Regulatory Sites Research Articles

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-ATPase (PSMC) Gene Family in the Light of Proteasome Inhibitor Resistance in Multiple Myeloma.

For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM.

Extracellular vesicles regulate the transmission of insulin resistance and redefine noncommunicable diseases.

Noncommunicable diseases (NCDs), such as diabetes and related neurological disorders, are considered to not be directly transmissible from one person to another. However, NCDs may be transmissible in vivo through extracellular vesicles (EVs). A long-term high-fat diet (HFD) can induce a series of health issues like hyperlipidemia, type 2 diabetes mellitus (T2DM), and diabetic peripheral neuropathy (DPN) due to insulin resistance. Multiple molecular signaling changes can stimulate insulin resistance, especially blocking insulin signaling by increased insulin resistance inducer (phosphorylation of negative regulatory sites of insulin receptor substrate (IRS) proteins) and decreased tyrosine phosphorylation of insulin receptor substrate (phosphorylation of positive regulatory sites of IRS), thus leading to reduced phosphorylation of AKT enzymes. Current efforts to treat T2DM and prevent its complications mainly focus on improving insulin sensitivity, enhancing insulin secretion, or supplementing exogenous insulin based on a common assumption that insulin resistance is noncommunicable. However, insulin resistance is transmissible within multiple tissues or organs throughout the body. Exploring the regulatory roles of EVs in developing insulin resistance may provide novel and effective preventive and therapeutic strategies.

Zinc controls operator affinity of human transcription factor YY1 by mediating dimerization via its N-terminal region

Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

New Insights Regarding Hemin Inhibition of the Purified Rat Brain 2-Oxoglutarate Carrier and Relationships with Mitochondrial Dysfunction.

A kinetic analysis of the transport assays on the purified rat brain 2-oxoglutarate/malate carrier (OGC) was performed starting from our recent results reporting about a competitive inhibitory behavior of hemin, a physiological porphyrin derivative, on the OGC reconstituted in an active form into proteoliposomes. The newly provided transport data and the elaboration of the kinetic equations show evidence that hemin exerts a mechanism of partially competitive inhibition, coupled with the formation of a ternary complex hemin-carrier substrate, when hemin targets the OGC from the matrix face. A possible interpretation of the provided kinetic analysis, which is supported by computational studies, could indicate the existence of a binding region responsible for the inhibition of the OGC and supposedly involved in the regulation of OGC activity. The proposed regulatory binding site is located on OGC mitochondrial matrix loops, where hemin could establish specific interactions with residues involved in the substrate recognition and/or conformational changes responsible for the translocation of mitochondrial carrier substrates. The regulatory binding site would be placed about 6 Å below the substrate binding site of the OGC, facing the mitochondrial matrix, and would allow the simultaneous binding of hemin and 2-oxoglutarate or malate to different regions of the carrier. Overall, the presented experimental and computational analyses help to shed light on the possible existence of the hemin-carrier substrate ternary complex, confirming the ability of the OGC to bind porphyrin derivatives, and in particular hemin, with possible consequences for the mitochondrial redox state mediated by the malate/aspartate shuttle led by the mitochondrial carriers OGC and AGC.

Evaluation of high-resolution predictions of fine particulate matter and its composition in an urban area using PMCAMx-v2.0

