Biology subjects

Kim, K.

Publications and source records attributed to Kim, K..

At least 19 recordsLinked to original sources

bioRxiv2018

Cullin3-RING ubiquitin ligases are intimately linked to the unfolded protein response of the endoplasmic reticulum

CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric a brac, Tramtrack and Broad-Complex (BTB)-domain proteins. KLHL12, a BTB-domain protein, appears to play an essential role in export of large cargo molecules like procollagen from the endoplasmic reticulum (ER). It has been suggested that CRL3KLHL12 mono-ubiquitinates SEC31 and mono-ubiquitinated SEC31 increases the dimension of a COPII coat to accommodate the large cargo molecules. As we examined this model, we found that functional CRL3KLHL12 was indeed critical for the assembly of large COPII structures. Interestingly, we noticed that CRL3KLHL12 influences collagen synthesis in human skin fibroblasts (HSFs). Our results also suggest that there is a CRL3KLHL12-independent collagen secretion route in HSFs. In addition, we found that CRL3KLHL12 strongly influences levels of sensors of the unfolded protein response (UPR). Different cell lines reacted differently to CUL3 depletion with respect to UPR regulation. This cell line-dependency appears to rely on a cell line-specific BTB-domain protein(s). Consistent with this idea, depletion of a muscle-specific BTB-domain protein KLHL41 recapitulated the effects of CUL3 depletion in C2C12 myotubes in UPR regulation. Based on these results we propose that CRL3KLHL12 and CRL3KLHL41 are regulators of the UPR.

cell biology↗

bioRxiv2018

Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.

biochemistry↗

bioRxiv2018

The GATOR complex regulates an essential response to meiotic double-stranded breaks in Drosophila

The TORC1 inhibitor GATOR1/SEACIT controls meiotic entry and early meiotic events in yeast. However, how metabolic pathways influence meiotic progression in metazoans remains poorly understood. Here we report that the TORC1 regulators GATOR1 and GATOR2 mediate a response to meiotic double-stranded breaks (DSBs) during Drosophila oogenesis. We find that meiotic DSBs trigger the activation of a GATOR1 dependent pathway that downregulates TORC1 activity in the female germline. In GATOR1 mutants, high TORC1 activity results in the delayed repair of meiotic DSBs and the hyperactivation of p53. Conversely, the GATOR2 component Mio is required to attenuate GATOR1 activity, to ensure that meiotic DSBs do not trigger a permanent growth arrest. Unexpectedly, we found that GATOR1 inhibits retrotransposon expression in the presence of meiotic DSBs in a pathway that functions in parallel to p53. Our studies have revealed a link between the GATOR complex, the repair of meiotic DSBs and retrotransposon expression

developmental biology↗

bioRxiv2018

Intracellular mass density increase is accompanying but not sufficient for stiffening and growth arrest of yeast cells

Many organisms, including yeast cells, bacteria, nematodes and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography and atomic force microscopy. We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

biophysics↗

bioRxiv2018

PBHoover and CigarRoller: a method for confident haploid variant calling on Pacific Biosciences data and its application to heterogeneous population analysis

MotivationSingle Molecule Real-Time (SMRT) sequencing has important and underutilized advantages that amplification-based platforms lack. Lack of systematic error (e.g. GC-bias), complete de novo assembly (including large repetitive regions) without scaffolding, can be mentioned. SMRT sequencing, however suffers from high random error rate and low sequencing depth (older chemistries). Here, we introduce PBHoover, software that uses a heuristic calling algorithm in order to make base calls with high certainty in low coverage regions. This software is also capable of mixed population detection with high sensitivity. PBHoovers CigarRoller attachment improves sequencing depth in low-coverage regions through CIGAR-string correction.\n\nResultsWe tested both modules on 348 M.tuberculosis clinical isolates sequenced on C1 or C2 chemistries. On average, CigarRoller improved percentage of usable read count from 68.9% to 99.98% in C1 runs and from 50% to 99% in C2 runs. Using the greater depth provided by CigarRoller, PBHoover was able to make base and variant calls 99.95% concordant with Sanger calls (QV33). PBHoover also detected antibiotic-resistant subpopulations that went undetected by Sanger. Using C1 chemistry, subpopulations as small as 9% of the total colony can be detected by PBHoover. This provides the most sensitive amplification-free molecular method for heterogeneity analysis and is in line with phenotypic methods sensitivity. This sensitivity significantly improves with the greater depth and lower error rate of the newer chemistries.\n\nAvailability and ImplementationExecutables are freely available under GNU GPL v3+ at http://www.gitlab.com/LPCDRP/pbhoover and http://www.gitlab.com/LPCDRP/CigarRoller. PBHoover is also available on bioconda: https://anaconda.org/bioconda/pbhoover.\n\nContactfaramarz@sdsu.edu

