Aida Razi

Doxorubicin encapsulated in stealth liposomes conferred with light-triggered drug release

Stealth liposomes can be used to extend the blood circulation time of encapsulated therapeutics. Inclusion of 2 molar % porphyrin-phospholipid (PoP) imparted optimal near infrared (NIR) light-triggered release of doxorubicin (Dox) from conventional sterically stabilized stealth liposomes. The type and amount of PoP affected drug loading, serum stability and drug release induced by NIR light. Cholesterol and PEGylation were required for Dox loading, but slowed light-triggered release. Dox in stealth PoP liposomes had a long circulation half-life in mice of 21.9 h and was stable in storage for months. Following intravenous injection and NIR irradiation, Dox deposition increased ∼ 7 fold in treated subcutaneous human pancreatic xenografts. Phototreatment induced mild tumor heating and complex tumor hemodynamics. A single chemophototherapy treatment with Dox-loaded stealth PoP liposomes (at 5-7 mg/kg Dox) eradicated tumors while corresponding chemo- or photodynamic therapies were ineffective. A low dose 3 mg/kg Dox phototreatment with stealth PoP liposomes was more effective than a maximum tolerated dose of free (7 mg/kg) or conventional long-circulating liposomal Dox (21 mg/kg). To our knowledge, Dox-loaded stealth PoP liposomes represent the first reported long-circulating nanoparticle capable of light-triggered drug release.

Lack of Adipocyte AMPK Exacerbates Insulin Resistance and Hepatic Steatosis through Brown and Beige Adipose Tissue Function

Brown (BAT) and white (WAT) adipose tissues play distinct roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a cellular energy sensor, but its role in regulating BAT and WAT metabolism is unclear. We generated an inducible model for deletion of the two AMPK β subunits in adipocytes (iβ1β2AKO) and found that iβ1β2AKO mice were cold intolerant and resistant to β-adrenergic activation of BAT and beiging of WAT. BAT from iβ1β2AKO mice had impairments in mitochondrial structure, function, and markers of mitophagy. In response to a high-fat diet, iβ1β2AKO mice more rapidly developed liver steatosis as well as glucose and insulin intolerance. Thus, AMPK in adipocytes is vital for maintaining mitochondrial integrity, responding to pharmacological agents and thermal stress, and protecting against nutrient-overload-induced NAFLD and insulin resistance.

Porphyrin-phospholipid liposomes with tunable leakiness

Drug bioavailability is a key consideration for drug delivery systems. When loaded with doxorubicin, liposomes containing 5 molar % porphyrin-phospholipid (HPPH liposomes) exhibited in vitro and in vivo serum stability that could be fine-tuned by varying the drug-to-lipid ratio. A higher drug loading ratio destabilized the liposomes, in contrast to standard liposomes which displayed an opposite and less pronounced trend. Following systemic administration of HPPH liposomes, near infrared laser irradiation induced vascular photodynamic damage, resulting in enhanced liposomal doxorubicin accumulation in tumors. In laser-irradiated tumors, the use of leaky HPPH liposomes resulted in improved doxorubicin bioavailability compared to stable standard liposomes. Using this approach, a single photo-treatment with 10mg/kg doxorubicin rapidly eradicated tumors in athymic nude mice bearing KB or MIA Paca-2 xenografts.

YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit

YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45SYphC and 44.5SYsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45SYphC and 44.5SYsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.

The C-terminal helix in the YjeQ zinc-finger domain catalyzes the release of RbfA during 30S ribosome subunit assembly.

YjeQ (also called RsgA) and RbfA proteins in Escherichia coli bind to immature 30S ribosome subunits at late stages of assembly to assist folding of the decoding center. A key step for the subunit to enter the pool of actively translating ribosomes is the release of these factors. YjeQ promotes dissociation of RbfA during the final stages of maturation; however, the mechanism implementing this functional interplay has not been elucidated. YjeQ features an amino-terminal oligonucleotide/oligosaccharide binding domain, a central GTPase module and a carboxy-terminal zinc-finger domain. We found that the zinc-finger domain is comprised of two functional motifs: the region coordinating the zinc ion and a carboxy-terminal α-helix. The first motif is essential for the anchoring of YjeQ to the 30S subunit and the carboxy-terminal α-helix facilitates the removal of RbfA once the 30S subunit reaches the mature state. Furthermore, the ability of the mature 30S subunit to stimulate YjeQ GTPase activity also depends on the carboxy-terminal α-helix. Our data are consistent with a model in which YjeQ uses this carboxy-terminal α-helix as a sensor to gauge the conformation of helix 44, an essential motif of the decoding center. According to this model, the mature conformation of helix 44 is sensed by the carboxy-terminal α-helix, which in turn stimulates the YjeQ GTPase activity. Hydrolysis of GTP is believed to assist the release of YjeQ from the mature 30S subunit through a still uncharacterized mechanism. These results identify the structural determinants in YjeQ that implement the functional interplay with RbfA.

