), compared with 3.0 days in patients who did not have an SSI. Use of laparoscopy was associated with significantly lower SSI rates, even after accounting for HDI. Conclusion The odds of SSI in children is nearly four times greater in low HDI compared with high HDI countries. Policies to reduce SSI should be prioritised as part of the wider global agenda. ©

ObjectiveCladribine tablets were tested against placebo in randomized controlled trials (RCTs). In this study, the effectiveness of cladribine vs other approved drugs in patients with relapsing-remitting MS (RRMS) was compared by matching RCT to observational data.MethodsData from the pivotal trial assessing cladribine tablets vs placebo (CLARITY) were propensity score matched to data from the Italian multicenter database i-MuST. This database included 3,150 patients diagnosed between 2010 and 2018 at 24 Italian MS centers who started a disease-modifying drug. The annualized relapse rate (ARR) over 2 years from treatment start and the 24-week confirmed disability progression were compared between patients treated with cladribine and other approved drugs (interferon, glatiramer acetate, fingolimod, natalizumab, and dimethyl fumarate), with comparisons with placebo as a reference. Treatment effects were estimated by the inverse probability weighting negative binomial regression model for ARR and Cox model for disability progression. The treatment effect has also been evaluated according to baseline disease activity.ResultsAll weighted baseline characteristics were well balanced between groups. All drugs tested had an effect vs placebo close to that detected in the RCT. Patients treated with cladribine had a significantly lower ARR compared with interferon (relapse ratio [RR] = 0.48; p < 0.001), glatiramer acetate (RR = 0.49; p < 0.001), and dimethyl fumarate (RR = 0.6; p = 0.001); a similar ARR to that with fingolimod (RR = 0.74; p = 0.24); and a significantly higher ARR than natalizumab (RR = 2.13; p = 0.014), confirming results obtained by indirect treatment comparisons from RCTs (network meta-analyses). The relative effect of cladribine tablets 10 mg (cumulative dose 3.5 mg/kg over 2 years) was higher in patients with high disease activity vs all treatments except fingolimod and natalizumab. Effects on disability progression were largely nonsignificant, probably due to lack of power for such analysis.ConclusionIn patients with RRMS, cladribine tablets showed lower ARR compared with matched patients who started interferon, glatiramer acetate, or dimethyl fumarate; was similar to fingolimod; and was higher than natalizumab. The beneficial effect of cladribine tablets was generally amplified in the subgroup of patients with high disease activity.Classification of evidenceThis study provides Class III evidence that for patients with RRMS, cladribine-treated patients had lower ARR compared with interferon, glatiramer acetate, or dimethyl fumarate; similar ARR compared with fingolimod; and higher ARR compared with natalizumab.

The Hsp70 family of chaperones works with its co-chaperones, the nucleotide exchange factors and J-domain proteins, to facilitate a multitude of cellular functions. Central players in protein homeostasis, these jacks-of-many-trades are utilized in a variety of ways because of their ability to bind with selective promiscuity to regions of their client proteins that are exposed when the client is unfolded, either fully or partially, or visits a conformational state that exposes the binding region in a regulated manner. The key to Hsp70 functions is that their substrate binding is transient and allosterically cycles in a nucleotide-dependent fashion between high- and low-affinity states. In the past few years, structural insights into the molecular mechanism of this allosterically regulated binding have emerged and provided deep insight into the deceptively simple Hsp70 molecular machine that is so widely harnessed by nature for diverse cellular functions. In this review, these structural insights are discussed to give a picture of the current understanding of how Hsp70 chaperones work.

Alemtuzumab is a humanized anti-CD52 monoclonal antibody used for the treatment of high activity relapsing multiple sclerosis (R-MS). The most common adverse event is an infusion reaction due to cytokine-release. Autoimmunity can arise from months to years after treatment and encompasses Grave’s disease and thrombocytopenia. Recent reports of stroke, heart attack, and arterial dissection after alemtuzumab administration, in some cases within hours of infusion, led the European Medicines Agency’s (EMA's) Pharmacovigilance Risk Assessment Committee (PRAC) to a safety review of treatment with alemtuzumab. We report a D-Dimer increasing with suspected associated pulmonary embolism in an RMS patient after the first alemtuzumab administration. D-dimer test is not mandatory after alemtuzumab treatment, but its possible increase should warn the physician to select the patients with lower cardiovascular and thrombosis risk.

