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

Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinin, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress-responsive genes in a poised state and regulates their expression under stress conditions. Furthermore, we show that upon artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly associated with the genes implicated in artemisinin resistance generation like BiP and TRiC chaperone. Interestingly, expression of genes bound by PfGCN5 was found to be upregulated during stress conditions. Moreover, inhibition of PfGCN5 in artemisinin-resistant parasites increases the sensitivity of the parasites to artemisinin treatment indicating its role in drug resistance generation. Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of stress-responses with a potential role in modulating artemisinin drug resistance and identify PfGCN5 as an important target against artemisinin-resistant parasites.</p

Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinin, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress-responsive genes in a poised state and regulates their expression under stress conditions. Furthermore, we show that upon artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly associated with the genes implicated in artemisinin resistance generation like BiP and TRiC chaperone. Interestingly, expression of genes bound by PfGCN5 was found to be upregulated during stress conditions. Moreover, inhibition of PfGCN5 in artemisinin-resistant parasites increases the sensitivity of the parasites to artemisinin treatment indicating its role in drug resistance generation. Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of stress-responses with a potential role in modulating artemisinin drug resistance and identify PfGCN5 as an important target against artemisinin-resistant parasites.</p

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