Abstract. Accurately predicting urban PM2.5 concentrations and composition has proved challenging in the past, partially due to the resolution limitations of computationally intensive chemical transport models (CTMs). Increasing the resolution of PM2.5 predictions is desired to support emissions control policy development and address issues related to environmental justice. A nested grid approach using the CTM PMCAMx-v2.0 was used to predict PM2.5 at increasing resolutions of 36 km × 36 km, 12 km × 12 km, 4 km × 4 km, and 1 km × 1 km for a domain largely consisting of Allegheny County and the city of Pittsburgh in southwestern Pennsylvania, US, during February and July 2017. Performance of the model in reproducing PM2.5 concentrations and composition was evaluated at the finest scale using measurements from regulatory sites as well as a network of low-cost monitors. Novel surrogates were developed to allocate emissions from cooking and on-road traffic sources to the 1 km × 1 km resolution grid. Total PM2.5 mass is reproduced well by the model during the winter period with low fractional error (0.3) and fractional bias (+0.05) when compared to regulatory measurements. Comparison with speciated measurements during this period identified small underpredictions of PM2.5 sulfate, elemental carbon (EC), and organic aerosol (OA) offset by a larger overprediction of PM2.5 nitrate. In the summer period, total PM2.5 mass is underpredicted due to a large underprediction of OA (bias = −1.9 µg m−3, fractional bias = −0.41). In the winter period, the model performs well in reproducing the variability between urban measurements and rural measurements of local pollutants such as EC and OA. This effect is less consistent in the summer period due to a larger fraction of long-range-transported OA. Comparison with total PM2.5 concentration measurements from low-cost sensors showed improvements in performance with increasing resolution. Inconsistencies in PM2.5 nitrate predictions in both periods are believed to be due to errors in partitioning between PM2.5 and PM10 modes and motivate improvements to the treatment of dust particles within the model. The underprediction of summer OA would likely be improved by updates to biogenic secondary organic aerosol (SOA) chemistry within the model, which would result in an increase of long-range transport SOA seen in the inner modeling domain. These improvements are obvious topics for future work towards model improvement. Comparison with regulatory monitors showed that increasing resolution from 36 to 1 km improved both fractional error and fractional bias in both modeling periods. Improvements at all types of measurement locations indicated an improved ability of the model to reproduce urban–rural PM2.5 gradients at higher resolutions.

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting.

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

Small-angle x-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model

In this work, we used small-angle x-ray and neutron scattering to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa transcriptional regulator MexR, a member of the multiple antibiotics resistance regulator (MarR) family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterization of both free and DNA-bound MarR proteins. Here, we show how combined neutron and x-ray small-angle scattering of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.

Mutant Allele of CD44 (rs8193C>T) and Pum2 Regulatory Element as A Prognosis Factor of Prostate Neoplasms: A Case-Control and In Silico Studies.

Expression of CD44 variant 6 (CD44v6) as a homing-associated cell adhesion molecule (HCAM), has proved to change most cancer cells. Aim of the study is the effect of mutant allele of CD44 (rs8193C>T) and Pum2 regulatory element as a prognosis factor of prostate neoplasms: a case-control and in silico studies in the Mazandaran province-Iran. In a case-control study, CD44-rs8193C>T genotyping of the 420 prostate neoplasms (210 benign prostatic hyperplasia (BPH) patients and 210 prostate cancer patients) and 150 healthy samples are performed by the touchdown polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The T mutant allele effects on the mRNA structure and cell pathways were also investigated in silico methods. Our results showed that the increase of T mutant allele frequency was significantly associated with BPH compared with prostate cancer. Furthermore, results showed TT genotype was significantly associated with BPH [odds ratio (OR)=0.572 and P=0.015], and also influenced the CD44v6 transcript secondary structure, miRNA binding, and regulatory element-binding site for Pum2 protein. Attachment of Pum2 to standard CD44 transcript may lead to transcript isoform-switching and shift-expression to a variety of CD44 isoforms, which can trigger some of the cell signaling pathways, such as Nanog-Stat, PKC-Nanog, and PKC-Twist. Based on this, the presence of the T mutant allele of CD44 (rs8193C>T) in the populations may create a regulatory element-binding site for Pum2. So, it could be known as a prognosis factor and prediction of prostate neoplasms. However, more comprehensive studies in different populations (with various ethnicities and large population sizes), and also CD44v6 gene expression studies in protein and transcript levels are required to confirm our data.

Calcium regulates acid-sensing ion channel 3 activation by competing with protons in the channel pore and at an allosteric binding site.

The extracellular Ca2+ concentration changes locally under certain physiological and pathological conditions. Such variations affect the function of ion channels of the nervous system and consequently also neuronal signalling. We investigated here the mechanisms by which Ca2+ controls the activity of acid-sensing ion channel (ASIC) 3. ASICs are neuronal, H+-gated Na+ channels involved in several physiological and pathological processes, including the expression of fear, learning, pain sensation and neurodegeneration after ischaemic stroke. It was previously shown that Ca2+ negatively modulates the ASIC pH dependence. While protons are default activators of ASIC3, this channel can also be activated at pH7.4 by the removal of the extracellular Ca2+. Two previous studies concluded that low pH opens ASIC3 by displacing Ca2+ ions that block the channel pore at physiological pH. We show here that an acidic residue, distant from the pore, together with pore residues, controls the modulation of ASIC3 by Ca2+. Our study identifies a new regulatory site in ASIC3 and demonstrates that ASIC3 activation involves an allosteric mechanism together with Ca2+ unbinding from the channel pore. We provide a molecular analysis of a regulatory mechanism found in many ion channels.

The Mitochondrial Protein MitoNEET as a Probe for the Allostery of Glutamate Dehydrogenase

The proteins glutamate dehydrogenase (GDH) and mitoNEET are both targets of drug development efforts to treat metabolic disorders, cancer, and neurodegenerative diseases. However, these two proteins differ starkly in the current knowledge about ligand binding sites. MitoNEET is a [2Fe-2S]-containing protein with no obvious binding site for small ligands observed in its crystal structures. In contrast, GDH is known to have a variety of ligands at multiple allosteric sites thereby leading to complex regulation in activity. In fact, while GDH can utilize either NAD(H) or NADP(H) for catalysis at the active site, only NAD(H) binds at a regulatory site to inhibit GDH activity. Previously, we found that mitoNEET forms a covalent bond with GDH in vitro and increases the catalytic activity of the enzyme. In this study we evaluated the effects of mitoNEET binding on the allosteric control of GDH conferred by inhibitors. We examined all effectors using NAD or NADP as the coenzyme to determine allosteric linkage by the NAD-binding regulatory site. We found that GDH activity, in the presence of the inhibitory palmitoyl-CoA and EGCG, can be rescued by mitoNEET, regardless of the coenzyme used. This suggests that mitoNEET rescues GDH by stabilizing the open conformation.

Oil source regulates intestinal health of hybrid grouper (♀ Epinephelus fuscoguttatus × ♂ E. lanceolatu): Structure, inflammation and microorganisms homeostasis

The price and yield of fish oil limits the development of aquaculture, plant oils as an alternative to fish oil can ensure the sustainable development of the aquaculture industry, but a series of negative effects caused by plant oils limit its application. To overcome these negative factors, it is important to further explore the regulatory sites and mechanisms of plant oils, particularly the intestinal tract as the digestive and absorptive site and one of the immune organs, and therefore the aim of the present study is to investigate the regulation of grouper intestinal structure, immune status and bacteriological homeostasis by different oil sources. Supplying 7 diets with fish oil, corn oil, sunflower oil, tea oil, olive oil, rice oil and mixed oil (above six equivalents were mixed) as lipid sources to juvenile hybrid grouper for 8 weeks, the intestinal morphological structure, intestinal inflammation and antioxidant-related factors and microbial composition and functional predictions were analyzed. The results showed that plant oils caused impaired intestinal structural integrity and inflammatory responses in grouper, as well as imbalance in intestinal microbial homeostasis, especially in the sunflower oil group, followed by corn oil. On the contrary, olive oil protected the intestinal structural integrity from damage, upregulated the expression of anti-inflammatory factors and antioxidant genes, and enhanced the relative abundance of intestinal probiotic bacteria, which had a protective effect on intestinal health. Above results implied that the structural damage and inflammatory response of intestine caused by most plant oils during fish oil replacement may be related to bacterial imbalance, while olive oil may have a protective effect on intestinal health and structure by increasing the relative abundance of intestinal probiotics, with potential for further in-depth exploration.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS‐CoV‐2 Papain‐Like Protease**

AbstractThe coronavirus papain‐like protease (PLpro) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS‐CoV‐2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS‐CoV‐2 PLpro and provid a novel site for allosteric inhibitors design.