The 70-kDa heat shock proteins (Hsp70s) are molecular chaperones that perform a wide range of critical cellular functions. They assist in the folding of newly synthesized proteins, facilitate assembly of specific protein complexes, shepherd proteins across membranes, and prevent protein misfolding and aggregation. Hsp70s perform these functions by a conserved mechanism that relies on allosteric cycles of nucleotide-modulated binding and release of client proteins. Current models for Hsp70 allostery have come from extensive study of the bacterial Hsp70, DnaK. Extending our understanding to eukaryotic Hsp70s is extremely important not only in providing a likely common mechanistic framework but also because of their central roles in cellular physiology. In this study, we examined the allosteric behaviors of the eukaryotic cytoplasmic Hsp70s, HspA1 and Hsc70, and found significant differences from that of DnaK. We found that HspA1 and Hsc70 favor a state in which the nucleotide-binding domain (NBD) and substrate-binding domain (SBD) are intimately docked significantly more as compared to DnaK. Past work established that the NBD-SBD interface and the helical lid-β-SBD interface govern the allosteric landscape of DnaK. Here, we identified sites on these interfaces that differ between eukaryotic cytoplasmic Hsp70s and DnaK. Our mutational analysis has revealed key evolutionary variations that account for the population shifts between the docked and undocked conformations. These results underline the tunability of Hsp70 functions by modulation of allosteric interfaces through evolutionary diversification and also suggest sites where the binding of small-molecule modulators could influence Hsp70 function.

Summary Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90., Graphical Abstract, Highlights • Antiparallel dimerization of Hsp70 is stabilized by PTMs • Hsp40 catalyzes Hsp70 dimerization and client transfer to Hsp70 • Hsp70 antiparallel dimerization is maintained in the client-loading complex • Addition of p23 induces transfer of GR onto Hsp90 and loss of Hop and Hsp70, Morgner et al. combine native mass spectrometry and chemical crosslinking to define the interactions of the Hsp70/90 chaperone system. They show that Hsp70 dimerization is antiparallel and stabilized by PTMs. They monitor the formation of chaperone complexes and discover a hexameric client-loading complex containing an Hsp70 dimer.

Hsp70 molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, and more specialized roles such as disassembly of particular protein complexes. It is a fascinating question to ask how the mechanism of these deceptively simple molecular machines is matched to their roles in these wide-ranging processes. The key is a combination of the nature of the recognition and binding of Hsp70 substrates and the impact of Hsp70 action on their substrates. In many cases, the binding, which relies on interaction with an extended, accessible short hydrophobic sequence, favors more unfolded states of client proteins. The ATP-mediated dissociation of the substrate thus releases it in a relatively less folded state for downstream folding, membrane translocation, or hand-off to another chaperone. There are cases, such as regulation of the heat shock response or disassembly of clathrin-coats, however, where binding of a short hydrophobic sequence selects conformational states of clients to favor their productive participation in a subsequent step. This Perspective discusses current understanding of how Hsp70 molecular chaperones recognize and act on their substrates and the relationships between these fundamental processes and the functional roles played by these molecular machines.

In this issue of Molecular Cell, Behnke et al. (2016) describe a novel cell-based peptide-binding assay and use it to analyze the binding specificities of the endoplasmic reticulum Hsp70 chaperone and its co-chaperones and to probe their different roles in protein quality control.

