Genetic and biochemical analyses of Hsp70-Hsp40 interactions in Saccharomyces cerevisiae provide insights into specificity and mechanisms of regulation

Heat shock proteins of 70kDa (Hsp70s) and their J domain-containing Hsp40 cofactors are conserved chaperone pairs that facilitate diverse cellular processes. One essential Hsp70 in the endoplasmic reticulum (ER) lumen, BiP (Kar2p in yeast), participates in polypeptide translocation into the ER, protein folding, and ER-associated degradation (ERAD). Like other Hsp70s, BiP contains an N-terminal ATPase domain, followed by a substrate binding domain and a C-terminal lid domain. To better define how substrate affinity and Hsp40 interaction affect BiP function, I constructed and characterized a mutation, R217A, in the putative J domain-interacting surface of yeast BiP. The mutation compromises ATPase stimulation by Sec63p, an Hsp40 required for translocation, but stimulation by Jem1p, an Hsp40 required for ERAD, is robust. In accordance with these data, yeast expressing R217A BiP exhibit translocation defects, but no ERAD defects, and a genetic interaction study using this mutant yielded data consistent with defects in translocation. In contrast, mutations in the substrate binding domain that either disrupt an ionic contact with the lid or remove this domain are deficient for peptide-stimulated ATPase activity. Expression of these mutants in yeast results in varying translocation and ERAD defects. Taken together, these data indicate that BiP can distinguish between its ER-resident cochaperones, and that optimal substrate binding is a key determinant of BiP function.Next, I tested the hypothesis that the functional specificity of Hsp70s is regulated by cognate Hsp40s. If this is true, one might expect divergent Hsp70-Hsp40 pairs to be unable to function in vivo. However, I discovered that a mammalian ER-lumenal Hsp40, ERdj3, when directed to the yeast cytosol, was able to rescue the temperature-sensitive growth phenotype of yeast containing mutant alleles in two cytosolic Hsp40s, HLJ1 and YDJ1. Moreover, ERdj3 activated the ATPase activity of Ssa1p, the yeast cytosolic Hsp70 that partners with Hlj1p and Ydj1p. Intriguingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect, even though they stimulated Ssa1p ATPase activity. These data suggest that the substrate binding properties of certain Hsp40s—not simply the formation of unique Hsp70-Hsp40 pairs—is critical to specify in vivo function

Portable Microfluidic Integrated Plasmonic Platform for Pathogen Detection

Timely detection of infectious agents is critical in early diagnosis and treatment of infectious diseases. Conventional pathogen detection methods, such as enzyme linked immunosorbent assay (ELISA), culturing or polymerase chain reaction (PCR) require long assay times, and complex and expensive instruments, which are not adaptable to point-of-care (POC) needs at resource-constrained as well as primary care settings. Therefore, there is an unmet need to develop simple, rapid, and accurate methods for detection of pathogens at the POC. Here, we present a portable, multiplex, inexpensive microfluidic-integrated surface plasmon resonance (SPR) platform that detects and quantifies bacteria, i.e., Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) rapidly. The platform presented reliable capture and detection of E. coli at concentrations ranging from ~105 to 3.2 × 107 CFUs/mL in phosphate buffered saline (PBS) and peritoneal dialysis (PD) fluid. The multiplexing and specificity capability of the platform was also tested with S. aureus samples. The presented platform technology could potentially be applicable to capture and detect other pathogens at the POC and primary care settings

Effect of therapeutic ultrasound on acoustically sensitive microcapsules

In the area of therapeutic ultrasound activated drug delivery, difficulties exist in designing a carrier that responds to ultrasound for triggering and imaging but also provides adequate treatment potential. In this paper, we report on a novel acoustically sensitive microcapsule reservoir that can be activated with therapeutic ultrasound for payload release and can be potentially tracked using imaging. It is being designed for increased longevity and is not planned for the circulation. Here, we describe its unique formulation and demonstrate effects of therapeutic ultrasound on it at 1MHz using a combined optical-acoustic setup on a microscope. We see membrane bulging and damage for small and large capsules with both continuous and pulsed ultrasound. We also show some preliminary work on understanding the mechanism behind these effects. The reservoirs show potential for future ultrasound activated release and imaging while being patent in form and function over several weeks

Structural changes and imaging signatures of acoustically sensitive microcapsules under ultrasound