The impact of recent improvements in cryo-electron microscopy technology on the understanding of bacterial ribosome assembly.

Abstract Cryo-electron microscopy (cryo-EM) had played a central role in the study of ribosome structure and the process of translation in bacteria since the development of this technique in the mid 1980s. Until recently cryo-EM structures were limited to ∼10 Å in the best cases. However, the recent advent of direct electron detectors has greatly improved the resolution of cryo-EM structures to the point where atomic resolution is now achievable. This improved resolution will allow cryo-EM to make groundbreaking contributions in essential aspects of ribosome biology, including the assembly process. In this review, we summarize important insights that cryo-EM, in combination with chemical and genetic approaches, has already brought to our current understanding of the ribosomal assembly process in bacteria using previous detector technology. More importantly, we discuss how the higher resolution structures now attainable with direct electron detectors can be leveraged to propose precise testable models regarding this process. These structures will provide an effective platform to develop new antibiotics that target this fundamental cellular process.

Design of hydrated porphyrin-phospholipid bilayers with enhanced magnetic resonance contrast

Computer simulations are used to design more hydrated bilayers, formed from amine-modified porphyrin-phospholipids (PoPs). Experiments confirm that the new constructs give rise to bilayers with greater water content. When chelated with manganese, amine-modified PoPs provide improved contrast for magnetic resonance and are safely used for imaging in vivo.

The cryo-EM structure of YjeQ bound to the 30S subunit suggests a fidelity checkpoint function for this protein in ribosome assembly

Significance Ribosome assembly in Escherichia coli is an extremely efficient process owing to the existence of assembly factors. Recent work indicates that some of these factors aid in the folding of the decoding center. The cryo-EM structure presented here includes an assembly factor testing the decoding fidelity of the mature 30S subunit before the particle is released to the pool of actively translating ribosomes. This finding reveals that in addition to their role as an assembly factor, these factors also may have a checkpoint function in the context of the mature ribosomal subunit. Understanding their specific functions may help identify key steps of the ribosome assembly pathway that will serve as molecular targets for new antibiotics. Recent work suggests that bacterial YjeQ (RsgA) participates in the late stages of assembly of the 30S subunit and aids the assembly of the decoding center but also binds the mature 30S subunit with high affinity. To determine the function and mechanisms of YjeQ in the context of the mature subunit, we determined the cryo-EM structure of the fully assembled 30S subunit in complex with YjeQ at 5.8-Å resolution. We found that binding of YjeQ stabilizes helix 44 into a conformation similar to that adopted by the subunit during proofreading. This finding indicates that, along with acting as an assembly factor, YjeQ has a role as a checkpoint protein, consisting of testing the proofreading ability of the 30S subunit. The structure also informs the mechanism by which YjeQ implements the release from the 30S subunit of a second assembly factor, called RbfA. Finally, it reveals how the 30S subunit stimulates YjeQ GTPase activity and leads to release of the protein. Checkpoint functions have been described for eukaryotic ribosome assembly factors; however, this work describes an example of a bacterial assembly factor that tests a specific translation mechanism of the 30S subunit.

Final touches and quality control on the assembly of the eukaryotic ribosome

One of the most fundamental processes of life is protein synthesis by the ribosome. Although much is known about the function and structure of this macromolecular complex, our understanding on its assembly is still vague. In this issue of The EMBO Journal, Malyutin et al ( ) provide a detailed picture of one of the latest assembly stages of the yeast 60S ribosomal subunit. The cryo‐EM map of the 60S‐Nmd3‐Lsg1‐Tif6 complex sheds new light on the function of Nmd3, Lsg1 and Tif6—and their release mechanisms—right before the 60S subunit joins the pool of actively translating ribosomes.

Capturing Near Atomic Resolution Snapshots of the Ribosome Assembly Process Using Direct Electron Detectors

Our research aims to understand the structure and function of new antimicrobial targets, developing them into tangible enzymes for intervention with new antibiotics. Current work focuses on the assembly process of the ribosome; a pathway of tremendous potential but that has not been explored as a target for antimicrobials yet. In recent years, or group and others found that the final steps in the ribosome assembly process are dedicated to the maturation of the functional sites [1-3]. Several protein factors bind at or near these sites ensuring they fold into their functional conformation. However, their specific functions or how these assembly factors assist maturation are still unknown [4]. Introduction of electron detectors in cryo-EM two years ago has revolutionized structural biology [5]. We are using these detectors to obtain structures of assembly intermediates alone and in complex with assembly factors to atomic resolution [6, 7]. These structures allow for amino acid level description of the molecular mechanism and the conformational changes that the assembly intermediates and ribosomal particles undergo upon interaction. This is critical information to fully understand the function of these assembly factors in the process of assembly of the ribosome and thus, for the development of new antibiotics targeting them. Overall, this work is impacting our ability to treat infectious diseases.