Biology subjects

Shrestha, S.

Publications and source records attributed to Shrestha, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intra-molecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

biochemistry↗

bioRxiv2023

Caloric restriction promotes beta cell longevity and delays aging and senescence by enhancing cell identity and homeostasis mechanisms.

Caloric restriction (CR) extends organismal lifespan and health span by improving glucose homeostasis mechanisms. How CR affects organellar structure and function of pancreatic beta cells over the lifetime of the animal remains unknown. Here, we used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis link this transcriptional phenotype to transcription factors involved in beta cell identity (Mafa) and homeostasis (Atf6). Imaging metabolomics further demonstrates that CR beta cells are more energetically competent. In fact, high-resolution light and electron microscopy indicates that CR reduces beta cell mitophagy and increases mitochondria mass, increasing mitochondrial ATP generation. Finally, we show that long-term CR delays the onset of beta cell aging and senescence to promote longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cells during aging and diabetes.

cell biology↗

bioRxiv2023

Analyses of cell wall synthesis in Clostridioides difficile reveal a diversification in cell division mechanisms in endospore-forming bacteria

Current models of bacterial cell division assume that the core synthases of the multiprotein divisome complex, FtsW-FtsI, are the primary drivers of septal peptidoglycan (PG) synthesis. These enzymes are typically encoded in the highly conserved division and cell wall (dcw) cluster and are considered to be universally essential for cell division. Here, we combine bioinformatics analyses with functional characterization in the pathogen Clostridioides difficile to show that dcw-encoded PG synthases have undergone a surprising specialization in the sole endospore-forming phylum, Firmicutes, to fulfill sporulation-specific roles. We describe a novel role for these enzymes in synthesizing septal PG during the sporulation-specific mode of cell division in C. difficile. Although these enzymes are directly regulated by canonical divisome components during this process, dcw-encoded PG synthases and their divisome regulators are unexpectedly dispensable for cell division during normal growth. Instead, C. difficile uses its sole bifunctional class A penicillin-binding protein (aPBP) to drive cell division, revealing a previously unreported role for this class of PG synthases as the core divisome enzyme. Collectively, our findings reveal how the emergence of endosporulation in the Firmicutes phylum was a key driver for the functional repurposing of an otherwise universally conserved cellular process such as cell division. Moreover, they indicate that C. difficile, and likely other clostridia, assemble a divisome that differs markedly from previously studied bacteria, thus representing an attractive, unique target for therapeutic purposes.

microbiology↗

bioRxiv2023

The MODY-associated TALK-1 L114P mutation causes islet α-cell overactivity and β-cell inactivity resulting in transient neonatal diabetes and glucose dyshomeostasis in adults

The gain-of-function mutation in the TALK-1 K+ channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of {beta}-cell electrical activity and glucose-stimulated insulin secretion. The KCNK16 gene encoding TALK-1 is the most abundant and {beta}-cell-restricted K+ channel transcript. To investigate the impact of KCNK16 L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1(ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment. Kcnk16 L114P increased whole-cell {beta}-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated Kcnk16 L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/545631v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@19031e9org.highwire.dtl.DTLVardef@1bccc49org.highwire.dtl.DTLVardef@803e1org.highwire.dtl.DTLVardef@36c584_HPS_FORMAT_FIGEXP M_FIG Graphical Summary C_FIG

physiology↗

bioRxiv2023

The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and Endocytic Machinery

The septin cytoskeleton plays a key role in the morphogenesis of the yeast mating projection, forming structures at the base of the projection. The yeast mating response uses the G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. Desensitization of the G, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. We hypothesized that Gpa1 would utilize known septin regulators to control septin organization. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the endocytic adaptor proteins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive G. We hypothesized that hyperactive G might increase the rate of endocytosis of a pheromone-responsive cargo, thereby altering where septins are localized. Mathematical modeling predicted that changes in endocytosis could explain the septin organizations we find in WT and mutant cells. Our results show that Gpa1-induced disorganization of septins requires clathrin-mediated endocytosis. Both the GPCR and the G are known to be internalized by clathrin-mediated endocytosis during the pheromone response. Deletion of the GPCR C-terminus to block internalization partially rescued septin organization. However, deleting the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the G delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity.