Nearly one third of the eukaryotic proteome traverses the secretory pathway and most of these proteins are N-glycosylated in the lumen of the endoplasmic reticulum. N-glycans fulfill multiple structural and biological functions, and are crucial for productive folding of many glycoproteins. N-glycosylation involves the attachment of an oligosaccharide to selected asparagine residues in the sequence N-X-S/T (X ≠ P), a motif known as an N-glycosylation’sequon’. Mutations that create novel sequons can cause disease due to the destabilizing effect of a bulky N-glycan. Thus, an analogous process must have occurred during evolution, whenever ancestrally cytosolic proteins were recruited to the secretory pathway. Here, we show that during evolution N-glycosylation triggered a dual selection pressure on secretory pathway proteins: while sequons were positively selected in solvent exposed regions, they were almost completely eliminated from buried sites. This process is one of the sharpest evolutionary signatures of secretory pathway proteins, and was therefore critical for the evolution of an efficient secretory pathway. Fil: López Medus, Máximo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Gomez, Gabriela Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina Fil: Zacchi, Lucia Florencia. University of Queensland; Australia Fil: Couto, Paula Monserrat. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Labriola, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Labanda, María Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Corti Bielsa, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Clerico, Eugenia. University of Massachusetts; Estados Unidos Fil: Schulz, Benjamin L.. University of Queensland; Australia Fil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Objective To map different areas of pain sensitivity and to determine the existence and/or pattern of referred pain from upon stimulating the sinonasal cavity. Study design Experimental human study. Methods Mechanical and electrical stimulations to various anatomical structures and areas of the nasal and sinus cavities were conducted on nine volunteers. Intensity, location and character of pain were recorded in all subjects. Results The postero-superior (cephalic) aspect of the nasal cavity, primarily the anterior face of the sphenoid sinus and the superior turbinate, were the most sensitive sites, and the antero-inferior (caudal) region was the least sensitive. Referred pain to the head and face was reported by several subjects. Conclusion Topographical differences in pain sensitivity exist in the sinonasal cavity. The phenomenon of referred pain from the nasal cavity was demonstrated.

Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.

Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including choice of site for ligation with retention of function, optimization of ligation yield, and purification from unreacted domains. Ligated semi-labeled DnaK was successfully produced with a Cys residue at position 383, and the ligated product harboring the Cys mutation was confirmed to be functional and identical to an expressed Cys-containing two-domain construct. The NMR spectrum of the segmentally labeled protein was considerably simplified, enabling unequivocal assignment and enhanced analysis of dynamics, as a prelude to exploring the energy landscape for allostery in the Hsp70 family.

Background The International Breast Cancer Study Group (IBCSG), in collaboration with Dana-Farber Cancer Institute, conducts the European (EU) cohort of the Helping Ourselves Helping Others (HOHO) study. HOHO is a prospective longitudinal cohort study investigating short and long-term treatment and fertility concerns in young women with breast cancer (BC): most previous studies have been retrospective. Patients and methods From Aug 2009-Jan 2016, 300 patients aged 18-40 yrs with early/advanced BC were enrolled within 6 mos of diagnosis in 18 Centers in Italy and Switzerland. Patients are surveyed every 6 mos the first 3 yrs and yearly for additional 7 yrs. 297 baseline surveys were evaluable. The baseline survey includes sociodemographic and treatment data, and a modified Fertility Issues Survey (including fertility concerns, preservation, outcome and impact on treatment decisions). Clinical data are collected yearly. We qualitatively compare the EU baseline data with the published US data (Ruddy et al, JCO 32:1151, 2014). Results Almost two thirds of women in both cohorts discussed future fertility with their doctors at diagnosis. Fertility concerns, desire for future pregnancy (both before disease onset and at time of survey) and steps to reduce infertility were all more common in the EU cohort. EU women were also more concerned about fertility when discussing treatment and about a possible relapse affecting their ability to care for future children. Conclusion Many young women with newly diagnosed BC have concerns about fertility, which may affect their treatment decisions. In the EU cohort fewer women had a stable relationship and children before diagnosis, potentially influencing their treatment decisions and encouraging fertility preservation. Concerns about a possible relapse could partly explain the decrease in their pregnancy desire after the disease. Differences between EU and US women seem to emerge: continued data collection will determine if they persist over time. The POSITIVE Trial (IBCSG 48-14/ALLIANCE 221405) will address safety and outcome of pregnancy after BC. USEUN (%)N (%)620 (100)297 (100)Age Citation Format: Pagani O, Bagnardi V, Ruggeri M, Bianco N, Gallerani E, Buser K, Giordano M, Gianni L, Rabaglio M, Freschi A, Cretella E, Clerico M, Amadori D, Simoncini E, Ciccarese M, Rauch D, Glaus A, Berardi R, Franzetti A, Ruddy KJ, Gelber S, Partridge AH, Colleoni M. HOHO study: How European and US young women cope with breast cancer and fertility concerns [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD6-04.

