Chromosomal 3q amplicon encodes essential regulators of secretory vesicles that drive secretory addiction in cancer.

Cancer cells exhibit heightened secretory states that drive tumor progression. Here, we identify a chromosome 3q amplicon that serves as a platform for secretory regulation in cancer. The 3q amplicon encodes multiple Golgi-resident proteins, including the scaffold Golgi integral membrane protein 4 (GOLIM4) and the ion channel ATPase Secretory Pathway Ca2+ Transporting 1 (ATP2C1). We show that GOLIM4 recruits ATP2C1 and Golgi phosphoprotein 3 (GOLPH3) to coordinate calcium-dependent cargo loading and Golgi membrane bending and vesicle scission. GOLIM4 depletion disrupts the protein complex, resulting in a secretory blockade that inhibits the progression of 3q-amplified malignancies. In addition to its role as a scaffold, GOLIM4 maintains intracellular manganese (Mn) homeostasis by binding excess Mn in the Golgi lumen, which initiates the routing of Mn-bound GOLIM4 to lysosomes for degradation. We show that Mn treatment inhibits the progression of multiple types of 3q-amplified malignancies by degrading GOLIM4, resulting in a secretory blockade that interrupts pro-survival autocrine loops and attenuates pro-metastatic processes in the tumor microenvironment. Potentially underlying the selective activity of Mn against 3q-amplified malignancies, ATP2C1 co-amplification increases Mn influx into the Golgi lumen, resulting in a more rapid degradation of GOLIM4. These findings show that functional cooperativity between co-amplified genes underlies heightened secretion and a targetable secretory addiction in 3q-amplified malignancies.

Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Metabolomic and Lipidomic Signature of Skeletal Muscle with Constitutively Active Mechanistic Target of Rapamycin Complex 1.

BACKGROUND: Regulation of mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in aging and nutrition. For example, caloric restriction reduces mTORC1 signaling and extends lifespan, whereas nutrient abundance and obesity increase mTORC1 signaling and reduce lifespan. Skeletal muscle-specific knockout (KO) of DEP domain-containing 5 protein (DEPDC5) results in constitutively active mTORC1 signaling, muscle hypertrophy and an increase in mitochondrial respiratory capacity. The metabolic profile of skeletal muscle, in the setting of hyperactive mTORC1 signaling, is not well known. OBJECTIVES: To determine the metabolomic and lipidomic signature in skeletal muscle from female and male wild-type (WT) and DEPDC5 KO mice. METHODS: Tibialis anterior (TA) muscles from WT and transgenic (conditional skeletal muscle-specific DEPDC5 KO) were obtained from female and male adult mice. Polar metabolites and lipids were extracted using a Bligh-Dyer extraction from 5 samples per group and identified and quantified by LC-MS/MS. Resulting analyte peak areas were analyzed with t-test, analysis of variance, and Volcano plots for group comparisons (e.g., WT compared with KO) and multivariate statistical analysis for genotype and sex comparisons. RESULTS: A total of 162 polar metabolites (organic acids, amino acids, and amines and acyl carnitines) and 1141 lipid metabolites were detected in TA samples by LC-MS/MS. Few polar metabolites showed significant differences in KO muscles compared with WT within the same sex group. P-aminobenzoic acid, ß-alanine, and dopamine were significantly higher in KO male muscle whereas erythrose-4-phosphate and oxoglutaric acid were significantly reduced in KO females. The lipidomic profile of the KO groups revealed an increase of muscle phospholipids and reduced triacylglycerol and diacylglycerol compared with the WT groups. CONCLUSIONS: Sex differences were detected in polar metabolome and lipids were dependent on genotype. The metabolomic profile of mice with hyperactive skeletal muscle mTORC1 is consistent with an upregulation of mitochondrial function and amino acid utilization for protein synthesis.

A large-scale LC-MS dataset of murine liver proteome from time course of heavy water metabolic labeling.

Metabolic stable isotope labeling with heavy water followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein turnover studies. Several algorithms and tools have been developed to determine the turnover rates of peptides and proteins from time-course stable isotope labeling experiments. The availability of benchmark mass spectrometry data is crucial to compare and validate the effectiveness of newly developed techniques and algorithms. In this work, we report a heavy water-labeled LC-MS dataset from the murine liver for protein turnover rate analysis. The dataset contains eighteen mass spectral data with their corresponding database search results from nine different labeling durations and quantification outputs from d2ome+ software. The dataset also contains eight mass spectral data from two-dimensional fractionation experiments on unlabeled samples.

