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Slo1 is a Ca2+- and voltage-activated K+ channel that underlies skeletal and smooth muscle contraction, audition, hormone secretion and neurotransmitter release. In mammals, Slol is regulated by auxiliary proteins that confer tissue-specific gating and pharmacological properties. This study presents cryo-EM structures of Slo1 in complex with the auxiliary protein, beta 4. Four beta 4, each containing two transmembrane helices, encircle Slot, contacting it through helical interactions inside the membrane. On the extracellular side, beta 4 forms a tetrameric crown over the pore. Structures with high and low Ca(2+ )concentrations show that identical gating conformations occur in the absence and presence of beta 4, implying that beta 4 serves to modulate the relative stabilities of 'pre-existing' conformations rather than creating new ones. The effects of beta 4 on scorpion toxin inhibition kinetics are explained by the crown, which constrains access but does not prevent binding.

Proteasome-mediated degradation of intracellular proteins is essential for cell function and survival. The proteasome-binding protein PI31 (Proteasomal Inhibitor of 31kD) promotes 265 assembly and functions as an adapter for proteasome transport in axons. As localized protein synthesis and degradation is especially critical in neurons, we generated a conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs). A cKO of PI31 in these neurons caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction. For both MNs and PCs, markers of proteotoxic stress preceded axonal degeneration and motor dysfunction, indicating a critical role for PI31 in neuronal homeostasis. The time course of the loss of MN and PC function in developing mouse central nervous system suggests a key role for PI31 in human neurodegenerative diseases.

From incoming sensory information, our brains make selections according to current behavioral goals. This process, selective attention, is controlled by parietal and frontal areas. Here, we show that another brain area, posterior inferotemporal cortex (PITd), also exhibits the defining properties of attentional control. We discovered this area with functional magnetic resonance imaging (fMRI) during an attentive motion discrimination task. Single-cell recordings from PITd revealed strong attentional modulation across 3 attention tasks yet no tuning to task-relevant stimulus features, like motion direction or color. Instead, PITd neurons closely tracked the subject's attention state and predicted upcoming errors of attentional selection. Furthermore, artificial electrical PITd stimulation controlled the location of attentional selection without altering feature discrimination. These are the defining properties of a feature-blind priority map encoding the locus of attention. Together, these results suggest area PITd, located strategically to gather information about object properties, as an attentional priority map.

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirustype-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TON motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNV5), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNV5 as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.

Bradykinin is a proinflammatory factor that mediates angioedema and inflammation in many diseases. It is a key player in some types of hereditary angioedema and is involved in septic shock, traumatic injury, Alzheimer's disease (AD), and stroke, among others. Activation of the plasma contact system leads to elevated levels of plasma kallikrein, which cleaves high molecular weight kininogen (HK) to release bradykinin. Drug development for bradykinin-meditated pathologies has focused on designing inhibitors to the enzymes that cleave HK (to prevent bradykinin release) or antagonists of endothelial bradykinin receptors (to prevent down-stream bradykinin action). Here we show a strategy to block bradykinin generation by using an HK antibody that binds to HK, preventing its cleavage and subsequent bradykinin release. We show that this antibody blocks dextran sodium sulfate-induced HK cleavage and bradykinin production. Moreover, while the pathogenic AD peptide beta-amyloid (A beta)42 cleaves HK and induces a dramatic increase in bradykinin production, our HK antibody blocked these events from occurring. These results may provide strategies for developing treatments for bradykinin-driven pathologies.

Infant maltreatment increases vulnerability to physical and mental disorders, yet specific mechanisms embedded within this complex infant experience that induce this vulnerability remain elusive. To define critical features of maltreatment-induced vulnerability, rat pups were reared from postnatal day 8 (PN8) with a maltreating mother, which produced amygdala and hippocampal deficits and decreased social behavior at PN13. Next, we deconstructed the maltreatment experience to reveal sufficient and necessary conditions to induce this phenotype. Social behavior and amygdala deficits (volume, neurogenesis, c-Fos, local field potential) required combined chronic high corticosterone and maternal presence (not maternal behavior). Hippocampal deficits were induced by chronic high corticosterone regardless of social context. Causation was shown by blocking corticosterone during maltreatment and suppressing amygdala activity during social behavior testing. These results highlight (1) that early life maltreatment initiates multiple pathways to pathology, each with distinct causal mechanisms and outcomes, and (2) the importance of social presence on brain development.

TRAAK is a membrane tension-activated K+ channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the 'leak' K+ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na+ channel availability for action potential propagation. We speculate on why nodes of Ranvier contain a mechanosensitive K+ channel.

DNA is both a fundamental building block of life and a fascinating natural polymer. The advent of single-molecule manipulation tools made it possible to exert controlled force on individual DNA molecules and measure their mechanical response. Such investigations elucidated the elastic properties of DNA and revealed its distinctive structural configurations across force regimes. In the meantime, a detailed understanding of DNA mechanics laid the groundwork for single-molecule studies of DNA-binding proteins and DNA-processing enzymes that bend, stretch, and twist DNA. These studies shed new light on the metabolism and transactions of nucleic acids, which constitute a major part of the cell's operating system. Furthermore, the marriage of single-molecule fluorescence visualization and force manipulation has enabled researchers to directly correlate the applied tension to changes in the DNA structure and the behavior of DNA-templated complexes. Overall, experimental exploitation of DNA mechanics has been and will continue to be a unique and powerful strategy for understanding how molecular machineries recognize and modify the physical state of DNA to accomplish their biological functions.

Protein-surface interactions play a pivotal role in processes as diverse as biomineralization, biofouling, and the cellular response to medical implants. In biomineralization processes, biomacromolecules control mineral deposition and architecture via complex and often unknown mechanisms. For studying these mechanisms, the formation of magnetite nanoparticles in magnetotactic bacteria has become an excellent model system. Most interestingly, nanoparticle morphologies have been discovered that defy crystallographic rules (e.g., in the species Desulfamplus magnetovallimortis strain BW-1). In certain conditions, this strain mineralizes bullet-shaped magnetite nanoparticles, which exhibit defined (111) crystal faces and are elongated along the [100] direction. We hypothesize that surface-specific protein interactions break the nanoparticle symmetry, inhibiting the growth of certain crystal faces and thereby favoring the growth of others. Screening the genome of BW-1, we identified Mad10 (Magnetosome-associated deep-branching) as a potential magnetite-binding protein. Using atomic force microscope (AFM)-based single-molecule force spectroscopy, we show that a Mad10-derived peptide, which represents the most conserved region of Mad10, binds strongly to (100)- and (111)-oriented single-crystalline magnetite thin films. The peptide-magnetite interaction is thus material- but not crystal-face-specific. It is characterized by broad rupture force distributions that do not depend on the retraction speed of the AFM cantilever. To account for these experimental findings, we introduce a three-state model that incorporates fast rebinding. The model suggests that the peptide-surface interaction is strong in the absence of load, which is a direct result of this fast rebinding process. Overall, our study sheds light on the kinetic nature of peptide-surface interactions and introduces a new magnetite-binding peptide with potential use as a functional coating for magnetite nanoparticles in biotechnological and biomedical applications.