bioinformatics↗

bioRxiv2018

Reassortment, positive selection, and the inter-segmental patterns of divergence and polymorphism in influenza virus H3N2

Reassortment in viruses with segmented genome is a major evolutionary process for their genetic diversity and adaptation. It is also crucial in generating different levels of sequence polymorphism among segments when positive selection occurs at different rates on them. Previous studies have detected intra-subtype reassortment events in human influenza H3N2 by between-segment incongruity in phylogenetic tree topology. Here, we quantitatively estimate the reassortment rate, probability that a pair of segments in a viral lineage become separated in a unit time, between hemmaglutinin (HA) and four non-antigenic segments (PB2, PB1, PA and NP) in human influenza virus H3N2. Using statistics that measure incongruity in tree topology or linkage disequilibrium between segments and performing simulations that are constrained to reproduce the various patterns of H3N2 molecular evolution, we infer that reassortment rate ranges between 0.001 and 0.01 assuming one generation to be 1/80 year. However, we find that a higher rate of reassortment is required to generate the observed pattern of ~40% less synonymous sequence polymorphism on HA relative to other non-HA segments, which results from recurrent selective sweeps by antigenic variants on the HA segment. Here, synonymous diversity was compared after correcting for difference in inferred mutation rates among segments, which we found significant. We also explored analytic approximations for inter-segmental difference in sequence diversity for a given reassortment rate to understand the underlying dynamics of recurrent positive selection. It is suggested that the effects of clonal interference and potentially demography-dependent rate of reassortment in the process of recurrent selective sweeps must be considered to fully explain the genomic pattern of diversity in H3N2 viruses.

evolutionary biology↗

bioRxiv2018

PTK2 regulates the UPS impairment via p62 phosphorylation in TDP-43 proteinopathy

TDP-43 proteinopathy is a common feature in a variety of neurodegenerative disorders including Amyotrophic lateral sclerosis (ALS) cases, Frontotemporal lobar degeneration (FTLD), and Alzheimers disease. However, the molecular mechanisms underlying TDP-43-induced neurotoxicity are largely unknown. In this study, we demonstrated that TDP-43 proteinopathy induces impairment in ubiquitin-proteasome system (UPS) evidenced by an accumulation of ubiquitinated proteins and reduction of proteasome activity in neuronal cells. Through kinase inhibitor screening, we identified PTK2 as a suppressor of neurotoxicity induced by UPS impairment. Importantly, PTK2 inhibition significantly reduces ubiquitin aggregates and attenuated TDP-43-induced cytotoxicity in Drosophila model of TDP-43 proteinopathy. We further identified that phosphorylation of p62 at serine 403 (p-p62S403), a key component in the autophagic degradation of poly-ubiquitinated proteins, is increased upon TDP-43 overexpression and dependent on activation of PTK2 in neuronal cells. Moreover, expressing a non-phosphorylated form of p62 (p62S403A) significantly represses accumulation of polyubiquitinated proteins and neurotoxicity induced by TDP-43 overexpression in neuronal cells. In addition, inhibition of TBK1, a kinase which phosphorylates S403 of p62, ameliorates neurotoxicity upon UPS impairment in neuronal cells. Taken together, our data suggest that activation of PTK2-TBK1-p62 axis plays a critical role in the pathogenesis of TDP-43 by regulating neurotoxicity induced by UPS impairment. Therefore, targeting PTK2-TBK1-p62 axis may represent a novel therapeutic intervention for neurodegenerative diseases with TDP-43 proteinopathy.

neuroscience↗

bioRxiv2018

AQ-seq: Accurate quantification of microRNAs and their variants

MicroRNAs (miRNAs) modulate diverse biological and pathological processes via post-transcriptional gene silencing. High-throughput small RNA sequencing (sRNA-seq) has been widely adopted to investigate the functions and regulatory mechanisms of miRNAs. However, accurate quantification has been limited owing to the severe ligation bias in conventional sRNA-seq methods. Here we present a high-throughput protocol, termed AQ-seq (accurate quantification by sequencing), that utilizes adapters with terminal degenerate sequences and a high concentration of polyethylene glycol (PEG), which removes the ligation bias during library preparation. By accurately measuring miRNAs and their variants (known as isomiRs), we identify alternatively processed miRNAs and correct the 5' end usage and strand preference that have been misannotated. We also uncover highly modified miRNAs that are uridylated and adenylated. Taken together, AQ-seq reveals the complexity of the miRNA isoform landscape, allowing us to refine miRNA annotation and to advance our understanding of miRNA regulation. Furthermore, AQ-seq can be adopted to improve other ligation-based sequencing methods including crosslinking-immunoprecipitation-sequencing (CLIP-seq) and ribosome profiling (Ribo-seq).

molecular biology↗

bioRxiv2018

Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex

Drugs that inhibit HIV-1 replication and prevent progression to AIDS do not eliminate HIV-1 proviruses from the chromosomes of long-lived CD4+ memory T cells. To escape eradication by these antiviral drugs, or by the host immune system, HIV-1 exploits poorly defined host factors that silence provirus transcription. These same factors, though, must be overcome by all retroviruses, including HIV-1 and other primate immunodeficiency viruses, in order to activate provirus transcription and produce new virus. Here we show that Vpx and Vpr, proteins from a wide range of primate immunodeficiency viruses, activate provirus transcription in human CD4+ T cells. Provirus activation required the DCAF1 adaptor that links Vpx and Vpr to the CUL4A/B ubiquitin ligase complex, but did not require degradation of SAMHD1, a well-characterized target of Vpx and Vpr. A loss-of-function screen for transcription silencing factors that mimic the effect of Vpx on provirus silencing identified all components of the Human Silencing Hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN), and MORC2. Vpx associated with the HUSH complex components and decreased steady-state levels of these proteins in a DCAF-dependent manner. Finally, vpx and FAM208A knockdown accelerated HIV-1 and SIVMAC replication kinetics in CD4+ T cells to a similar extent, and HIV-2 replication required either vpx or FAM208A disruption. These results demonstrate that the HUSH complex restricts transcription of primate immunodeficiency viruses and thereby contributes to provirus latency. To counteract this restriction and activate provirus expression, primate immunodeficiency viruses encode Vpx and Vpr proteins that degrade HUSH complex components.

molecular biology↗

bioRxiv2018

Dynamic embedding of salience coding in hippocampal spatial maps

Hippocampal CA1 neurons participate in dynamic ensemble codes for space and memory. Prominent features of the environment are represented by an increased density of place cells, but cellular principles governing the formation and plasticity of such disproportionate maps are unknown. We thus imaged experience-dependent long-term changes in spatial representations at the cellular level in the CA1 deep sublayer in mice learning to navigate in a virtual-reality environment. The maps were highly dynamic but gradually stabilized as over-representations for motivational (reward) and environmental (landmark) salience emerged in different time courses by selective consolidation of relevant spatial representations. Relocation of the reward extensively reorganized pre-formed maps by a mechanism involving rapid recruitment of cells from the previous location followed by their re-stabilization, indicating that a subset of neurons encode reward-related information. The distinct properties of these CA1 cells may provide a substrate by which salient experience forms lasting and adaptable memory traces.