The ultrasound drug delivery field is actively designing new agents that would obviate the problems of just using microbubbles for drug delivery. Microbubbles have very short circulation time (minutes), low payload and large size (2–10 μm), all of these aspects are not ideal for systemic drug delivery. However, microbubble carriers provide excellent image contrast and their use for image guidance can be exploited. In this paper, we suggest an alternative approach by developing acoustically sensitive microcapsule reservoirs that have future applications for treating large ischemic tumors through intratumoral therapy. We call these agents Acoustically Sensitized Microcapsules (ASMs) and these are not planned for the circulation. ASMs are very simple in their formulation, robust and reproducible. They have been designed to offer high payload (because of their large size), be acoustically sensitive and reactive (because of the Ultrasound Contrast Agents (UCAs) encapsulated) and mechanically robust for future injections/implantations within tumors. We describe three different aspects – (1) effect of therapeutic ultrasound; (2) mechanical properties and (3) imaging signatures of these agents. Under therapeutic ultrasound, the formation of a cavitational bubble was seen prior to rupture. The time to rupture was size dependent. Size dependency was also seen when measuring mechanical properties of these ASMs. % Alginate and permeability also affected the Young’s modulus estimates. For study of imaging signatures of these agents, we show six schemes. For example, with harmonic imaging, tissue phantoms and controls did not generate higher harmonic components. Only ASM phantoms created a harmonic signal, whose sensitivity increased with applied acoustic pressure. Future work includes developing schemes combining both sonication and imaging to help detect ASMs before, during and after release of drug substance

Interaction of the intrinsically disordered C-terminal domain of the sesbania mosaic virus RNA-dependent RNA polymerase with the viral protein P10 in vitro: modulation of the oligomeric state and polymerase activity

The RNA-dependent RNA polymerase (RdRp) of sesbania mosaic virus (SeMV) was previously shown to interact with the viral protein P10, which led to enhanced polymerase activity. In the present investigation, the equilibrium dissociation constant for the interaction between the two proteins was determined to be 0.09 mu M using surface plasmon resonance, and the disordered C-terminal domain of RdRp was shown to be essential for binding to P10. The association with P10 brought about a change in the oligomeric state of RdRp, resulting in reduced aggregation and increased polymerase activity. Interestingly, unlike the wild-type RdRp, C-terminal deletion mutants (C del 43 and C del 72) were found to exist predominantly as monomers and were as active as the RdRp-P10 complex. Thus, either the deletion of the C-terminal disordered domain or its masking by binding to P10 results in the activation of polymerase activity. Further, deletion of the C-terminal 85 residues of RdRp resulted in complete loss of activity. Mutation of a conserved tyrosine (RdRp Y480) within motif E, located between 72 and 85 residues from the C-terminus of RdRp, rendered the protein inactive, demonstrating the importance of motif E in RNA synthesis in vitro

Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics

A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure–dynamics–function relationships in the catalysis of the NADH-dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modelling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate-limiting step for turnover which is not the case in FucO catalysed alcohol oxidation. We further propose that a water molecule in the active site – hydrogen bonded to a conserved histidine (H267) and the 2′-hydroxyl of the coenzyme ribose – functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side-chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity

The Fe2+-dependent E. coli enzyme FucO catalyzes the reversible interconversion of short-chain (S)-lactaldehyde and (S)-1,2-propanediol, using NADH and NAD+ as cofactors, respectively. Laboratory-directed evolution experiments have been carried out previously using phenylacetaldehyde as the substrate for screening catalytic activity with bulky substrates, which are very poorly reduced by wild-type FucO. These experiments identified the N151G/L259V double mutant (dubbed DA1472) as the most active variant with this substrate via a two-step evolutionary pathway, in which each step consisted of one point mutation. Here the crystal structures of DA1472 and its parent D93 (L259V) are reported, showing that these amino acid substitutions provide more space in the active site, though they do not cause changes in the main-chain conformation. The catalytic activity of DA1472 with the physiological substrate (S)-lactaldehyde and a series of substituted phenylacetaldehyde derivatives were systematically quantified and compared with that of wild-type as well as with the corresponding point-mutation variants (N151G and L259V). There is a 9000-fold increase in activity, when expressed as kcat/KM values, for DA1472 compared with wild-type FucO for the phenylacetaldehyde substrate. The crystal structure of DA1472 complexed with a non-reactive analog of this substrate (3,4-dimethoxyphenylacetamide) suggests the mode of binding of the bulky group of the new substrate. These combined structure–function studies therefore explain the dramatic increase in catalytic activity of the DA1472 variant for bulky aldehyde substrates. The structure comparisons also suggest why the active site in which Fe2+ is replaced by Zn2+ is not able to support catalysis