EMT activates exocytotic Rabs to coordinate invasion and immunosuppression in lung cancer.

Epithelial-to-mesenchymal transition (EMT) underlies immunosuppression, drug resistance, and metastasis in epithelial malignancies. However, the way in which EMT orchestrates disparate biological processes remains unclear. Here, we identify an EMT-activated vesicular trafficking network that coordinates promigratory focal adhesion dynamics with an immunosuppressive secretory program in lung adenocarcinoma (LUAD). The EMT-activating transcription factor ZEB1 drives exocytotic vesicular trafficking by relieving Rab6A, Rab8A, and guanine nucleotide exchange factors from miR-148a-dependent silencing, thereby facilitating MMP14-dependent focal adhesion turnover in LUAD cells and autotaxin-mediated CD8+ T cell exhaustion, indicating that cell-intrinsic and extrinsic processes are linked through a microRNA that coordinates vesicular trafficking networks. Blockade of ZEB1-dependent secretion reactivates antitumor immunity and negates resistance to PD-L1 immune checkpoint blockade, an important clinical problem in LUAD. Thus, EMT activates exocytotic Rabs to drive a secretory program that promotes invasion and immunosuppression in LUAD.

Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection.

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.

Quantifying label enrichment from two mass isotopomers increases proteome coverage for in vivo protein turnover using heavy water metabolic labeling.

Heavy water metabolic labeling followed by liquid chromatography coupled with mass spectrometry is a powerful high throughput technique for measuring the turnover rates of individual proteins in vivo. The turnover rate is obtained from the exponential decay modeling of the depletion of the monoisotopic relative isotope abundance. We provide theoretical formulas for the time course dynamics of six mass isotopomers and use the formulas to introduce a method that utilizes partial isotope profiles, only two mass isotopomers, to compute protein turnover rate. The use of partial isotope profiles alleviates the interferences from co-eluting contaminants in complex proteome mixtures and improves the accuracy of the estimation of label enrichment. In five different datasets, the technique consistently doubles the number of peptides with high goodness-of-fit characteristics of the turnover rate model. We also introduce a software tool, d2ome+, which automates the protein turnover estimation from partial isotope profiles.

Designer molecules of the synaptic organizer MDGA1 reveal 3D conformational control of biological function.

MDGAs (MAM domain-containing glycosylphosphatidylinositol anchors) are synaptic cell surface molecules that regulate the formation of trans-synaptic bridges between neurexins (NRXNs) and neuroligins (NLGNs), which promote synaptic development. Mutations in MDGAs are implicated in various neuropsychiatric diseases. MDGAs bind NLGNs in cis on the postsynaptic membrane and physically block NLGNs from binding to NRXNs. In crystal structures, the six immunoglobulin (Ig) and single fibronectin III domains of MDGA1 reveal a striking compact, triangular shape, both alone and in complex with NLGNs. Whether this unusual domain arrangement is required for biological function or other arrangements occur with different functional outcomes is unknown. Here, we show that WT MDGA1 can adopt both compact and extended 3D conformations that bind NLGN2. Designer mutants targeting strategic molecular elbows in MDGA1 alter the distribution of 3D conformations while leaving the binding affinity between soluble ectodomains of MDGA1 and NLGN2 intact. In contrast, in a cellular context, these mutants result in unique combinations of functional consequences, including altered binding to NLGN2, decreased capacity to conceal NLGN2 from NRXN1ß, and/or suppressed NLGN2-mediated inhibitory presynaptic differentiation, despite the mutations being located far from the MDGA1-NLGN2 interaction site. Thus, the 3D conformation of the entire MDGA1 ectodomain appears critical for its function, and its NLGN-binding site on Ig1-Ig2 is not independent of the rest of the molecule. As a result, global 3D conformational changes to the MDGA1 ectodomain via strategic elbows may form a molecular mechanism to regulate MDGA1 action within the synaptic cleft.

Alternative splicing of the SUMO1/2/3 transcripts affects cellular SUMOylation and produces functionally distinct SUMO protein isoforms.