neuroscience↗

bioRxiv2018

Lrfn2-mutant mice display suppressed synaptic plasticity and inhibitory synapse development and abnormal social communication and startle response

SALM1, also known as LRFN2, is a PSD-95-interacting synaptic adhesion molecule implicated in the regulation of NMDA receptor (NMDAR) clustering largely based on in vitro data, although its in vivo functions remain unclear. Here, we found that mice lacking SALM1/LRFN2 (Lrfn2-/- mice) show a normal density of excitatory synapses but altered excitatory synaptic function, including enhanced NMDAR-dependent synaptic transmission but suppressed NMDAR-dependent synaptic plasticity in the hippocampal CA1 region. Unexpectedly, SALM1 expression is detected in both glutamatergic and GABAergic neurons, and Lrfn2-/- CA1 pyramidal neurons show decreases in the density of inhibitory synapses and frequency of spontaneous inhibitory synaptic transmission. Behaviorally, ultrasonic vocalization was suppressed in Lrfn2-/- pups separated from their mothers, and acoustic startle was enhanced, but locomotion, anxiety-like behavior, social interaction, repetitive behaviors, and learning and memory were largely normal in adult Lrfn2-/- mice. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, and social communication and startle behaviors in mice.\n\nSignificance StatementSynaptic adhesion molecules regulate synapse development and function, which govern neural circuit and brain functions. The SALM/LRFN family of synaptic adhesion proteins consists of five known members whose in vivo functions are largely unknown. Here we characterized mice lacking SALM1/LRFN2 (SALM1 knockout) known to associate with NMDA receptors and found that these mice showed altered NMDA receptor-dependent synaptic transmission and plasticity, as expected, but unexpectedly also exhibited suppressed inhibitory synapse development and synaptic transmission. Behaviorally, SALM1 knockout pups showed suppressed ultrasonic vocalization upon separation from their mothers, and SALM1 knockout adults showed enhanced responses to loud acoustic stimuli. These results suggest that SALM1/LRFN2 regulates excitatory synapse function, inhibitory synapse development, social communication, and acoustic startle behavior.

neuroscience↗

bioRxiv2017

Heartfelt Face Perception via the Interoceptive Pathway - a MEG study

The somatic marker hypothesis proposes that the cortical representation of visceral signals is a crucial component of emotion processing. No previous study has investigated the causal relationships among brain regions that process visceral information during emotional perception. In this magnetoencephalography study of 32 healthy subjects, heartbeat-evoked potentials (HEPs), which reflect the cortical processing of heartbeats, were modulated by the perception of a sad face. The modulation effect was localized to the prefrontal cortices, the globus pallidus, and an interoceptive network including the right anterior insula (RAI) and anterior cingulate cortex (RACC). Importantly, our Granger causality analysis provides the first evidence for increased causal flow of heartbeat information from the RAI to the RACC during sad face perception. Moreover, this HEP modulation effect was neither an artefact nor an effect of visual-evoked potentials. These findings provide important progress in the understanding of brain-body interactions during emotion processing.

neuroscience↗

bioRxiv2017

Correlative three-dimensional fluorescence and refractive index tomography: bridging the gap between molecular specificity and quantitative bioimaging

Optical diffraction tomography (ODT) provides label-free three-dimensional (3D) refractive index (RI) measurement of biological samples. However, due to the nature of the RI values of biological specimens, ODT has limited access to molecular specific information. Here, we present an optical setup combining ODT with three-channel 3D fluorescence microscopy, to enhance the molecular specificity of the 3D RI measurement. The 3D RI distribution and 3D deconvoluted fluorescence images of HeLa cells and NIH-3T3 cells are measured, and the cross-correlative analysis between RI and fluorescence of live cells are presented.

cell biology↗

bioRxiv2017

Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines

HIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, these individuals have chronic inflammation associated with heightened risk of cardiovascular pathology. HIV-1 establishes proviruses in long-lived CD4+ memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Though the majority of proviruses that persist during antiviral therapy are defective for production of infectious virions, many are expressed, raising the possibility that the HIV-1provirus or its transcripts contribute to ongoing inflammation. Here we found that the HIV-1 provirus activated innate immune signaling in isolated dendritic cells, macrophages, and CD4+ T cells. Immune activation required transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. If rev was provided in trans, all HIV-1 coding sequences were dispensable for activation except those cis-acting sequences required for replication or splicing. These results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens.

