Proteases and their inhibitors as prognostic factors for high-grade serous ovarian cancer

Ovarian carcinoma is one of the most lethal malignancies, but only very few prognostic biomarkers are known. The degradome, comprising proteases, protease non-proteolytic homologues and inhibitors, have been involved in the prognosis of many cancer types, including ovarian carcinoma. The prognostic significance of the whole degradome family has not been specifically studied in high-grade serous ovarian cancer. A targeted DNA microarray known as the CLIP-CHIP microarray was used to identify potential prognostic factors in ten high-grade serous ovarian cancer women who had early recurrence (<1.6 years) or late/no recurrence after first line surgery and chemotherapy. In women with early recurrence, we identified seven upregulated genes (TMPRSS4, MASP1/3, SPC18, PSMB1, IGFBP2, CFI – encoding Complement Factor I – and MMP9) and one down-regulated gene (ADAM-10). Using immunohistochemistry, we evaluated the prognostic effect of these 8 candidate genes in an independent cohort of 112 high-grade serous ovarian cancer women. Outcomes were progression, defined according to CA-125 criteria, and death. Multivariate Cox proportional hazard regression models were done to estimate the associations between each protein and each outcome. High ADAM-10 expression (intensity of 2–3) was associated with a lower risk of progression (adjusted hazard ratio (HR): 0.51; 95% confidence interval (CI): 0.29-0.87). High complement factor I expression (intensity 2–3) was associated with a higher risk of progression (adjusted HR: 2.30, 95% CI: 1.17–4.53) and death (adjusted HR: 3.42; 95% CI: 1.72–6.79). Overall, we identified the prognostic value of two proteases, ADAM-10 and complement factor I, for high-grade serous ovarian cancer which could have clinical significance.     

Proteasome inhibitors and Smac mimetics cooperate to induce cell death in diffuse large B-cell lymphoma by stabilizing NOXA and triggering mitochondrial apoptosis

Copy number gains and increased expression levels of cellular Inhibitor of Apoptosis protein (cIAP)1 and cIAP2 have been identified in primary diffuse large B-cell lymphoma (DLBCL) tissues. Second mitochondria-derived activator of caspases (Smac) mimetics were designed to antagonize IAP proteins. However, since their effect as single agents is limited, combination treatment represents a strategy for their clinical development. Therefore, we investigated the Smac mimetic BV6 in combination with proteasome inhibitors and analyzed the molecular mechanisms of action. We discovered that BV6 treatment sensitizes DLBCL cells to proteasome inhibition. We show a synergistic decrease in cell viability and induction of apoptosis by BV6/Carfilzomib (CFZ) treatment, which was confirmed by calculation of combination index (CI) and Bliss score. BV6 and CFZ acted together to trigger activation of BAX and BAK, which facilitated cell death, as knockdown of BAX and BAK significantly reduced BV6/CFZ-mediated cell death. Activation of BAX and BAK was accompanied by loss of mitochondrial membrane potential (MMP) and activation of caspases. Pretreatment with the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) rescued BV6/CFZ-induced cell death, confirming caspase dependency. Treatment with CFZ alone or in combination with BV6 caused accumulation of NOXA, which was required for cell death, as gene silencing by siRNA or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated NOXA inactivation inhibited BV6/CFZ-induced cell death. Together, these experiments indicate that BV6 and CFZ cooperatively induce apoptotic cell death via the mitochondrial pathway. These findings emphasize the role of Smac mimetics for sensitizing DLBCL cells to proteasome inhibition with important implications for further (pre)clinical studies.     

Efficacy of the HSP70 inhibitor PET-16 in multiple myeloma

Multiple myeloma (MM) is a common and largely incurable blood cancer for which new treatment options are needed, as resistance to current modalities is an issue. Additionally, because this tumor type often resides in a hypoxic niche of the bone marrow, new therapeutics that remain effective even under hypoxic conditions are sought. Because of the secretory nature of MM cells they are uniquely under proteotoxic stress, and we hypothesized that these tumor cells may alleviate this stress by upregulating the major stress-induced cytosolic form of the chaperone HSP70. In this work we test the efficacy of the HSP70 inhibitor PET-16 for MM. We show that MM cell lines express significant levels of HSP70, and further that inhibition of HSP70 causes decreased viability and apoptosis, along with proteotoxic stress, as assessed by the accumulation of poly-ubiquitylated proteins. Importantly, we show that growth of these tumor cells under hypoxic conditions has no effect on the ability of PET-16 to be cytotoxic. The HSP70 inhibitor PET-16 should thus be considered further for pre-clinical analyses of efficacy in MM.     