Delivery of Active AKT1 to Human Cells

Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Thr308, Ser473), yet cell stimulation also activates many other kinases. To produce cells with specific AKT1 activity, we developed a novel system to deliver active AKT1 to human cells. We recently established a method to produce AKT1 phospho-variants from Escherichia coli with programmed phosphorylation. Here, we fused AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) protein. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308 induced selective phosphorylation of the known AKT1 substrate GSK-3α, but not GSK-3β, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Ser240/244. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on AKT1 activity.

Mechanism of KIT gene regulation by GATA1 lacking the N-terminal domain in Down syndrome–related myeloid disorders

Children with Down syndrome (DS) are at high risk of transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). GATA1 mutations are detected in almost all TAM and ML-DS samples, with exclusive expression of short GATA1 protein (GATA1s) lacking the N-terminal domain (NTD). However, it remains to be clarified how GATA1s is involved with both disorders. Here, we established the K562 GATA1s (K562-G1s) clones expressing only GATA1s by CRISPR/Cas9 genome editing. The K562-G1s clones expressed KIT at significantly higher levels compared to the wild type of K562 (K562-WT). Chromatin immunoprecipitation studies identified the GATA1-bound regulatory sites upstream of KIT in K562-WT, K562-G1s clones and two ML-DS cell lines; KPAM1 and CMK11-5. Sonication-based chromosome conformation capture (3C) assay demonstrated that in K562-WT, the − 87 kb enhancer region of KIT was proximal to the − 115 kb, − 109 kb and + 1 kb region, while in a K562-G1s clone, CMK11-5 and primary TAM cells, the − 87 kb region was more proximal to the KIT transcriptional start site. These results suggest that the NTD of GATA1 is essential for proper genomic conformation and regulation of KIT gene expression, and that perturbation of this function might be involved in the pathogenesis of TAM and ML-DS.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS-CoV-2 Papain-Like Protease.

The coronavirus papain-like protease (PLpro ) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS-CoV-2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS-CoV-2 PLpro and provid a novel site for allosteric inhibitors design.

Regulation of light energy conversion between linear and cyclic electron flow within photosystem II controlled by the plastoquinone/quinol redox poise.

Ultrapurified Photosystem II complexes crystalize as uniform microcrystals (PSIIX) of unprecedented homogeneity that allow observation of details previously unachievable, including the longest sustained oscillations of flash-induced O2 yield over > 200 flashes and a novel period-4.7 water oxidation cycle. We provide new evidence for a molecular-based mechanism for PSII-cyclic electron flow that accounts for switching from linear to cyclic electron flow within PSII as the downstream PQ/PQH2 pool reduces in response to metabolic needs and environmental input. The model is supported by flash oximetry of PSIIX as the LEF/CEF switch occurs, Fourier analysis of O2 flash yields, and Joliot-Kok modeling. The LEF/CEF switch rebalances the ratio of reductant energy (PQH2) to proton gradient energy (H+o/H+i) created by PSII photochemistry. Central to this model is the requirement for a regulatory site (QC) with two redox states in equilibrium with the dissociable secondary electron carrier site QB. Both sites are controlled by electrons and protons. Our evidence fits historical LEF models wherein light-driven water oxidation delivers electrons (from QA-) and stromal protons through QB to generate plastoquinol, the terminal product of PSII-LEF in vivo. The new insight is the essential regulatory role of QC. This site senses both the proton gradient (H+o/H+i) and the PQ pool redox poise via e-/H+ equilibration with QB. This information directs switching to CEF upon population of the protonated semiquinone in the Qc site (Q-H+)C, while the WOC is in the reducible S2 or S3 states. Subsequent photochemical primary charge separation (P+QA-) forms no (QH2)B, but instead undergoes two-electron backward transition in which the QC protons are pumped into the lumen, while the electrons return to the WOC forming (S1/S2). PSII-CEF enables production of additional ATP needed to power cellular processes including the terminal carboxylation reaction and in some cases PSI-dependent CEF.