Substantial increases in the conjugation of the main human SUMO paralogs, SUMO1, SUMO2, and SUMO3, are observed upon exposure to different cellular stressors, and such increases are considered important to facilitate cell survival to stress. Despite their critical cellular role, little is known about how the levels of the SUMO modifiers are regulated in the cell, particularly as it relates to the changes observed upon stress. Here we characterize the contribution of alternative splicing towards regulating the expression of the main human SUMO paralogs under normalcy and three different stress conditions, heat-shock, cold-shock, and Influenza A Virus infection. Our data reveal that the normally spliced transcript variants are the predominant mature mRNAs produced from the SUMO genes and that the transcript coding for SUMO2 is by far the most abundant of all. We also provide evidence that alternatively spliced transcripts coding for protein isoforms of the prototypical SUMO proteins, which we refer to as the SUMO alphas, are also produced, and that their abundance and nuclear export are affected by stress in a stress- and cell-specific manner. Additionally, we provide evidence that the SUMO alphas are actively synthesized in the cell as their coding mRNAs are found associated with translating ribosomes. Finally, we provide evidence that the SUMO alphas are functionally different from their prototypical counterparts, with SUMO1α and SUMO2α being non-conjugatable to protein targets, SUMO3α being conjugatable but targeting a seemingly different subset of protein from those targeted by SUMO3, and all three SUMO alphas displaying different cellular distributions from those of the prototypical SUMOs. Thus, alternative splicing appears to be an important contributor to the regulation of the expression of the SUMO proteins and the cellular functions of the SUMOylation system.

EMT-activated secretory and endocytic vesicular trafficking programs underlie a vulnerability to PI4K2A antagonism in lung cancer.

Hypersecretory malignant cells underlie therapeutic resistance, metastasis, and poor clinical outcomes. However, the molecular basis for malignant hypersecretion remains obscure. Here, we showed that epithelial-mesenchymal transition (EMT) initiates exocytic and endocytic vesicular trafficking programs in lung cancer. The EMT-activating transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) executed a PI4KIIIß-to-PI4KIIα (PI4K2A) dependency switch that drove PI4P synthesis in the Golgi and endosomes. EMT enhanced the vulnerability of lung cancer cells to PI4K2A small-molecule antagonists. PI4K2A formed a MYOIIA-containing protein complex that facilitated secretory vesicle biogenesis in the Golgi, thereby establishing a hypersecretory state involving osteopontin (SPP1) and other prometastatic ligands. In the endosomal compartment, PI4K2A accelerated recycling of SPP1 receptors to complete an SPP1-dependent autocrine loop and interacted with HSP90 to prevent lysosomal degradation of AXL receptor tyrosine kinase, a driver of cell migration. These results show that EMT coordinates exocytic and endocytic vesicular trafficking to establish a therapeutically actionable hypersecretory state that drives lung cancer progression.

Functional excitatory to inhibitory synaptic imbalance and loss of cognitive performance in people with Alzheimer's disease neuropathologic change.

Individuals at distinct stages of Alzheimer's disease (AD) show abnormal electroencephalographic activity, which has been linked to network hyperexcitability and cognitive decline. However, whether pro-excitatory changes at the synaptic level are observed in brain areas affected early in AD, and if they are emergent in MCI, is not clearly known. Equally important, it is not known whether global synaptic E/I imbalances correlate with the severity of cognitive impairment in the continuum of AD. Measuring the amplitude of ion currents of human excitatory and inhibitory synaptic receptors microtransplanted from the hippocampus and temporal cortex of cognitively normal, mildly cognitively impaired and AD individuals into surrogate cells, we found regional differences in pro-excitatory shifts of the excitatory to inhibitory (E/I) current ratio that correlates positively with toxic proteins and degree of pathology, and impinges negatively on cognitive performance scores. Using these data with electrophysiologically anchored analysis of the synapto-proteome in the same individuals, we identified a group of proteins sustaining synaptic function and those related to synaptic toxicity. We also found an uncoupling between the function and expression of proteins for GABAergic signaling in the temporal cortex underlying larger E/I and worse cognitive performance. Further analysis of transcriptomic and in situ hybridization datasets from an independent cohort across the continuum of AD confirm regional differences in pro-excitatory shifts of the E/I balance that correlate negatively with the most recent calibrated composite scores for memory, executive function, language and visuospatial abilities, as well as overall cognitive performance. These findings indicate that early shifts of E/I balance may contribute to loss of cognitive capabilities in the continuum of AD clinical syndrome.