cell biology↗

bioRxiv2023

Tyramine promotes colon cancer risk and development by inducing DNA damage and inflammation

High dietary consumption of processed meat is associated with increased colorectal cancer (CRC) risk, but mechanistic links remain largely unknown. Tyramine is a biogenic amine found in processed food and a gut bacterial product from tyrosine. However, the impact of tyramine on gut health has not been studied. We found that tyramine induced necrosis and promoted cell proliferation and DNA damage in HCT116 cells. Ingestion of tyramine increased colonic tumor size, intestinal cell proliferation and inflammation (e.g., increased mRNA expression of IL-17A and a higher number of Ly6G+ neutrophils) in ApcMin/+ mice. Furthermore, tyramine-treated wild-type mice exhibited visible adenomas and significantly enhanced intestinal tissue DNA damage, together with altered gene pathways involved in epithelial barrier function. In addition, natural killer cell numbers were lower and polymorphonuclear-myeloid derived suppressor cells were higher in tumors from tyramine-treated ApcMin/+ mice, suggesting a suppressive anti-tumor immune response. Thus, tyramine not only increases CRC risk, but also facilitates tumor development. Modulating the levels of tyramine in food and monitoring high-risk individuals could aid in better prognosis and management of CRC.

cancer biology↗

bioRxiv2023

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc via CRISPRi led to faster growth and uptake of glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only). IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatics making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatics present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

microbiology↗

bioRxiv2023

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate

Human induced pluripotent stem cell (iPSCs)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies (EBs) in an ultra-low attachment (ULA) 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95 - 100%. By differentiating the EBs on the pillar plate, we achieved robust generation of cerebral organoids with a coefficient of variation (CV) below 19%. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

bioengineering↗

bioRxiv2023

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases

Catalytic signaling outputs of protein kinases are dynamically regulated by an array of structural mechanisms, including allosteric interactions mediated by intrinsically disordered segments flanking the conserved catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of microtubule-associated proteins characterized by a flexible C-terminal autoregulatory tail segment that varies in length across the various human DCLK isoforms. However, the mechanism whereby these isoform-specific variations contribute to unique modes of autoregulation is not well understood. Here, we employ a combination of statistical sequence analysis, molecular dynamics simulations and in vitro mutational analysis to define hallmarks of DCLK family evolutionary divergence, including analysis of splice variants within the DCLK1 sub-family, which arise through alternative codon usage and serve to supercharge the inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs that readily distinguish DCLKs from all other Calcium Calmodulin Kinases (CAMKs), and a Swiss-army assembly of distinct motifs that tether the C-terminal tail to conserved ATP and substrate-binding regions of the catalytic domain to generate a scaffold for auto-regulation through C-tail dynamics. Consistently, deletions and mutations that alter C-terminal tail length or interfere with co-conserved interactions within the catalytic domain alter intrinsic protein stability, nucleotide/inhibitor-binding, and catalytic activity, suggesting isoform-specific regulation of activity through alternative splicing. Our studies provide a detailed framework for investigating kinome-wide regulation of catalytic output through cis-regulatory events mediated by intrinsically disordered segments, opening new avenues for the design of mechanistically-divergent DCLK1 modulators, stabilizers or degraders.

evolutionary biology↗

bioRxiv2023

A Pillar and Perfusion Plate Platform for Robust Human Organoid Culture and Analysis

Human organoids have potential to revolutionize in vitro disease modeling by providing multicellular architecture and function that are similar to those in vivo. This innovative and evolving technology, however, still suffers from assay throughput and reproducibility to enable high-throughput screening (HTS) of compounds due to cumbersome organoid differentiation processes and difficulty in scale-up and quality control. Using organoids for HTS is further challenged by lack of easy-to-use fluidic systems that are compatible with relatively large organoids. Here, we overcome these challenges by engineering "microarray three-dimensional (3D) bioprinting" technology and associated pillar and perfusion plates for human organoid culture and analysis. High-precision, high-throughput stem cell printing and encapsulation techniques were demonstrated on a pillar plate, which was coupled with a complementary deep well plate and a perfusion well plate for static and dynamic organoid culture. Bioprinted cells and spheroids in hydrogels were differentiated into liver and intestine organoids for in situ functional assays. The pillar/perfusion plates are compatible with standard 384-well plates and HTS equipment, and thus may be easily adopted in current drug discovery efforts.