Exploring the mono-/bistability range of positively autoregulated signaling systems in the presence of competing transcription factor binding sites.

Binding of transcription factor (TF) proteins to regulatory DNA sites is key to accurate control of gene expression in response to environmental stimuli. Theoretical modeling of transcription regulation is often focused on a limited set of genes of interest, while binding of the TF to other genomic sites is seldom considered. The total number of TF binding sites (TFBSs) affects the availability of TF protein molecules and sequestration of a TF by TFBSs can promote bistability. For many signaling systems where a graded response is desirable for continuous control over the input range, biochemical parameters of the regulatory proteins need be tuned to avoid bistability. Here we analyze the mono-/bistable parameter range for positively autoregulated two-component systems (TCSs) in the presence of different numbers of competing TFBSs. TCS signaling, one of the major bacterial signaling strategies, couples signal perception with output responses via protein phosphorylation. For bistability, competition for TF proteins by TFBSs lowers the requirement for high fold change of the autoregulated transcription but demands high phosphorylation activities of TCS proteins. We show that bistability can be avoided with a low phosphorylation capacity of TCSs, a high TF affinity for the autoregulated promoter or a low fold change in signaling protein levels upon induction. These may represent general design rules for TCSs to ensure uniform graded responses. Examining the mono-/bistability parameter range allows qualitative prediction of steady-state responses, which are experimentally validated in the E. coli CusRS system.

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-Atpase (PSMC) Gene Family Transmit Proteasome Inhibitor Resistance in Multiple Myeloma By Impacting the ADP/ATP Binding Pocket

Background For the treatment of Multiple Myeloma (MM), proteasome inhibitors (PIs) are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients e.g., mutations in the PI binding domain PSMB5. However, the proteasome is a large and complex machinery and its mechanistic understanding remains incomplete. In this study, we unravel the role of the six ATPases of the 19S proteasome regulator (PSMC1-6) in MM resistance. Methods As an example of cancer evolution and the acquisition of PI resistance, we conducted an in-depth analysis of an index patient applying deep sequencing of immunoglobulin rearrangements (IG), FISH, and WES in serial samples at MGUS, newly diagnosed MM, and five later timepoints including two relapses. Then we expand towards generic meta-analyses of PSMC alterations in our own and other published datasets comprising 1,499 newly diagnosed (NDMM) and 447 PI pretreated MM (PMM) patients for WGS/WES. For RNA sequencing, we analyzed 16 normal plasma cells (NPCs) and 360 NDMM patients. Furthermore, we explore the structural impact of PSMC single nucleotide variants on the proteasome and their relevance for PI resistance using 3D modeling with Chimera and Pymol. Results The WES analysis of our index patient uncovers a PSMC2 Y429S mutation acquired at the second relapse after intensive bortezomib-containing therapy. We functionally confirm this variant to mediate PI resistance in the cell line RPMI 8226. In the meta-analysis, a total of 36 mutations were identified (mutation incidence: 1.85%). The PSMC5 G152S mutation was found in three independent patients, and a G152R in a fourth patient, thus, representing a potential hotspot. Based on 3D structural projections, the identified mutations can be clustered into three main groups: first, mutations affecting the intrinsic conformation of the respective subunit; second, mutations perturbing the PSMC protomer interactions and the proper folding/functioning of the whole complex; and third, mutations impairing the ADP/ATP binding pocket (Figure). Nine mutations belong to the third category interacting with the adenine ring or the phosphate groups of the ATP. The expression analysis reveals a significant upregulation of PSMC3 (P=0.003), PSMC4 (P≤0.001), and PSMC5 (P=0.043) from NPC to NDMM. Conclusions The 19S hexameric AAA ATPase ring of the 26S proteasome is responsible for unfolding and introducing the substrates into the catalytic 20S complex. PSMC gene alterations are structurally often located in regulatory sites for activity e.g. impacting the ADP/ATP binding pocket. The following activation of the 26S proteasome increases the PI tolerance and represents a new PI resistance mechanism. Moreover, changes in the expression level of the PSMC members seem to play a role not only in the development of PI resistance but already in the development of MM, probably by increasing the protein turnover capacity of myeloma cells. This study opens the way to explore new classes of proteasome inhibitors avoiding this kind of mechanism of resistance. Figure:Mutations in the'PSMC' subunits of the 19S Base AAA-ATPase hexamer accumulate in proximity to the ATP/ADP binding pockets. The backbone of the PSMC hexamer is displayed as a ribbon diagram, mutations (red) and ADP (blue) as 3D spheres (PDB 5GJR). The mutations cluster in areas responsible for proper folding and functioning of the complex or in proximity to the ADP/ATP binding pockets, exemplarily shown for PSMC1, PSMC2, and PSMC5. ADP is presented as a wireframe stick in light green (PDB 5GJQ). The PSMC2Y429S mutation of the index patient is indicated in yellow. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RNA-binding proteins of KHDRBS and IGF2BP families control the oncogenic activity of MLL-AF4