Analysis of Golgi Secretory Functions in Cancer.

Cancer cells utilize secretory pathways for paracrine signaling and extracellular matrix remodeling to facilitate directional cell migration, invasion, and metastasis. The Golgi apparatus is a central secretory signaling hub that is often deregulated in cancer. Here we described technologies that utilize microscopic, biochemical, and proteomic approaches to analyze Golgi secretory functions in genetically heterogeneous cancer cell lines.

INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex.

Oral-facial-digital (OFD) syndromes are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. Mutations within 12 cilia-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline variants in INTS13, a subunit of the Integrator complex. This multiprotein complex associates with RNA Polymerase II and cleaves nascent RNA to modulate gene expression. We determined that INTS13 utilizes its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline variants p.S652L and p.K668Nfs*9. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos leads to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions between components of the Integrator complex.

Comparative genomic and biochemical analyses identify a collagen galactosylhydroxylysyl glucosyltransferase from Acanthamoeba polyphaga mimivirus.

Humans and Acanthamoeba polyphaga mimivirus share numerous homologous genes, including collagens and collagen-modifying enzymes. To explore this homology, we performed a genome-wide comparison between human and mimivirus using DELTA-BLAST (Domain Enhanced Lookup Time Accelerated BLAST) and identified 52 new putative mimiviral proteins that are homologous with human proteins. To gain functional insights into mimiviral proteins, their human protein homologs were organized into Gene Ontology (GO) and REACTOME pathways to build a functional network. Collagen and collagen-modifying enzymes form the largest subnetwork with most nodes. Further analysis of this subnetwork identified a putative collagen glycosyltransferase R699. Protein expression test suggested that R699 is highly expressed in Escherichia coli, unlike the human collagen-modifying enzymes. Enzymatic activity assay and mass spectrometric analyses showed that R699 catalyzes the glucosylation of galactosylhydroxylysine to glucosylgalactosylhydroxylysine on collagen using uridine diphosphate glucose (UDP-glucose) but no other UDP-sugars as a sugar donor, suggesting R699 is a mimiviral collagen galactosylhydroxylysyl glucosyltransferase (GGT). To facilitate further analysis of human and mimiviral homologous proteins, we presented an interactive and searchable genome-wide comparison website for quickly browsing human and Acanthamoeba polyphaga mimivirus homologs, which is available at RRID Resource ID: SCR_022140 or https://guolab.shinyapps.io/app-mimivirus-publication/ .

Comparative Analysis of Co-Cultured Amniotic Cell-Conditioned Media with Cell-Free Amniotic Fluid Reveals Differential Effects on Epithelial-Mesenchymal Transition and Myofibroblast Activation.

Myofibroblast activation is a cellular response elicited by a variety of physiological or pathological insults whereby cells initiate a coordinated response intended to eradicate the insult and then revert back to a basal state. However, an underlying theme in various disease states is persistent myofibroblast activation that fails to resolve. Based on multiple observations, we hypothesized that the secreted factors harvested from co-culturing amniotic stem cells might mimic the anti-inflammatory state that cell-free amniotic fluid (AF) elicits. We optimized an amnion epithelial and amniotic fluid cell co-culture system, and tested this hypothesis in the context of myofibroblast activation. However, we discovered that co-cultured amniotic cell conditioned media (coACCM) and AF have opposing effects on myofibroblast activation: coACCM activates the epithelial-mesenchymal transition (EMT) and stimulates gene expression patterns associated with myofibroblast activation, while AF does the opposite. Intriguingly, extracellular vesicles (EVs) purified from AF are necessary and sufficient to activate EMT and inflammatory gene expression patterns, while the EV-depleted AF potently represses these responses. In summary, these data indicate that coACCM stimulates myofibroblast activation, while AF represses it. We interpret these findings to suggest that coACCM, AF, and fractionated AF represent unique biologics that elicit different cellular responses that are correlated with a wide variety of pathological states, and therefore could have broad utility in the clinic and the lab.

Impad1 and Syt11 work in an epistatic pathway that regulates EMT-mediated vesicular trafficking to drive lung cancer invasion and metastasis.

Lung cancer is a highly aggressive and metastatic disease responsible for approximately 25% of all cancer-related deaths in the United States. Using high-throughput in vitro and in vivo screens, we have previously established Impad1 as a driver of lung cancer invasion and metastasis. Here we elucidate that Impad1 is a direct target of the epithelial microRNAs (miRNAs) miR-200 and miR∼96 and is de-repressed during epithelial-to-mesenchymal transition (EMT); thus, we establish a mode of regulation of the protein. Impad1 modulates Golgi apparatus morphology and vesicular trafficking through its interaction with a trafficking protein, Syt11. These changes in Golgi apparatus dynamics alter the extracellular matrix and the tumor microenvironment (TME) to promote invasion and metastasis. Inhibiting Impad1 or Syt11 disrupts the cancer cell secretome, regulates the TME, and reverses the invasive or metastatic phenotype. This work identifies Impad1 as a regulator of EMT and secretome-mediated changes during lung cancer progression.

Quantitative proteomic analytic approaches to identify metabolic changes in the medial prefrontal cortex of rats exposed to space radiation.

NASA's planned mission to Mars will result in astronauts being exposed to ∼350 mSv/yr of Galactic Cosmic Radiation (GCR). A growing body of data from ground-based experiments indicates that exposure to space radiation doses (approximating those that astronauts will be exposed to on a mission to Mars) impairs a variety of cognitive processes, including cognitive flexibility tasks. Some studies report that 33% of individuals may experience severe cognitive impairment. Translating the results from ground-based rodent studies into tangible risk estimates for astronauts is an enormous challenge, but it would be germane for NASA to use the vast body of data from the rodent studies to start developing appropriate countermeasures, in the expectation that some level of space radiation (SR) -induced cognitive impairment could occur in astronauts. While some targeted studies have reported radiation-induced changes in the neurotransmission properties and/or increased neuroinflammation within space radiation exposed brains, there remains little information that can be used to start the development of a mechanism-based countermeasure strategy. In this study, we have employed a robust label-free mass spectrometry (MS) -based untargeted quantitative proteomic profiling approach to characterize the composition of the medial prefrontal cortex (mPFC) proteome in rats that have been exposed to 15 cGy of 600 MeV/n28Si ions. A variety of analytical techniques were used to mine the generated expression data, which in such studies is typically hampered by low and variable sample size. We have identified several pathways and proteins whose expression alters as a result of space radiation exposure, including decreased mitochondrial function, and a further subset of proteins differs in rats that have a high level of cognitive performance after SR exposure in comparison with those that have low performance levels. While this study has provided further insight into how SR impacts upon neurophysiology, and what adaptive responses can be invoked to prevent the emergence of SR-induced cognitive impairment, the main objective of this paper is to outline strategies that can be used by others to analyze sub-optimal data sets and to identify new information.

Chemically targeting the redox switch in AP1 transcription factor ΔFOSB.

The AP1 transcription factor ΔFOSB, a splice variant of FOSB, accumulates in the brain in response to chronic insults such as exposure to drugs of abuse, depression, Alzheimer's disease and tardive dyskinesias, and mediates subsequent long-term neuroadaptations. ΔFOSB forms heterodimers with other AP1 transcription factors, e.g. JUND, that bind DNA under control of a putative cysteine-based redox switch. Here, we reveal the structural basis of the redox switch by determining a key missing crystal structure in a trio, the ΔFOSB/JUND bZIP domains in the reduced, DNA-free form. Screening a cysteine-focused library containing 3200 thiol-reactive compounds, we identify specific compounds that target the redox switch, validate their activity biochemically and in cell-based assays, and show that they are well tolerated in different cell lines despite their general potential to bind to cysteines covalently. A crystal structure of the ΔFOSB/JUND bZIP domains in complex with a redox-switch-targeting compound reveals a deep compound-binding pocket near the DNA-binding site. We demonstrate that ΔFOSB, and potentially other, related AP1 transcription factors, can be targeted specifically and discriminately by exploiting unique structural features such as the redox switch and the binding partner to modulate biological function despite these proteins previously being thought to be undruggable.

QTQTN motif upstream of the furin-cleavage site plays a key role in SARS-CoV-2 infection and pathogenesis.

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here, we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated, and disruption of its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site-the FCS, loop length, and glycosylation-are required for efficient SARS-CoV-2 replication and pathogenesis.