Abstract Salmonella utilizes translocated virulence proteins (termed effectors) to promote host cell invasion. The effector SopD contributes to invasion by promoting scission of the plasma membrane, ...generating Salmonella -containing vacuoles. SopD is expressed in all Salmonella lineages and plays important roles in animal models of infection, but its host cell targets are unknown. Here we show that SopD can bind to and inhibit the small GTPase Rab10, through a C-terminal GTPase activating protein (GAP) domain. During infection, Rab10 and its effectors MICAL-L1 and EHBP1 are recruited to invasion sites. By inhibiting Rab10, SopD promotes removal of Rab10 and recruitment of Dynamin-2 to drive scission of the plasma membrane. Together, our study uncovers an important role for Rab10 in regulating plasma membrane scission and identifies the mechanism used by a bacterial pathogen to manipulate this function during infection.
A weak screening hit with suboptimal physicochemical properties was optimized against PFKFB3 kinase using critical structure-guided insights. The resulting compounds demonstrated high selectivity ...over related PFKFB isoforms and modulation of the target in a cellular context. A selected example demonstrated exposure in animals following oral dosing. Examples from this series may serve as useful probes to understand the emerging biology of this metabolic target.
Macroautophagy is a highly conserved eukaryotic cellular pathway involving the engulfment of macromolecules, organelles, and invading microbes by a double-membrane compartment and subsequent ...lysosomal degradation. The mechanisms that regulate macroautophagy, and the interaction of its components with other cellular pathways, have remained unclear. Here, we performed proximity-dependent biotin identification (BioID) on 39 core human macroautophagy proteins, identifying over 700 unique high confidence proximity interactors with new putative connections between macroautophagic and essential cellular processes. Of note, we identify members of the OSBPL (oxysterol binding protein like) family as Atg8-family protein interactors. We subsequently conducted comprehensive screens of the OSBPL family for LC3B-binding and roles in xenophagy and aggrephagy. OSBPL7 and OSBPL11 emerged as novel lipid transfer proteins required for macroautophagy of selective cargo. Altogether, our proximity interaction map provides a valuable resource for the study of autophagy and highlights the critical role of membrane contact site proteins in the pathway.
BioID: proximity-dependent biotin identification; GO: gene ontology; OSBPL: oxysterol binding protein like; VAPA: VAMP associated protein A; VAPB: VAMP associated protein B and C
Abstract
RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the ...Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (
RREB1
) is the common deleted gene in multiple 6p-interstitial microdeletion cases.
Rreb1
hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome.
Rreb1
haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of
SIN3A
and mutations in
KDM1A
cause syndromes similar to
RREB1
haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.
Lung cancer is the leading cause of cancer mortality worldwide, with squamous cell carcinoma (SQCC) being the second most common form. SQCCs are thought to originate in bronchial basal cells through ...an injury response to smoking, which results in this stem cell population committing to hyperplastic squamous rather than mucinous and ciliated fates. Copy number gains in SOX2 in the region of 3q26–28 occur in 94% of SQCCs, and appear to act both early and late in disease progression by stabilizing the initial squamous injury response in stem cells and promoting growth of invasive carcinoma. Thus, anti-SOX2 targeting strategies could help treat early and/or advanced disease. Because SOX2 itself is not readily druggable, we sought to characterize SOX2 binding partners, with the hope of identifying new strategies to indirectly interfere with SOX2 activity. We now report the first use of proximity-dependent biotin labeling (BioID) to characterize the SOX2 interactome in vivo. We identified 82 high confidence SOX2-interacting partners. An interaction with the coactivator EP300 was subsequently validated in both basal cells and SQCCs, and we demonstrate that EP300 is necessary for SOX2 activity in basal cells, including for induction of the squamous fate. We also report that EP300 copy number gains are common in SQCCs and that growth of lung cancer cell lines with 3q gains, including SQCC cells, is dependent on EP300. Finally, we show that EP300 inhibitors can be combined with other targeted therapeutics to achieve more effective growth suppression. Our work supports the use of BioID to identify interacting protein partners of nondruggable oncoproteins such as SOX2, as an effective strategy to discover biologically relevant, druggable targets.
The geographical range of Zika virus (ZIKV) has expanded dramatically in recent decades, but no antiviral drugs or vaccines are available for the treatment or prevention of ZIKV infection. Here we ...map host cell protein interactions for each of the ten ZIKV polypeptides, generating a protein network comprising >3000 interactions. Mining this rich dataset, we highlight multiple examples of how ZIKV may usurp or disrupt the function of host organelles and uncover an important role for peroxisomes in ZIKV infection.
