Normal-Tension Glaucoma Complicated by a Giant Internal Carotid-Ophthalmic Artery Aneurysm.

Purpose. We describe a patient with normal tension glaucoma (NTG) of several years whose management was complicated by the presence of a giant internal carotid-ophthalmic artery aneurysm. Observations. A 72-year-old woman presented to our glaucoma clinic with accelerated deterioration of her vision in her left eye (OS) over a 1-month period. Her ophthalmic history was most notable for bilateral NTG diagnosed 3 years prior which had been treated with several laser trabeculoplasty OS and topical bimatoprost 0.01% eye drops in both eyes (OU). Upon evaluation, her visual acuity OS had worsened, and visual field (VF) testing showed extensive progressive losses temporally and pericentrally OS over a year with stable IOP measurements and no neurological complaints. Given her atypical NTG progression, she was referred for an urgent neurological evaluation which revealed an unruptured giant left internal carotid-ophthalmic aneurysm. Following the successful treatment of the aneurysm with platinum coils, she continued to demonstrate additional bilateral ophthalmic changes including further progression of VF loss and RNFL thinning OS > OD on follow-up. Conclusion and Importance. Overall, this report describes a unique complication in the management of a patient with chronic bilateral NTG in the form of a giant internal carotid-ophthalmic aneurysm. Moreover, it highlights the need for clinicians to maintain a degree of suspicion for compressive lesions of the optic nerve when presented with atypical progression of VFs and/or visual acuity loss in glaucomatous patients.

Photoswitchable Isoprenoid Lipids Enable Optical Control of Peptide Lipidation.

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.

Temperature-Responsive Nano-Biomaterials from Genetically Encoded Farnesylated Disordered Proteins.

Despite broad interest in understanding the biological implications of protein farnesylation in regulating different facets of cell biology, the use of this post-translational modification to develop protein-based materials and therapies remains underexplored. The progress has been slow due to the lack of accessible methodologies to generate farnesylated proteins with broad physicochemical diversities rapidly. This limitation, in turn, has hindered the empirical elucidation of farnesylated proteins' sequence-structure-function rules. To address this gap, we genetically engineered prokaryotes to develop operationally simple, high-yield biosynthetic routes to produce farnesylated proteins and revealed determinants of their emergent material properties (nano-aggregation and phase-behavior) using scattering, calorimetry, and microscopy. These outcomes foster the development of farnesylated proteins as recombinant therapeutics or biomaterials with molecularly programmable assembly.

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences.

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid "Cxx" C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.

Engineering reversible cell-cell interactions using enzymatically lipidated chemically self-assembled nanorings.

Multicellular biology is dependent on the control of cell-cell interactions. These concepts have begun to be exploited for engineering of cell-based therapies. Herein, we detail the use of a multivalent lipidated scaffold for the rapid and reversible manipulation of cell-cell interactions. Chemically self-assembled nanorings (CSANs) are formed via the oligomerization of bivalent dihydrofolate reductase (DHFR2) fusion proteins using a chemical dimerizer, bis-methotrexate. With targeting proteins fused onto the DHFR2 monomers, the CSANs can target specific cellular antigens. Here, anti-EGFR or anti-EpCAM fibronectin-DHFR2 monomers incorporating a CAAX-box sequence were enzymatically prenylated, then assembled into the corresponding CSANs. Both farnesylated and geranylgeranylated CSANs efficiently modified the cell surface of lymphocytes and remained bound to the cell surface with a half-life of >3 days. Co-localization studies revealed a preference for the prenylated nanorings to associate with lipid rafts. The presence of antigen targeting elements in these bifunctional constructs enabled them to specifically interact with target cells while treatment with trimethoprim resulted in rapid CSAN disassembly and termination of the cell-cell interactions. Hence, we were able to determine that activated PBMCs modified with the prenylated CSANs caused irreversible selective cytotoxicity toward EGFR-expressing cells within 2 hours without direct engagement of CD3. The ability to disassemble these nanostructures in a temporally controlled manner provides a unique platform for studying cell-cell interactions and T cell-mediated cytotoxicity. Overall, antigen-targeted prenylated CSANs provide a general approach for the regulation of specific cell-cell interactions and will be valuable for a plethora of fundamental and therapeutic applications.