bioengineering↗

bioRxiv2023

Simulating Rumen Conditions using an Anaerobic Dynamic Membrane Bioreactor to Enhance Hydrolysis of Lignocellulosic Biomass

An anaerobic dynamic membrane bioreactor (AnDMBR) was used to mimic rumen conditions to enhance the hydrolysis of lignocellulosic materials and the production of volatile fatty acids (VFA) when treating food waste. The AnDMBR was inoculated with cow rumen content and operated at a 0.5-day hydraulic retention time, 2 to 4-day solids retention time, a temperature of 39 {degrees}C, and a pH of 6.3, characteristics similar to those of a rumen. An operational strategy of repeating settle-permeate-mix cycles was utilized to simulate the regurgitation of ruminant animals. Removal rates of neutral detergent fiber (NDF) and acid detergent fiber (ADF) of 58.9 {+/-} 8.4 % and 69.0 {+/-} 8.6 %, respectively, and a VFA yield of 0.55 {+/-} 0.12 g VFA/g CODfed were observed when operating at an organic loading rate of 18 {+/-} 2 kg VS/m3/day. Sequencing results of both 16S rRNA gene and 16S rRNA revealed that a microbial community resistant to both disruption and reinoculation developed six weeks after inoculation. Up to 66.7 {+/-} 5.7 % of the active microbial populations (based on 16S rRNA sequence data) and 51.0 {+/-} 7.0 % of the total microbial populations (based on 16S rRNA gene sequence data) present in the rumen-mimicking AnDMBR originated from the rumen inoculum. This study offers a reliable strategy to leverage the unique features of a rumen; the rumen-mimicking AnDMBR achieved high hydrolysis and acidogenesis rates even when treating substrates drastically different from those fed to ruminants.

bioengineering↗

bioRxiv2023

A pillar/perfusion plate enhances cell growth, reproducibility, throughput, and user friendliness in dynamic 3D cell culture

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from the necrotic core due to limited nutrient and oxygen diffusion and waste removal and have limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids have been loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow rates were maintained within perfusion wells, and the pillar plate could be separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in dynamic 3D cell culture.

bioengineering↗

bioRxiv2022

DNA methylation at the suppressor of cytokine signaling 3 (SOCS3) gene influences height in childhood

Human height is strongly influenced by genetics but the contribution of modifiable epigenetic factors is under-explored, particularly in low and middle-income countries (LMIC). We investigated links between blood DNA methylation and child height in four LMIC cohorts (n=1927) and identified a robust association at three CpGs in the suppressor of cytokine signalling 3 (SOCS3) gene which replicated in a high-income country cohort (n=879). SOCS3 methylation (SOCS3m) - height associations were independent of genetic effects. Mendelian randomization analysis confirmed a causal effect of SOCS3m on height. In longitudinal analysis in a LMIC cohort, SOCS3m explained a maximum 9.5% of height variance in mid-childhood while the variance explained by height polygenic risk score increased from birth to 21 years (2% to 18%). Childrens SOCS3m was associated with prenatal maternal folate and socio-economic status. In-vitro characterization confirmed a regulatory effect of SOCS3m on gene expression. Our findings suggest that epigenetic modifications may play an important role in driving child height in LMIC.

genomics↗

bioRxiv2022

Single-spore germination analyses reveal that Clostridioides difficile uses parallel pathways to sense distinct classes of co-germinants during spore germination.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+ in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. ImportanceClostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

microbiology↗

bioRxiv2022

COMBINATION THERAPIES TARGETING ALK-ABERRANT NEUROBLASTOMA IN PRECLINICAL MODELS.