Display omitted
Highlights
•BioID and IP-MS were conducted to generate a global ZIKV-host protein interactome•Interactome consists of >3000 high confidence ZIKV-host protein interactions•Data mining indicates that ZIKV proteins interact with multiple host cell organelles•An important role for peroxisomes in ZIKV infection is uncovered
Zika virus (ZIKV) is a membrane enveloped Flavivirus with a positive strand RNA genome, transmitted by Aedes mosquitoes. The geographical range of ZIKV has dramatically expanded in recent decades resulting in increasing numbers of infected individuals, and the spike in ZIKV infections has been linked to significant increases in both Guillain-Barré syndrome and microcephaly. Although a large number of host proteins have been physically and/or functionally linked to other Flaviviruses, very little is known about the virus-host protein interactions established by ZIKV. Here we map host cell protein interaction profiles for each of the ten polypeptides encoded in the ZIKV genome, generating a protein topology network comprising 3033 interactions among 1224 unique human polypeptides. The interactome is enriched in proteins with roles in polypeptide processing and quality control, vesicle trafficking, RNA processing and lipid metabolism. >60% of the network components have been previously implicated in other types of viral infections; the remaining interactors comprise hundreds of new putative ZIKV functional partners. Mining this rich data set, we highlight several examples of how ZIKV may usurp or disrupt the function of host cell organelles, and uncover an important role for peroxisomes in ZIKV infection.
Depolarized mitochondria can be degraded via mitophagy, a selective form of autophagy. The RAB GTPase RAB7A was recently shown to play a key role in this process. RAB7A regulates late endocytic ...trafficking under normal growth conditions but is translocated to the mitochondrial surface following depolarization. However, how RAB7A activity is regulated during mitophagy is not understood. Here, using a proximity-dependent biotinylation approach (miniTurbo), we identified C5orf51 as a specific interactor of GDP-locked RAB7A. C5orf51 also interacts with the RAB7A guanine nucleotide exchange factor (GEF) complex members MON1 and CCZ1. In the absence of C5orf51, localization of RAB7A on depolarized mitochondria is compromised and the protein is degraded by the proteasome. Furthermore, depletion of C5orf51 also inhibited ATG9A recruitment to depolarized mitochondria. Together, these results indicate that C5orf51 is a positive regulator of RAB7A in its shuttling between late endosomes and mitochondria to enable mitophagy.
Abbreviations: ATG9A: autophagy related 9A; Baf A
1
: bafilomycin A
1
; BioID: proximity-dependent biotin identification; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CCZ1: CCZ1 homolog, vacuolar protein trafficking and biogenesis associated; DQ-BSA: dye quenched-bovine serum albumin; FYCO1: FYVE and coiled-coil domain autophagy adaptor 1; GAP: GTPase activating protein; GEF: guanine nucleotide exchange factor; KO: knockout; LRPPRC: leucine rich pentatricopeptide repeat containing; MG132: carbobenzoxy-Leu-Leu-leucinal; MON1: MON1 homolog, secretory trafficking associated; mtDNA: mitochondrial DNA; PINK1: PTEN induced kinase 1; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RMC1: regulator of MON1-CCZ1; TBC1D15: TBC1 domain family member 15; TBC1D17: TBC1 domain family member 17; TOMM20: translocase of outer mitochondrial membrane 20; WDR91: WD repeat domain 91; WT: wild type.
The N = 28 shell closure has been investigated via the 46Ar(d,p)47Ar transfer reaction in inverse kinematics. Energies and spectroscopic factors of the neutron p(3/2), p(1/2), and f(5/2) states in ...47Ar were determined and compared to those of the 49Ca isotone. We deduced a reduction of the N = 28 gap by 330(90) keV and spin-orbit weakenings of approximately 10(2) and 45(10)% for the f and p states, respectively. Such large variations for the f and p spin-orbit splittings could be accounted for by the proton-neutron tensor force and by the density dependence of the spin-orbit interaction, respectively. This contrasts with the picture of the spin-orbit interaction as a surface term only.
•Both spatial and temporal factors efficiently explained moose-vehicle collisions.•Collisions occurred more at night, dusk and dawn for moose and white-tailed deer.•Collisions occurred more in months ...during which cervid movement rate was higher.•We found an interaction between slope and elevation on collision risk with moose.•Road sinuosity and % of mature coniferous stands increased collision risk with deer.
Mitigation strategies for wildlife-vehicle collisions require sufficient knowledge about why, where and when collisions occur in order to be an efficient tool to improve public safety. Collisions with cervids are known to be influenced by spatial factors such as topography and forest cover. However, temporal changes in animal and motorist behaviors are often overlooked although they can increase the odds of cervid-vehicle collisions. Consequently, we evaluated potential factors influencing the spatiotemporal distribution of 450 collisions with moose and white-tailed deer that occurred between 1990 and 2015 along the 100-km long highway in southeastern Québec, Canada. Both spatial and temporal factors efficiently explained moose-vehicle collisions but not collisions with white-tailed deer, suggesting that the latter occurred more randomly along the highway. The risk of moose-vehicle collisions was mainly modulated by topographic and habitat variables, as the interactions between slope and elevation and slope and distance to suitable moose habitats had a strong effect on collision risk. Road sinuosity and the proportion of mature coniferous stands around the collision site positively influenced deer-vehicle collisions. A temporal increase in collision numbers was noted in different biological periods during which movement rates are known to be higher (e.g. post-winter dispersal and rut). These results suggest that cervid movement is the main factor influencing collision risk and frequency. Our results indicate that mitigation strategies aimed at decreasing the probability of collision with cervids must be species-specific and should focus more closely on animal movement.
PDF