Circulating inhibitory serpins regulate important physiological processes including inflammation. Misfolding and polymerization of serpins in the endoplasmic reticulum (ER) can lead to both loss-of-function and gain-of-function diseases. The topology of the two-domain, non-sequential serpin fold is complicated with both the αβ and mainly β domains containing regions from near the N- and the C-termini. Despite these complications recent molecular dynamics simulations of serpin folding suggest that significant amounts of local secondary structure forms and rearranges for long-range contact formation resulting in the folded, active serpin. This folding pathway requires a balance between local structural stability and the flexibility/dynamics needed for long-range contact formation. To help test this proposed mechanism, we expressed and characterized N- to C-terminal fragments of the canonical, 394 residue serpin α1-antitrypsin (Alpha1). Even the shortest, 150 residue long, fragment showed cooperative unfolding and surprising stability with a denaturation midpoint of 1.9 M guanidinium hydrochloride. Nonetheless, the far-UV circular dichroism (CD) spectra differ from the spectra predicted for the full-length, native protein. Similarly, fluorescence emission of the longest fragment, 1-323, exhibited the hyper-fluorescent signature of the unfolding/folding intermediate observed during equilibrium denaturation or refolding of full-length Alpha1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to better define the structure of the fragments. After 10 seconds of deuteration, significant protection is observed in parts of the N-terminal region of the αβ domain, and for longer fragments in some of the β strands that form the core of the β domain. These results suggest that surprisingly stable local structures can be formed by fragments of the serpin Alpha1 and are consistent with models for serpin folding derived from molecular dynamics simulations. The results also suggest that significant amounts of serpin structure might form co-translationally.

Knowledge about the balance between heritable and nonheritable risk in multiple sclerosis (MS) is based on twin studies in high-prevalence areas. In a study that avoided ascertainment limitations and directly compared continental Italy (medium-prevalence) and Sardinia (high-prevalence), we ascertained 216 pairs from 34,549 patients. This gives a twinning rate of 0.62% among MS patients, significantly less than that of the general population. In continental Italy, probandwise concordance was 14.5% (95% confidence interval, 5.1-23.8) for monozygotic and 4.0% (95% confidence interval, 0.8-7.1) for dizygotic twins. Results in Sardinia resemble those in northern populations but in limited numbers. Monozygotic concordance was 22.2% (95% confidence interval, 0-49.3) probandwise, but no concordant dizygotic pairs were identified. A questionnaire on 80 items possibly related to disease cause was administered to 70 twin pairs, 135 sporadic patients, and 135 healthy volunteers. Variables positively (7) or negatively (2) associated with predisposition and concordance in twins largely overlapped and were mainly linked to infection. If compared with previous studies, our data demonstrate that penetrance in twins appears to correlate with MS prevalence. They highlight the relevance of nonheritable variables in Mediterranean areas. The apparent underrepresentation of MS among Italian twins draws attention to protective factors, shared by twins, that may influence susceptibility.

DnaK is a molecular chaperone that helps protein folding and protects cells from stress. The allosteric mechanism of DnaK enables ATP binding to the N-terminal nucleotide-binding domain (NBD) to alter substrate affinity to the C-terminal substrate-binding domain (SBD) and in turn substrate binding enhances ATP hydrolysis. Cycling between ATP-bound and ADP/substrate-bound states requires DnaK to visit an “ATP+substrate” state with high ATPase activity and fast on/off kinetics of substrate binding. The short interdomain linker transduces the allosteric signal between NBD-SBD and the β-SBD-α-lid interfaces. We used pulsed dipolar ESR and ion mobility mass spectroscopy technologies to characterize the conformational ensembles of DnaK in its allosteric states to better understand how the nucleotide and substrates modulate the allosteric landscape. We found that DnaK has a narrow distance distribution in ATP-bound state, but broader distance distributions in all other states exhibit multiple coexisting components. The ATP+substrate ensemble reflects the tug-of-war between the forces of NBD-SBD interaction driven by the binding of nucleotides, and the force of β-SBD-α-lid interaction driven by the binding of substrates. The ATP+substrate state contains 24% of docked and 76% of undocked conformers. The ADP+substrate state has a smaller fraction of docked conformers and an additional species, which we may represent as a “domain rotamer” around the unbound linker. Rotation of the NBD and SBD around the interdomain linker may play an important role in the allosteric mechanism. The ATP+substrate state releases the SBD's helical lid from the NBD bound in the ATP-bound state to an SBD bound position and a “free” position; and the ADP+substrate state pushes the equilibrium from the free position to the SBD bound position. The allosteric states can be modulated by mutations to dissect the energetic contributions.

