Ex vivo drug testing of patient-derived lung organoids to predict treatment responses for personalized medicine.

Lung cancer is the leading cause of global cancer-related mortality resulting in âˆ¼ 1.8 million deaths annually. Systemic, molecular targeted, and immune therapies have provided significant improvements of survival outcomes for patients. However, drug resistance usually arises and there is an urgent need for novel therapy screening and personalized medicine. 3D patient-derived organoid (PDO) models have emerged as a more effective and efficient alternative for ex vivo drug screening than 2D cell culture and patient-derived xenograft (PDX) models. In this review, we performed an extensive search of lung cancer PDO-based ex vivo drug screening studies. Lung cancer PDOs were successfully established from fresh or bio-banked sections and/or biopsies, pleural effusions and PDX mouse models. PDOs were subject to ex vivo drug screening with chemotherapy, targeted therapy and/or immunotherapy. PDOs consistently recapitulated the genomic alterations and drug sensitivity of primary tumors. Although sample sizes of the previous studies were limited and some technical challenges remain, PDOs showed great promise in the screening of novel therapy drugs. With the technical advances of high throughput, tumor-on-chip, and combined microenvironment, the drug screening process using PDOs will enhance precision care of lung cancer patients.

Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination.

While macrophage phagocytosis is an immune defense mechanism against invading cellular organisms, cancer cells expressing the CD47 ligand send forward signals to repel this engulfment. Here we report that the reverse signaling using CD47 as a receptor additionally enhances a pro-survival function of prostate cancer cells under phagocytic attack. Although low CD47-expressing cancer cells still allow phagocytosis, the reverse signaling delays the process, leading to incomplete digestion of the entrapped cells and subsequent tumor hybrid cell (THC) formation. Viable THCs acquire c-Myc from parental cancer cells to upregulate both M1- and M2-like macrophage polarization genes. Consequently, THCs imitating dual macrophage features can confound immunosurveillance, gaining survival advantage in the host. Furthermore, these cells intrinsically express low levels of androgen receptor and its targets, resembling an adenocarcinoma-immune subtype of metastatic castration-resistant prostate cancer. Therefore, phagocytosis-generated THCs may represent a potential target for treating the disease.

AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are integral to the development of complex tumor microenvironments (TMEs) and can execute disparate cellular programs in response to extracellular cues. However, upstream signaling processes underpinning this phenotypic plasticity remain to be elucidated. Here, we report that concordant AXL-STAT3 signaling in TAMs is triggered by lung cancer cells or cancer-associated fibroblasts in the cytokine milieu. This paracrine action drives TAM differentiation toward a tumor-promoting "M2-like" phenotype with upregulation of CD163 and putative mesenchymal markers, contributing to TAM heterogeneity and diverse cellular functions. One of the upregulated markers, CD44, mediated by AXL-IL-11-pSTAT3 signaling cascade, enhances macrophage ability to interact with endothelial cells and facilitate formation of primitive vascular networks. We also found that AXL-STAT3 inhibition can impede the recruitment of TAMs in a xenograft mouse model, thereby suppressing tumor growth. These findings suggest the potential application of AXL-STAT3-related markers to quantitatively assess metastatic potential and inform therapeutic strategies in lung cancer.

Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM Imaging.

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and ß-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and ß-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.

Soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence and brain microvascular dysfunction in a mouse model of tauopathy.

Vascular mechanisms of Alzheimer's disease (AD) may constitute a therapeutically addressable biological pathway underlying dementia. We previously demonstrated that soluble pathogenic forms of tau (tau oligomers) accumulate in brain microvasculature of AD and other tauopathies, including prominently in microvascular endothelial cells. Here we show that soluble pathogenic tau accumulates in brain microvascular endothelial cells of P301S(PS19) mice modeling tauopathy and drives AD-like brain microvascular deficits. Microvascular impairments in P301S(PS19) mice were partially negated by selective removal of pathogenic soluble tau aggregates from brain. We found that similar to trans-neuronal transmission of pathogenic forms of tau, soluble tau aggregates are internalized by brain microvascular endothelial cells in a heparin-sensitive manner and induce microtubule destabilization, block endothelial nitric oxide synthase (eNOS) activation, and potently induce endothelial cell senescence that was recapitulated in vivo in microvasculature of P301S(PS19) mice. Our studies suggest that soluble pathogenic tau aggregates mediate AD-like brain microvascular deficits in a mouse model of tauopathy, which may arise from endothelial cell senescence and eNOS dysfunction triggered by internalization of soluble tau aggregates.

A Role for the Proteasome Alpha2 Subunit N-Tail in Substrate Processing.

The proteolytic active sites of the 26S proteasome are sequestered within the catalytic chamber of its 20S core particle (CP). Access to this chamber is through a narrow channel defined by the seven outer α subunits. In the resting state, the N-termini of neighboring α subunits form a gate blocking access to the channel. The attachment of the activators or regulatory particles rearranges the blocking α subunit N-termini facilitating the entry of substrates. By truncating or mutating each of the participating α N-termini, we report that whereas only a few N-termini are important for maintaining the closed gate, all seven N-termini participate in the open gate. Specifically, the open state is stabilized by a hydrogen bond between an invariant tyrosine (Y) in each subunit with a conserved aspartate (D) in its counterclockwise neighbor. The lone exception is the α1-α2 pair leaving a gap in the ring circumference. The third residue (X) of this YD(X) motif aligns with the open channel. Phenylalanine at this position in the α2 subunit comes in direct contact with the translocating substrate. Consequently, deletion of the α2 N-terminal tail attenuates proteolysis despite the appearance of an open gate state. In summary, the interlacing N-terminal YD(X) motifs regulate both the gating and translocation of the substrate.