BackgroundALK activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1-2% of cases. Lorlatinib, a third generation ALK inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data has suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. AimsTo study the preclinical activity of ALK inhibitors alone and in combination with chemotherapy or idasanutlin. MethodsWe compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient derived xenografts (PDX). ResultsLorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with the highest ALK expression. In this PDX lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. ConclusionOur study suggests that in neuroblastoma, high ALK expression could be associated with response to lorlatinib and either chemotherapy or idasanutlin. The synergy between MDM2 inhibition and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma. STATEMENT OF TRANSLATIONAL RELEVANCENeuroblastoma is a pediatric tumor of the developing sympathetic nervous system. Around 50% of high-risk neuroblastoma patients are curable. Mutations or amplification of Anaplastic Lymphoma Kinase (ALK) have emerged as a marker with which to further risk-stratify patients. The ALK inhibitor lorlatinib will soon be used alongside chemotherapy in upfront treatment of high-risk patients with ALK-aberrant disease. In this preclinical study, we used a panel of ALK aberrant neuroblastoma models to evaluate ALK inhibitors focusing on lorlatinib in combination with conventional chemotherapy and the small molecule MDM2 inhibitor idasanutlin. In both approaches we found synergy in models with high basal ALK expression without MAPK pathway alterations. We conclude that in neuroblastoma the level of ALK expression could be an additional biomarker predictive of clinical response to ALK inhibitors.

cancer biology↗

bioRxiv2022

Molecular basis of the TRAP complex function in ER protein biogenesis

The Translocon Associated Protein (TRAP) complex resides in the endoplasmic reticulum (ER) membrane and interacts with the Sec translocon and the translating ribosome to facilitate biogenesis of secretory and membrane proteins1-4. TRAP is essential for the secretion of many hormones, and its key role in the production of the hormone peptide insulin has been particularly well established5,6. The mechanism by which TRAP engages ribosomes and the translocon to facilitate translocation of protein clients in the secretory pathway is not clear. Here, we reveal the molecular architecture of the mammalian TRAP complex and how it engages the translating ribosome associated with Sec61 translocon on the ER membrane. The TRAP complex is anchored to the ribosome via a long tether and its position relative to the ribosome and the translocon is further stabilized by a finger-like loop. This spatial arrangement positions a cradle-like lumenal domain of TRAP below the protein conducting pore of the translocon for interactions with translocated nascent chains. The biological importance of these key interactions is evident by structure-guided TRAP mutations in C. elegans that lead to growth deficits associated with increased ER stress and defects in insulin secretion. Our findings elucidate the molecular basis of the TRAP complex in the biogenesis and translocation of proteins at the ER.

molecular biology↗

bioRxiv2022

Evolutionary and cellular analysis of the dark pseudokinase PSKH2

Pseudokinases, so named because they lack one or more conserved canonical amino acids that define their catalytically-active relatives, have evolved a variety of biological functions in both prokaryotic and eukaryotic organisms. Human PSKH2 is closely related to the canonical kinase PSKH1, which maps to the CAMK family of protein kinases. Primates encode PSKH2 in the form of a pseudokinase, which is predicted to be catalytically inactive due to loss of the invariant catalytic Asp residue. Although the biological role(s) of vertebrate PSKH2s remains unclear, we previously identified species-level adaptions in PSKH2 that have led to the appearance of kinase or pseudokinase variants in vertebrate genomes alongside a canonical PSKH1 paralog. In this paper we confirm that, as predicted, PSKH2 lacks detectable protein phosphotransferase activity, and exploit structural informatics, biochemistry and cellular proteomics to begin to characterise vertebrate PSKH2 orthologues. AlphaFold 2-based structural analysis predicts functional roles for both the PSKH2 N- and C-regions that flank the pseudokinase domain core, and cellular truncation analysis confirms that the N-terminal domain, which contains a conserved myristoylation site, is required for both stable human PSKH2 expression and localisation to a membrane-rich subcellular fraction containing mitochondrial proteins. Using mass spectrometry-based proteomics, we confirm that human PSKH2 is part of a cellular mitochondrial protein network, and that its expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system. HSP90 interactions are mediated through binding to the PSKH2 C-terminal tail, leading us to predict that this region might act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signalling.

biochemistry↗

bioRxiv2022

Emerging Roles of Importin alpha/beta and EB1 in Differential Regulation of Kif18B Astral Microtubule Destabilization

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin /{beta} as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin /{beta} interaction disrupted Kif18B localization without affecting aster size. In vitro, importin /{beta} increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin /{beta} have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin /{beta}-spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran-regulation is important not only to control molecular motor function near chromatin but also provides a spatial control mechanism to modulate MT binding of NLS-containing spindle assembly factors.

cell biology↗