microbiology↗

bioRxiv2017

Questioning unexpected CRISPR off-target mutations in vivo

To the Editor To the Editor References Recently, Schaefer et al.1 reported that whole genome sequencing (WGS) of two Cas9-treated, gene-corrected mice and a wild-type control mouse unveiled 1,397 single-nucleotide variations (SNVs) and 117 small insertions and deletions (indels) present commonly in the two Cas9-treated mice \"but absent in the uncorrected control\" and from a database of mouse SNVs and indels. There was essentially no sequence homology between the on-target site and these SNVs and indel sites, most of which lacked a protospacer-adjacent motif (PAM) sequence, suggesting that these variations were both small guide RNA (sgRNA)-independent and Cas9-independent, respectively. Nevertheless, the authors made a bold claim that these variations were caused by CRISPR-Cas9 without validating these unexpected off-target effects even at a single SNV or indel site by ...

genomics↗

bioRxiv2017

Visualization And Label-Free Quantification Of Microfluidic Mixing Using Quantitative Phase Imaging

Microfluidic mixing plays a key role in various fields, including biomedicine and chemical engineering. To date, although various approaches for imaging microfluidic mixing have been proposed, they provide only quantitative imaging capability and require for exogenous labeling agents. Quantitative phase imaging techniques, however, circumvent these problems and offer label-free quantitative information about concentration maps of microfluidic mixing. We present the quantitative phase imaging of microfluidic mixing in various types of PDMS microfluidic channels with different geometries; the feasibility of the present method was validated by comparing it with the results obtained by theoretical calculation based on Ficks law.

bioengineering↗

bioRxiv2017

The Toxoplasma centrocone houses cell cycle regulatory factors

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor, essential for chromosome replication 1 (ECR1), that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures. ECR1 was discovered by complementation of a temperature sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40{degrees}C similar to a ts-mutant carrying a defect in topoisomerase. ECR1 is a 52kDa protein containing divergent RING and TRAF-Sina like zinc-binding domains that is dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the centrocone compartment of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughters resolve from the mother, ECR1 is down regulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the CDK-related kinase, TgCrk5, which shares a similar cell cycle expression and localization during tachyzoite replication. Altogether, the results of this study suggest ECR1 may be a unique E3 ligase that regulates DNA licensing and other mitotic processes. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.\n\nIMPORTANCEParasites of the apicomplexan family are important causes of human disease including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole genome sequencing because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of well-studied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model. An example of this approach is described here with the discovery of a putative E3 ligase/protein kinase mechanism involved in regulating chromosome replication and mitotic processes of asexual stage parasites.

microbiology↗

bioRxiv2017

Label-free identification of non-activated lymphocytes using three-dimensional refractive index tomography and machine learning

Identification of lymphocyte cell types is crucial for understanding their pathophysiologic roles in human diseases. Current methods for discriminating lymphocyte cell types primarily relies on labelling techniques with magnetic beads or fluorescence agents, which take time and have costs for sample preparation and may also have a potential risk of altering cellular functions. Here, we present label-free identification of non-activated lymphocyte subtypes using refractive index tomography. From the measurements of three-dimensional refractive index maps of individual lymphocytes, the morphological and biochemical properties of the lymphocytes are quantitatively retrieved. Machine learning methods establish an optimized classification model using the retrieved quantitative characteristics of the lymphocytes to identify lymphocyte subtypes at the individual cell level. We show that our approach enables label-free identification of three lymphocyte cell types (B, CD4+ T, and CD8+ T lymphocytes) with high specificity and sensitivity. The present method will be a versatile tool for investigating the pathophysiological roles of lymphocytes in various diseases including cancers, autoimmune diseases, and virus infections.

immunology↗