Faculty Bibliography

Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants^1-3. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space^4-9. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline-proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.

Response to cancer immunotherapy in primary versus metastatic disease has not been well-studied. We found primary pancreatic ductal adenocarcinoma (PDA) is responsive to diverse immunotherapies whereas liver metastases are resistant. We discovered divergent immune landscapes in each compartment. Compared to primary tumor, liver metastases in both mice and humans are infiltrated by highly anergic T cells and MHCII^loIL10⁺ macrophages that are unable to present tumor-antigen. Moreover, a distinctive population of CD24⁺CD44^-CD40^- B cells dominate liver metastases. These B cells are recruited to the metastatic milieu by Muc1^hiIL18^hi tumor cells, which are enriched >10-fold in liver metastases. Recruited B cells drive macrophage-mediated adaptive immune-tolerance via CD200 and BTLA. Depleting B cells or targeting CD200/BTLA enhanced macrophage and T-cell immunogenicity and enabled immunotherapeutic efficacy of liver metastases. Our data detail the mechanistic underpinnings for compartment-specific immunotherapy-responsiveness and suggest that primary PDA models are poor surrogates for evaluating immunity in advanced disease.

Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.

Brugada syndrome is a heritable channelopathy characterized by a peculiar electrocardiogram (ECG) pattern and increased risk of cardiac arrhythmias and sudden death. The arrhythmias originate because of an imbalance between the repolarizing and depolarizing currents that modulate the cardiac action potential. Even if an overt structural cardiomyopathy is not typical of Brugada syndrome, fibrosis and structural changes in the right ventricle contribute to a conduction slowing, which ultimately facilitates ventricular arrhythmias. Currently, Mendelian autosomal dominant transmission is detected in less than 25% of all clinical confirmed cases. Although 23 genes have been associated with the condition, only SCN5A, encoding the cardiac sodium channel, is considered clinically actionable and disease causing. The limited monogenic inheritance has pointed toward new perspectives on the possible complex genetic architecture of the disease, involving polygenic inheritance and a polygenic risk score that can influence penetrance and risk stratification. Expected final online publication date for the Annual Review of Genomics and Human Genetics, Volume 23 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Genetic evidence has increasingly linked lysosome dysfunction to an impaired autophagy-lysosomal pathway (ALP) flux in Alzheimer disease (AD) although the relationship of these abnormalities to other pathologies is unclear. In our recent investigation on the origin of impaired autophagic flux in AD, we established the critical early role of defective lysosomes in 5 mouse AD models. To assess in vivo alterations of autophagy and ALP vesicle acidification, we expressed eGFP-mRFP-LC3 specifically in neurons. We discovered that autophagy dysfunction in these models arises from exceptionally early failure of autolysosome/lysosome acidification, which then drives downstream AD pathogenesis. Extreme autophagic stress in compromised but still intact neurons causes AVs containing toxic APP metabolites, Aβ/β-CTFs, to pack into huge blebs and protrude from the perikaryon membrane. Most notably, AVs also coalesce with ER tubules and yield fibrillar β-amyloid within these tubules. Collectively, amyloid immunoreactivity within these intact neurons assumes the appearance of amyloid-plaques and, indeed, their eventual death transforms them into extracellular plaque lesions. Quantitative analysis confirms that neurons undergoing this transformation are the principal source of β-amyloid-plaques in APP-AD models. These findings prompt reconsideration of the conventionally accepted sequence of events in plaque formation and may help explain the inefficacy of Aβ/amyloid vaccine therapies.

The cell envelope of Gram-negative bacteria is composed of an inner membrane, outer membane, and an intervening periplasmic space. How the outer membrane lipids are trafficked and assembled there, and how the asymmetry of the outer membrane is maintained is an area of intense research. The Mla system has been implicated in the maintenance of lipid asymmetry in the outer membrane, and is generally thought to drive the removal of mislocalized phospholipids from the outer membrane and their retrograde transport to the inner membrane. At the heart of the Mla pathway is a structurally unique ABC transporter complex in the inner membrane, called MlaFEDB. Recently, an explosion of cryo-EM studies has begun to shed light on the structure and lipid translocation mechanism of MlaFEDB, with many parallels to other ABC transporter families, including human ABCA and ABCG, as well as bacterial lipopolysaccharide and O-antigen transporters. Here we synthesize information from all available structures, and propose a model for lipid trafficking across the cell envelope by MlaFEDB.

BACKGROUND:gene, which encodes the PKP2 protein (plakophilin-2). METHODS:studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: CONCLUSIONS:

Human gut commensals are increasingly suggested to impact non-communicable diseases, such as inflammatory bowel diseases (IBD), yet their targeted suppression remains a daunting unmet challenge. In four geographically distinct IBD cohorts (n = 537), we identify a clade of Klebsiella pneumoniae (Kp) strains, featuring a unique antibiotics resistance and mobilome signature, to be strongly associated with disease exacerbation and severity. Transfer of clinical IBD-associated Kp strains into colitis-prone, germ-free, and colonized mice enhances intestinal inflammation. Stepwise generation of a lytic five-phage combination, targeting sensitive and resistant IBD-associated Kp clade members through distinct mechanisms, enables effective Kp suppression in colitis-prone mice, driving an attenuated inflammation and disease severity. Proof-of-concept assessment of Kp-targeting phages in an artificial human gut and in healthy volunteers demonstrates gastric acid-dependent phage resilience, safety, and viability in the lower gut. Collectively, we demonstrate the feasibility of orally administered combination phage therapy in avoiding resistance, while effectively inhibiting non-communicable disease-contributing pathobionts.