Hsp70s comprise a group of highly conserved chaperones essential to cellular homeostasis. They take part in a wide array of processes, including their key roles in protein homeostasis in which they guard the proteome by binding partially folded proteins, thus protecting them from aggregation and allowing them to fold. Hsp70s work through binding and release of hydrophobic protein segments at the C-terminal substrate-binding domain (SBD), the affinity of which is modulated by ATP binding and hydrolysis to the N-terminal nucleotide-binding domain (NBD): When ATP binds to the NBD, the SBD has low affinity for substrate; when ATP is hydrolyzed and ADP is bound to the NBD, the SBD has high substrate affinity. This allosteric mechanism is regulated by co-chaperones: J proteins deliver substrates to Hsp70s and accelerate ATP hydrolysis; nucleotide exchange factors facilitate exchange of ADP by ATP. In humans, Hsp70 members are expressed in all cell types and many subcellular compartments. The functional diversity of Hsp70s is stunning, including shepherding of nascent chains, protein translocation across membranes, disassembly of complexes and recovery of proteins from aggregates. Some Hsp70s focus on housekeeping functions, while others are highly expressed under stress, promote cell survival and are essential for the cell to cope with adverse conditions. Consequently, Hsp70s are crucial for tumor cells to thrive and constitute a promising target for cancer treatments; in addition, Hsp70s have ameliorative effects on protein misfolding diseases. This review describes structural features and cellular functions of Hsp70s that are relevant to design of Hsp70-targeted drugs.

Three patients with typical (common) migraine unresponsive to conventional therapy were evaluated with complete otolaryngologic examination, diagnostic nasal endoscopy, and coronal sinus computed tomography (CT). Enlargement of the superior turbinate due to pneumatization, with accompanying mucosal contact, was found in each case. Application of topical or injected anesthetic to the superior turbinate lessened or relieved headache, and subsequent endoscopic sinonasal surgery succeeded in providing significant headache relief (follow-up of 6 to 14 months). This is the first report of superior turbinate variations/abnormalities causing migraine, or indeed any type of headache. A discussion of the mechanism of referred pain from mucosal contact is offered after a discussion of each case.

Headache associated with acute sinusitis is a well-recognized entity; the diagnosis is easily made due to the associated nasal and sinus symptoms. However, the phenomenon of referred headache from chronic sinusitis or intranasal abnormalities or both without upper respiratory symptoms is not well understood. Only recently have the nasal and sinus cavities been adequately visualized by both the human eye and radiographic techniques; a fact that may account for the historic neglect in considering this region a factor in headache etiology. Modern techniques employed in the workup of sinusitis, namely the use of rigid nasal endoscopes and coronal-plane CT scanning, have greatly enhanced the clinician's ability to evaluate and diagnose pathology in this area. This report describes a series of patients presenting with various primary headache syndromes without significant nasal or sinus symptoms who failed to respond to conventional antiheadache therapy. On nasal endoscopic and coronal CT examinations, various intranasal and sinus abnormalities were found (either anatomic variations or subclinical inflammation). Medical and/or surgical therapy addressing the sinonasal pathology resulted in improvement in every case, ranging from decreased severity of attacks to total resolution of headaches. A model explaining the possible mechanism of referred vascular-type headache from sinus and nasal origin is proposed.

SummaryThe allosteric mechanism of Hsp70 molecular chaperones enables ATP binding to the N-terminal nucleotide-binding domain (NBD) to alter substrate affinity to the C-terminal substrate-binding domain (SBD) and substrate binding to enhance ATP hydrolysis. Cycling between ATP-bound and ADP/substrate-bound states requires Hsp70s to visit a state with high ATPase activity and fast on/off kinetics of substrate binding. We have trapped this “allosterically active” state for the E. coli Hsp70, DnaK, and identified how interactions among the NBD, the β subdomain of the SBD, the SBD α-helical lid, and the conserved hydrophobic interdomain linker enable allosteric signal transmission between ligand-binding sites. Allostery in Hsp70s results from an energetic tug-of-war between domain conformations and formation of two orthogonal interfaces: between the NBD and SBD, and between the helical lid and the β subdomain of the SBD. The resulting energetic tension underlies Hsp70 functional properties and enables them to be modulated by ligands and cochaperones and “tuned” through evolution.

