Faculty Bibliography

Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.

Mitochondrial DNA double-strand breaks (mtDSBs) lead to the degradation of circular genomes and a reduction in copy number; yet, the cellular response in human cells remains elusive. Here, using mitochondrial-targeted restriction enzymes, we show that a subset of cells with mtDSBs exhibited defective mitochondrial protein import, reduced respiratory complexes, and loss of membrane potential. Electron microscopy confirmed the altered mitochondrial membrane and cristae ultrastructure. Intriguingly, mtDSBs triggered the integrated stress response (ISR) via the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by DELE1 and heme-regulated eIF2α kinase (HRI). When ISR was inhibited, the cells experienced intensified mitochondrial defects and slower mtDNA recovery post-breakage. Lastly, through proteomics, we identified ATAD3A-a membrane-bound protein interacting with nucleoids-as potentially pivotal in relaying signals from impaired genomes to the inner mitochondrial membrane. In summary, our study delineates the cascade connecting damaged mitochondrial genomes to the cytoplasm and highlights the significance of the ISR in maintaining mitochondrial homeostasis amid genome instability.

Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr¹³¹, allowing its interaction with V₁ subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.

BACKGROUND:Human infants develop IgG responses to dietary antigens during the first 2 years of life. Yet, the source of these antibodies is unclear. In previous studies we reported on the thymus as a unique functional niche for plasma cells (PCs) specific to environmental antigens. OBJECTIVE:We sought to examine whether PCs specific to dietary antigens are detected in the infant thymus. METHODS:We tested IgG reactivity to 112 food antigens and allergens in the serum of 20 neonates and infants using microarrays. The presence of PC-secreting IgG specific to the most prominent antigens was then assessed among thymocytes in the same cohort. Using an LC-MS proteomics approach, we looked for traces of these antigens in the thymus. RESULTS:Our studies first confirmed that cow's milk proteins are prevalent targets of serum IgG in early life. Subjects with the highest serum IgG titers to cow's milk proteins also harbored IgG-producing PCs specific to the same antigens in the thymic niche. Furthermore, we detected multiple peptide fragments of cow's milk antigens in the thymus. Lastly, we verified that both serum IgG and IgG secreted by thymic PCs recognized the peptide epitopes found in the thymus. CONCLUSIONS:Our studies reveal the presence of antibody-secreting PCs specific to common dietary antigens in the infant thymus. The presence of these antigens in the thymus suggested that activation and differentiation of specific PCs occurred in this organ. Further studies are now warranted to evaluate the possible implication of these cells in tolerance to dietary antigens.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host^1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis^1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids^4-7 and cholesterol^1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

Proteomic studies of human Alzheimer's disease brain tissue have potential to identify protein changes that drive disease, and to identify new drug targets. Here, we analyse 38 published Alzheimer's disease proteomic studies, generating a map of protein changes in human brain tissue across thirteen brain regions, three disease stages (preclinical Alzheimer's disease, mild cognitive impairment, advanced Alzheimer's disease), and proteins enriched in amyloid plaques, neurofibrillary tangles, and cerebral amyloid angiopathy. Our dataset is compiled into a searchable database (NeuroPro). We found 848 proteins were consistently altered in 5 or more studies. Comparison of protein changes in early-stage and advanced Alzheimer's disease revealed proteins associated with synapse, vesicle, and lysosomal pathways show change early in disease, but widespread changes in mitochondrial associated protein expression change are only seen in advanced Alzheimer's disease. Protein changes were similar for brain regions considered vulnerable and regions considered resistant. This resource provides insight into Alzheimer's disease brain protein changes and highlights proteins of interest for further study.