Chromosomal translocation generates the MLL-AF4 fusion gene, which causes acute leukemia of multiple lineages. MLL-AF4 is a strong oncogenic driver that induces leukemia without additional mutations and is the most common cause of pediatric leukemia. However, establishment of a murine disease model via retroviral transduction has been difficult owning to a lack of understanding of its regulatory mechanisms. Here, we show that MLL-AF4 protein is post-transcriptionally regulated by RNA-binding proteins, including those of KHDRBS and IGF2BP families. MLL-AF4 translation is inhibited by ribosomal stalling, which occurs at regulatory sites containing AU-rich sequences recognized by KHDRBSs. Synonymous mutations disrupting the association of KHDRBSs result in proper translation of MLL-AF4 and leukemic transformation. Consequently, the synonymous MLL-AF4 mutant induces leukemia in vivo. Our results reveal that post-transcriptional regulation critically controls the oncogenic activity of MLL-AF4; these findings might be valuable in developing novel therapies via modulation of the activity of RNA-binding proteins.

Phosphorylation of S11 in PHR1 negatively controls its transcriptional activity.

Plant responses to phosphate starvation (-Pi) are very well characterized at the biochemical and molecular levels. The expression of thousands of genes is modified under this stress condition, depending on the action of Phosphate starvation response 1 (PHR1). Existing data indicate that neither the PHR1 transcript nor the quantity or localization of its protein increase during nutrient stress, raising the question of how its activity is regulated. Here, we present data showing that SnRK1 kinase is able to phosphorylate some phosphate starvation response proteins (PSRs), including PHR1. Based on a model of the three-dimensional structure of the catalytic subunit SnRK1α1, docking simulations predicted the binding modes of peptides from PHT1;8, PHO1 and PHR1 with SnRK1. PHR1 recombinant protein interacted in vitro with the catalytic subunits SnRK1α1 and SnRK1α2. A BiFC assay corroborated the in vivo interaction between PHR1 and SnRK1α1 in the cytoplasm and nucleus. Analysis of phosphorylated residues suggested the presence of one phosphorylated site containing the SnRK1 motif at S11, and mutation in this residue disrupted the incorporation of 32 P, suggesting that it is a major phosphorylation site. Electrophoretic mobility shift assay results indicated that the binding of PHR1 to P1BS motifs was not influenced by phosphorylation. Importantly, transient expression assays in Arabidopsis protoplasts showed a decrease in PHR1 activity in contrast with the S11A mutant, suggesting a role for Ser11 as a negative regulatory phosphorylation site. Taken together, these findings suggest that phosphorylation of PHR1 at Ser11 is a mechanism to control the PHR1-mediated adaptive response to -Pi.