Background The purpose of the study was to evaluate the benefit of adjuvant chemotherapy (AC) versus surgery alone in patients with muscle-invasive bladder cancer (MIBC). Patients and methods One hundred and ninety-four patients with pT2G3, pT3–4, N0–2 transitional cell bladder carcinoma were randomly allocated to control (92 patients) or to four courses of AC (102 patients). These latter patients were further randomly assigned to receive gemcitabine 1000 mg/m2 days 1, 8 and 15 and cisplatin 70 mg/m2 day 2 or gemcitabine as above plus cisplatin 70 mg/m2 day 15, every 28 days. Results At a median follow-up of 35 months, the 5-year overall survival (OS) was 48.5%, with no difference between the two arms [P = 0.24, hazard ratio (HR) 1.29, 95% confidence interval (CI) 0.84–1.99]. Mortality hazard was significantly correlated with Nodes (N) and Tumor (T) stage. The control and AC arms had comparable disease-free survival (42.3% and 37.2%, respectively; P = 0.70, HR 1.08, 95% CI 0.73–1.59). Only 62% of patients received the planned cycles. A significant higher incidence of thrombocytopenia was observed in patients receiving cisplatin on day 2 (P = 0.006). A similar global quality of life was observed in the two arms. Conclusion The study was underpowered to demonstrate that AC with cisplatin and gemcitabine improves OS and disease-free survival in patients with MIBC.

BACKGROUND The EORTC Head and Neck Cancer Cooperative Group conducted a randomized comparison of cisplatin, methotrexate, bleomycin and vincristine (CABO) versus cisplatin and 5-fluorouracil (CF) versus cisplatin (C) in chemotherapy naive patients with recurrent or metastatic squamous cell carcinoma of the head and neck. The primary objectives of this study were to investigate whether the CF regimen was in anyway superior to the CABO regimen and to detect any superiority of these two combinations over cisplatin alone. PATIENTS AND METHODS Three hundred eighty-two patients were randomized to one of three treatments: (1) methotrexate (40 mg/m2) days 1 and 15, bleomycin (10 mg) and vincristine (2 mg) days 1, 8 and 15, cisplatin (50 mg/m2) day 4, repeated every 21 days, (2) cisplatin (100 mg/m2) and 5-FU (1 g/m2 x 4), repeated every 21 days, and (3) cisplatin (50 mg/m2) days 1 and 8, repeated every 28 days. After 3 cycles, all responding and stable disease patients in the three arms of the study continued with cisplatin alone. RESULTS The overall response rates to CABO (34%) and CF (31%) were superior to C (15%) (p < 0.001, p = 0.003, respectively). In addition, complete response rate to CABO (9.5%) was superior to that of C (2.5%) (p = 0.02), and also superior to that of CF (1.7%) (p = 0.01). Response was associated with performance status and prior treatment, but by multivariate analysis treatment type was the important determinant of response (p = 0.0006). Although CABO and CF were superior to C with respect to time to progression within the first 6 to 8 months after randomization, there was no overall difference in progression-free survival or survival between the three arms of the study. Both hematologic and non-hematologic toxicity were worse in the combination chemotherapy arms. CONCLUSION We conclude that the CF regimen has no advantage over the CABO regimen, which in fact showed a higher complete response rate. Both combinations showed improved response rates but also more toxicity and no improvement in overall survival in comparison with cisplatin alone.

Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Forster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH

Molecular aspects of the circadian clock in the cyanobacterium Synechococcus elongatus have been described in great detail. Three-dimensional structures have been determined for the three proteins, KaiA, KaiB and KaiC, that constitute a central oscillator of the clock. Moreover, a temperature-compensated circadian rhythm of KaiC phosphorylation can be reconstituted in vitro with the addition of KaiA, KaiB and ATP. These data suggest a relatively simple circadian system in which a single oscillator provides temporal information for all downstream processes. However, in vivo the situation is more complex, and additional components contribute to the maintenance of a normal period, the resetting of relative phases of circadian oscillations, and the control of rhythms of gene expression. We show here that two well-studied promoters in the S. elongatus genome report different circadian periods of expression under a given set of conditions in wild-type as well as mutant genetic backgrounds. Moreover, the period differs between these promoters with respect to modulation by light intensity, growth phase, and the presence or absence of a promoter-recognition subunit of RNA polymerase. These data contrast sharply with the current clock model in which a single Kai-based oscillator governs circadian period. Overall, these findings suggest that complex interactions among the circadian oscillator, perhaps other oscillators, and other cellular machinery result in a clock that is plastic and sensitive to the environment and to the physiological state of the cell.

Photoaffinity crosslinking comprises a group of invaluable techniques used to investigate in detail a binding interaction between two polypeptides. As the diverse photo crosslinking techniques available display inherent differences, the method of choice will provide specific information about a particular system under study. We used two complementary crosslinking approaches: photo-induced crosslinking of unmodified proteins (PICUP) and benzophenone-mediated (BPM) crosslinking to extensively examine the interaction between the signal recognition particle (SRP) and signal sequences. Signal peptide binding by SRP presents a central puzzle in the protein targeting process because signal sequences must be recognized with fidelity but lack strict primary structural homology. The concurrent use of PICUP and BPM crosslinking to link signal peptides to E. coli SRP allowed us to explore the crosslinking pattern resulting from using different crosslinking chemistries, varying the position of the photoprobe in the hydrophobic core of the signal sequence, and shifting the crosslinking reactive group away from the signal peptide backbone. By PICUP, signal peptides crosslinked exclusively to the NG domain of the SRP protein Ffh, regardless of the position of the reactive residue. Benzophenone-modified amino acids preferentially crosslinked the signal peptide to the C-terminal (M) domain of Ffh. We conclude that signal peptide binding is largely mediated by the M domain. Importantly, our data also indicate intimate, at least transient, contacts between the hydrophobic core of the signal peptide and the NG domain. These results reopen the possibility of a direct involvement of the NG domain in signal sequence recognition.

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self-contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these "zipcodes" for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence-binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future.

Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The "plastic" genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation. Here we describe different approaches to mutagenize the chromosome of S. elongatus, including insertional mutagenesis, hit-and-run allele replacement, rps12-mediated gene replacement, and regulated expression of genes from ectopic sites, the neutral sites of the S. elongatus genome.

The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying prokaryotic circadian rhythms. Although S. elongatus does not display an easily measurable overt circadian behavior, its gene expression is under circadian control; hence, a "behavior" is created by linking a cyanobacterial promoter to either the bacterial luxAB or firefly luc luciferase genes to create reporter fusions whose activity can be easily monitored by bioluminescence. Numerous vectors have been created in our lab for introducing luciferase reporter genes into the S. elongatus chromosome. A choice of methods and equipment to detect light production from the luciferase fusions provides a means for high-throughput, automated mutant screens as well as testing rhythms from two promoter fusions within the same cell culture.

Synechococcus elongatus PCC 7942 is an excellent model system for studying the molecular mechanism of the circadian clock in cyanobacteria. The "plastic" genetic characteristics of this organism have facilitated the development of various methods for mutagenesis of its chromosome. These methods are based on homologous recombination between the chromosome and foreign DNA, introduced to the cyanobacteria by either transformation or conjugation. Here we describe different approaches to mutagenize the chromosome of S. elongatus, including insertional mutagenesis, hit-and-run allele replacement, rps12-mediated gene replacement, and regulated expression of genes from ectopic sites, the neutral sites of the S. elongatus genome.

The unicellular cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying prokaryotic circadian rhythms. Although S. elongatus does not display an easily measurable overt circadian behavior, its gene expression is under circadian control; hence, a "behavior" is created by linking a cyanobacterial promoter to either the bacterial luxAB or firefly luc luciferase genes to create reporter fusions whose activity can be easily monitored by bioluminescence. Numerous vectors have been created in our lab for introducing luciferase reporter genes into the S. elongatus chromosome. A choice of methods and equipment to detect light production from the luciferase fusions provides a means for high-throughput, automated mutant screens as well as testing rhythms from two promoter fusions within the same cell culture.