OBJECTIVE:High-fat and low-carbohydrate diets can reduce seizure frequency in some treatment-resistant epilepsy patients, including the more flexible modified Atkins diet (MAD), which is more palatable, mimicking fasting and inducing high ketone body levels. Low-carbohydrate diets may shift brain energy production, particularly impacting neuron- and astrocyte-linked metabolism. METHODS:We evaluated the effect of short-term MAD on molecular mechanisms in adult epilepsy patients from surgical brain tissue and plasma compared to control participants consuming a nonmodified higher carbohydrate diet (n = 6 MAD, mean age = 43.7 years, range = 21-53, diet for average 10 days; n = 10 control, mean age = 41.9 years, range = 28-64). RESULTS: = .48). Brain proteomics and RNAseq identified few differences, including 2.75-fold increased hippocampal MT-ND3 and trends (p < .01, false discovery rate > 5%) in hippocampal nicotinamide adenine dinucleotide (NADH)-related signaling pathways (activated oxidative phosphorylation and inhibited sirtuin signaling). SIGNIFICANCE/CONCLUSIONS:Short-term MAD was associated with metabolic differences in plasma and resected epilepsy brain tissue when compared to control participants, in combination with trending expression changes observed in hippocampal NADH-related signaling pathways. Future studies should evaluate how brain molecular mechanisms are altered with long-term MAD in a larger cohort of epilepsy patients, with correlations to seizure frequency, epilepsy syndrome, and other clinical variables. [Clinicaltrials.gov NCT02565966.].

OBJECTIVE:Identify molecular mechanisms in brain tissue of Rasmussen encephalitis (RE) when compared to people with non-RE epilepsy (PWE) and control cases using whole exome sequencing (WES), RNAseq, and proteomics. METHODS:Frozen brain tissue (ages 2-19 years) was obtained from control autopsy (n=14), surgical PWE (n=10), and surgical RE cases (n=27). We evaluated WES variants in RE associated with epilepsy, seizures, RE, and human leukocyte antigens (HLAs). Differential expression was evaluated by RNAseq (adjusted p<0.05) and label-free quantitative mass spectrometry (false discovery rate<5%) in the three groups. RESULTS:, z=5.61). Proteomics detected fewer altered targets. SIGNIFICANCE/CONCLUSIONS:In RE, we identified activated immune signaling pathways and immune cell type annotation enrichment that suggest roles of the innate and adaptive immune responses, as well as HLA variants that may increase vulnerability to RE. Follow up studies could evaluate cell type density and subregional localization associated with top targets, clinical history (neuropathology, disease duration), and whether modulating crosstalk between dendritic and natural killer cells may limit disease progression.

INTRODUCTION/UNASSIGNED:Alzheimer's disease (AD) and epilepsy are reciprocally related. Among sporadic AD patients, clinical seizures occur in 10-22% and subclinical epileptiform abnormalities occur in 22-54%. Cognitive deficits, especially short-term memory impairments, occur in most epilepsy patients. Common neurophysiological and molecular mechanisms occur in AD and epilepsy. The choroid plexus undergoes pathological changes in aging, AD, and epilepsy, including decreased CSF turnover, amyloid beta (Aβ), and tau accumulation due to impaired clearance and disrupted CSF amino acid homeostasis. This pathology may contribute to synaptic dysfunction in AD and epilepsy. METHODS/UNASSIGNED:= 12) using laser capture microdissection (LCM) followed by label-free quantitative mass spectrometry on the choroid plexus adjacent to the hippocampus at the lateral geniculate nucleus level. RESULTS/UNASSIGNED: DISCUSSION/UNASSIGNED:We found altered signaling pathways in the choroid plexus of severe AD cases and many correlated changes in the protein expression of cell metabolism pathways in AD and epilepsy cases. The shared molecular mechanisms should be investigated further to distinguish primary pathogenic changes from the secondary ones. These mechanisms could inform novel therapeutic strategies to prevent disease progression or restore normal function. A focus on dual-diagnosed AD/epilepsy cases, specific epilepsy syndromes, such as temporal lobe epilepsy, and changes across different severity levels in AD and epilepsy would add to our understanding.