Genetic and pharmacologic proteasome augmentation ameliorates Alzheimer's-like pathology in mouse and fly APP overexpression models.

The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer's disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome's increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.

Contacts with Macrophages Promote an Aggressive Nanomechanical Phenotype of Circulating Tumor Cells in Prostate Cancer.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of "mechanical fitness" in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell-macrophage cocultures revealed that macrophages promoted epithelial-mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. SIGNIFICANCE: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

New Peptide-Based Pharmacophore Activates 20S Proteasome.

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the α1/α2 pocket of the core proteasome α ring as the binding site of TAT peptides. We postulate that the TATs' pharmacophore consists of an N-terminal basic pocket-docking "activation anchor" connected via a ß turn inducer to a C-terminal "specificity clamp" that binds on the proteasome α surface. By allosteric effects-including destabilization of the proteasomal gate-the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

Single-Cell Proteomic Profiling Identifies Combined AXL and JAK1 Inhibition as a Novel Therapeutic Strategy for Lung Cancer.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFß signaling and induced JAK1-STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1-STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFß, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFß, and JAK1-STAT3 signal activation in select tumors that may be targeted by combined AXL-JAK1 inhibition. SIGNIFICANCE: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

Inhibitory Interplay of SULT2B1b Sulfotransferase with AKR1C3 Aldo-keto Reductase in Prostate Cancer.

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3ß-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.

Pipecolic esters as minimized templates for proteasome inhibition.

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.

Small Molecule Modulation of Proteasome Assembly.

The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S-20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different proteasome complexes.

Targeting Protein-Protein Interactions in the Ubiquitin-Proteasome Pathway.

The ubiquitin-proteasome pathway (UPP) is a major venue for controlled intracellular protein degradation in Eukaryota. The machinery of several hundred proteins is involved in recognizing, tagging, transporting, and cleaving proteins, all in a highly regulated manner. Short-lived transcription factors, misfolded translation products, stress-damaged polypeptides, or worn-out long-lived proteins, all can be found among the substrates of UPP. Carefully choreographed protein-protein interactions (PPI) are involved in each step of the pathway. For many of the steps small-molecule inhibitors have been identified and often they directly or indirectly target PPI. The inhibitors may destabilize intracellular proteostasis and trigger apoptosis. So far this is the most explored option used as an anticancer strategy. Alternatively, substrate-specific polyubiquitination may be regulated for a precise intervention aimed at a particular metabolic pathway. This very attractive opportunity is moving close to clinical application. The best known drug target in UPP is the proteasome: the end point of the journey of a protein destined for degradation. The proteasome alone is a perfect object to study the mechanisms and roles of PPI on many levels. This giant protease is built from multisubunit modules and additionally utilizes a service from transient protein ligands, for example, delivering substrates. An elaborate set of PPI within the highest-order proteasome assembly is involved in substrate recognition and processing. Below we will outline PPI involved in the UPP and discuss the growing prospects for their utilization in pharmacological interventions.

TGF-ß signal rewiring sustains epithelial-mesenchymal transition of circulating tumor cells in prostate cancer xenograft hosts.

Activation of TGF-ß signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-ß signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-ß signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII-edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro. Therefore, combined targeting of TGF-ß and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α1-Proteinase Inhibitor.

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography.

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency.

Targeting Protein-Protein Interactions in the Proteasome Super-Assemblies.

Protein-protein interactions (PPI) are at the center of molecular mechanisms of life. The protein ligands convene for regulation of biological function: adding, enhancing or inhibiting activity, for assistance in structural integrity or to enable subsequent PPI. All these general roles of PPI are represented in the proteasome, the giant proteolytic factory universally present in human cells. The proteasome is a renowned target for anti-cancer drugs and a considered target for drugs curbing inflammation. The essential function of the proteasome, the degradation of a majority of intracellular proteins via the ubiquitin-proteasome pathway, relies on proper interactions between multiple subunits of the enzyme and between multiple modules forming distinct super-assemblies covered by the "proteasome" name. The interface regions between constitutive, alternative or transient protein components of the proteasome provide a rich platform for design of drugs with potentially very diverse actions. Still, the resource remains largely untapped since all proteasome-targeting drugs used so far in humans are classical competitive inhibitors blocking catalytic centers. In this review, we will discuss the opportunities and challenges of targeting PPI in the hub enzyme for intracellular protein catabolism, the proteasome.

A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition.

The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31years) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although Heat shock protein 72 kDa (HSP72) and Heat shock protein 40 kDa (Homolog of bacterial DNAJ1) (HSP40(Hdj1)) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging.

Dissecting a role of a charge and conformation of Tat2 peptide in allosteric regulation of 20S proteasome.

Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators.