Faculty Bibliography

Seminal fluid (SF) RNA is essential for embryonic development, but the mechanism by which extracellular RNA is transferred from SF to sperm remains unclear. Here, we identify a role for functional amyloid fibrils in SF that bind and traffic RNA to mammalian sperm and, upon fertilization, to the zygote. We show that highly cationic peptides in SF phase separate with RNA to form amyloid fibrils. Cryo-electron tomography (cryo-ET), volume electron microscopy (volume EM), and fluorescence live-cell imaging reveal that these amyloid fibrils are internalized by mammalian sperm. Furthermore, biophysical and in vitro fertilization (IVF) experiments show that amyloid-bound RNA reaches the zygote, where molecular chaperones can control RNA dissociation from amyloid fibrils. Our work uncovers a mechanism for RNA trafficking from SF to the zygote, establishing amyloid fibrils as potential tools for RNA delivery during fertilization.

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope-labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter, we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover.

Arrhythmogenic cardiomyopathy is a heritable cardiac disorder commonly linked to alterations in desmosomal proteins. While desmosomal dysfunction is a recognized pathogenic mechanism, the early molecular remodeling that precedes clinical manifestations remains unclear. Here we used a mouse model with inducible cardiomyocyte-specific knockout of Pkp2 (encoding plakophilin-2) and performed time-resolved mass spectrometry-based proteomics to map disease progression from early PKP2 loss to structural remodeling. Molecular changes preceded phenotypic abnormalities, and spatial asymmetry was evident, with earlier and more pronounced proteome remodeling in the right ventricle despite comparable PKP2 depletion in both ventricles. Downregulation of protein networks spanning desmosomes, sarcomeres and mitochondria suggests their functional coupling, with mitochondrial remodeling emerging as an early and progressively worsening feature. Intervention with honokiol attenuated changes across the major protein networks, highlighting mitochondrial pathways as promising therapeutic targets and honokiol as a potential strategy to delay arrhythmogenic cardiomyopathy progression. Our findings provide critical insights into the concealed stage of disease progression and identify molecular targets for early intervention.

Primary cilia are signaling hubs that influence nearly every aspect of cell physiology. Neuronal primary cilia were recently found to form extensive attachments to glia in the mammalian brain, but it is not known how these attachments form or what functions they serve. Defects in cilia cause profound neurological disorders, highlighting the importance of understanding the specialized functions of neuronal cilia. Here, we use a genetic screen in Caenorhabditis elegans to determine how a stereotyped cilia-glia attachment forms and how the attachment affects ciliary calcium signaling. We find that two sensory neurons (URX and BAG) use their cilia to attach to a specific glial partner (ILsoL/R). This attachment requires BUG-1, a large secreted protein that localizes precisely to the cilia-glia attachment site. In the absence of BUG-1, neuronal cilia are present but always fail to attach to the glial cell. As a result, the URX cilia do not undergo activity-dependent remodeling seen in wild-type neurons and the BAG cilia exhibit reduced sensory stimulus-evoked calcium entry and defects in calcium clearance. Our work shows that cilia can select specific glial partners for attachment, identifies a novel protein that mediates these attachments, and demonstrates the functional consequences of loss of cilia-glia attachment on neuronal signaling. We propose that neuronal primary cilia act not only as antennae to receive long-range signals but also as points of close-range cell-cell contact, through which glia could modulate neuronal signaling.

The glomerulus, a unique capillary network in the nephron, filters an entire blood volume approximately 300 times a day. Specialized epithelial cells known as podocytes form a critical component of the glomerular filtration barrier, and diseases linked to podocyte injury include minimal change disease, focal segmental glomerulosclerosis, and diabetic kidney disease. Because podocytes are terminally differentiated, their ability to respond to external stress is critical. One class of proteins, molecular chaperones, protects against cell stress by preventing protein misfolding, assisting in protein refolding, maintaining protein solubility, and modulating protein translation. We previously found that depletion of one such molecular chaperone, GRP170, in the renal tubule induces the unfolded stress response (UPR), a stress-responsive pathway originating in the endoplasmic reticulum (ER), and causes apoptosis and acute kidney injury in mice. Here we report on a new constitutive podocyte-specific GRP170 knockout mouse (GRP170^Pd-/-). Surprisingly, GRP170^Pd-/- mice were born healthy. Within a month, however, these mice exhibited profound glomerular injury manifesting as proteinuria, hypoalbuminemia, hyperlipidemia, and kidney injury. Concomitant with glomerular injury, we observed increased expression of the pro-apoptotic UPR target, CHOP, and altered ER morphology in podocytes. Our new model not only defines a protective role for GRP170 against glomerular injury but provides a new avenue to test the therapeutic potential of small molecule UPR modulators to treat glomerular diseases.

The extent to which conserved neural circuit architectures depend on shared molecular specification programs remains unclear. Here, we address this question by examining the somatosensory system of the little skate, Leucoraja erinacea, an early-diverging vertebrate that retains ancestral features of both finned and limb-based body plans. We show that core features of somatosensory circuit organization, including laminar organization of the spinal cord and dorsally restricted targeting of sensory afferents, are deeply conserved. Unexpectedly, the molecular programs specifying dorsal root ganglion (DRG) sensory subtypes diverge extensively from those of mammals. Although DRG neuron subtype specification and spinal connectivity rely on target-derived cues, skates employ distinct neurotrophin receptor and transcription factor identity codes. These findings support a model in which conserved spinal circuit architectures provide a stable scaffold that leverages flexible sensory neuron specification programs, enabling the evolutionary diversification of vertebrate somatosensory systems.

Atsttrin, a therapeutic derivative of progranulin, promotes tissue repair via TNFR2-mediated anti-inflammatory and regenerative signaling. Our group developed an injectable, thermoresponsive graphene oxide-polyethylene glycol/collagen type I hydrogel for sustained and localized delivery of Atsttrin and systematically evaluated its therapeutic efficacy in vitro and in vivo models. The hydrogel demonstrated favorable rheological properties, controlled degradation, high porosity, and sustained Atsttrin release over seven weeks. In vitro cytocompatibility assays confirmed its non-cytotoxic nature, which was further corroborated by long-term in vivo safety assessments indicating the absence of organ toxicity up to three months post-administration. Functional studies using wild-type and 14-3-3ε knockout bone marrow-derived mesenchymal stem cells revealed that Atsttrin significantly enhanced chondrogenic and osteogenic differentiation in WT cells, effects that were markedly reduced in KO cells. Hydrogel-encapsulated Atsttrin elicited more robust regenerative responses compared to administration of free Atsttrin, underscoring the critical role of controlled release in enhancing therapeutic efficacy. In vivo, Atsttrin-loaded hydrogels significantly accelerated fracture healing in both WT and diabetic mice, with diminished efficacy in 14-3-3ε-deficient models. Collectively, these results support an important role for 14-3-3ε in mediating the regenerative effects of Atsttrin and demonstrate the potential of the GO-PEG/Collagen I hydrogel as a localized delivery platform for bone repair.

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, a highly interconnected system of over 3000 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental functional unit in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, hindering its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. In the other manuscripts in this series, we survey the components of the proteostasis network that make up (1) the machineries for protein synthesis, protein folding, protein trafficking into and out of organelles, and organelle-specific degradation pathways and (2) the ubiquitin-proteasome system. Here, we have compiled a curated list of over 850 unique high-confidence components of the autophagy-lysosome pathway, the other major protein degradation system in human cells. This resource will facilitate the rigorous investigation of autophagy-lysosome pathway dysfunction as a driver of proteostasis imbalance in aging and disease.

Cyclin D-CDK4/6 complexes drive cell-cycle entry through an RB-E2F-dependent transcriptional program, but how they coordinate proliferation with the membrane and organelle protein supply required for growth is unclear. We identify DeSI1 as a cyclin D-CDK4/6 substrate whose phosphorylation at S25 converts a latent homodimer into an active monomeric deubiquitylase that recognizes hydrophobic proteins such as those bearing transmembrane domains and mitochondrial targeting sequences. Phosphorylated, active DeSI1 extends the lifetime of newly synthesized hydrophobic proteins that support membrane and mitochondrial capacity. Constitutive DeSI1 activation uncouples this proteostatic program from metabolic supply, creating a cytidine-nucleotide supply-demand imbalance associated with impaired CTP-dependent phospholipid homeostasis, cardiolipin depletion, and mitochondrial decompensation -defects that cytidine reverses. In mice, constitutive DeSI1 activation causes progressive cerebellar degeneration with membrane-protein accumulation, respiratory-chain loss, and phospholipid depletion. These findings define a post-translational mechanism coupling cell-cycle entry to membrane and mitochondrial capacity, and reveal the cost of uncoupling this program from its metabolic support.

Tafazzin is a phospholipid-lysophospholipid acyltransferase that remodels fatty acids of mitochondrial phospholipids. To study the tafazzin mechanism, we expressed and purified the enzyme, measured its kinetics with different pairs of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) species, and simulated the reactions in a mathematical model based on the mass-action law. Variations in chain length and PC:LPC ratio showed that tafazzin reacts with micelles but not with bilayers. In the presence of micellar substrates (LPC + short-chain PC), transacylation rates measured at different substrate ratios suggested similar affinities for PC and LPC. Surprisingly, tafazzin catalyzed both transacylation and hydrolysis of fatty acids, the former preferring oleoyl over palmitoyl and the latter preferring palmitoyl over oleoyl groups. To study the remodeling of cardiolipin, we measured the reaction of trilinoleoyl-monolyso-cardiolipin with palmitoyl-oleoyl-PC and simulated it in a mathematical model. Simulations predicted and measurements confirmed a large number of bidirectional transacylations between the initial substrates, between substrates and products, and between different products. This led to a gradual diversification of the lipid composition from 2 to more than 15 species. Discrepancies between observed and simulated compositions demonstrated transacylation preference for oleoyl over palmitoyl groups. The data are consistent with a bidirectional ping-pong mechanism that operates through a single low-affinity binding site and suggest that transacylation specificity can at least in part be explained by selective loss of fatty acids through hydrolysis.