Exogenous Hsp70 delays senescence and improves cognitive function in aging mice

Molecular chaperone Heat Shock Protein 70 (Hsp70) plays an important protective role in various neurodegenerative disorders often associated with aging, but its activity and availability in neuronal tissue decrease with age. Here we explored the effects of intranasal administration of exogenous recombinant human Hsp70 (eHsp70) on lifespan and neurological parameters in middle-aged and old mice. Long-term administration of eHsp70 significantly enhanced the lifespan of animals of different age groups. Behavioral assessment after 5 and 9 mo of chronic eHsp70 administration demonstrated improved learning and memory in old mice. Likewise, the investigation of locomotor and exploratory activities after eHsp70 treatment demonstrated a significant therapeutic effect of this chaperone. Measurements of synaptophysin show that eHsp70 treatment in old mice resulted in larger synaptophysin-immunopositive areas and higher neuron density compared with control animals. Furthermore, eHsp70 treatment decreased accumulation of lipofuscin, an aging-related marker, in the brain and enhanced proteasome activity. The potential of eHsp70 intranasal treatment to protect synaptic machinery in old animals offers a unique pharmacological approach for various neurodegenerative disorders associated with human aging.Heat shock proteins (HSPs) serve to maintain intracellular protein homeostasis and have been shown to prevent protein damage during aging in different animal models (1). HSPs are required for longevity (2, 3), and a number of studies suggest that longer-lived species have higher constitutive expression of HSPs (4–7). Consistent with this finding, overexpression of HSP genes increased longevity in Drosophila, Caenorhabditis elegans, and vertebrates (1, 8, 9). Hsp70 is the major cytoprotective molecular chaperone with many different functions in the cell (10–12). Observations suggest that genetic variants of the Hsp70 family contribute to longevity in a wide range of organisms (9, 13, 14). Its defensive role in multiple neurodegenerative disorders (15, 16) can be explained by the multifaceted action of this protein. Indeed, the induction of Hsp70 has been shown to diminish oxidative stress damage (17, 18), suppress apoptosis (19), support proteasomal and lysosomal functioning (20), suppress toxic protein aggregation such as Aβ (21), inhibit proinflammatory signaling (22), and increase survival of endogenous neural progenitor cells (21). Notwithstanding Hsp70’s importance, its chaperone activity, as well as the rate of its synthesis and induction in response to stimuli, decreases in neurons with age (3, 6, 21, 22), suggesting that a pharmacological approach aiming to recover this chaperone in the aging brain may counter neurodegeneration.To our knowledge, the effect of exogenous HSPs on longevity has not yet been investigated. We previously showed that intranasally injected Hsp70 rapidly entered the brain of wild-type mice and was transported within neurons (23, 24). Furthermore, chronic Hsp70 treatment ameliorated multiple behavioral and molecular disturbances in two models of Alzheimer’s disease (AD)-type neurodegeneration (23). In this study, we explored the geroprotection potential of recombinant exogenous Hsp70 (eHsp70) in healthy mice. For all of the described experiments, we used highly pure LPS-free human eHsp70 (25), which rules out a possibility of confounding inflammatory responses associated with contaminated Hsp70. Our results demonstrate that long-term intranasal administration of human eHsp70 improves longevity and ameliorates aging-related behavioral deficits and molecular alterations to synaptic structure in the brains of aging mice.     

Differential global structural changes in the core particle of yeast and mouse proteasome induced by ligand binding

Two clusters of configurations of the main proteolytic subunit β5 were identified by principal component analysis of crystal structures of the yeast proteasome core particle (yCP). The apo-cluster encompasses unliganded species and complexes with nonpeptidic ligands, and the pep-cluster comprises complexes with peptidic ligands. The murine constitutive CP structures conform to the yeast system, with the apo-form settled in the apo-cluster and the PR-957 (a peptidic ligand) complex in the pep-cluster. In striking contrast, the murine immune CP classifies into the pep-cluster in both the apo and the PR-957–liganded species. The two clusters differ essentially by multiple small structural changes and a domain motion enabling enclosure of the peptidic ligand and formation of specific hydrogen bonds in the pep-cluster. The immune CP species is in optimal peptide binding configuration also in its apo form. This favors productive ligand binding and may help to explain the generally increased functional activity of the immunoproteasome. Molecular dynamics simulations of the representative murine species are consistent with the experimentally observed configurations. A comparison of all 28 subunits of the unliganded species with the peptidic liganded forms demonstrates a greatly enhanced plasticity of β5 and suggests specific signaling pathways to other subunits.Among the many factors involved in protein degradation through the ubiquitin-proteasome pathway, the core particle (CP) 20S proteasome plays the key role of the protease component. With the regulatory particle (RP), it forms a complex that selectively degrades ubiquitin-protein conjugates (1, 2). The CP in eukaryotes is a multisubunit complex composed of four stacked heptameric rings: two identical outer rings formed by seven different α subunits and two identical inner rings formed by seven different β subunits. The α_1–7β_1–7β_1–7α_1–7 organization defines a cylindrical structure (3). The α-rings control substrate entry into the lumen of the particle, where it is processed at the peptidolytic active centers, which are located at the inner walls of the β rings, specifically at subunits β1, β2, and β5. These active subunits are characterized by an N-terminal Thr residue. The other four β subunits have unprocessed N-terminal propeptides and are enzymatically inactive.All three active subunits share a common peptide hydrolyzing mechanism with two main steps (4): (i) the positioning of the substrate peptide in the active site by antiparallel alignment in between segments 47–49 and 21 of the active β subunits and (ii) peptide bond cleavage initiated by a nucleophilic attack of the hydroxyl group of the N-terminal Thr1 on the carbonyl carbon atom of the scissile peptide. Sequence diversity among β subunits endows them with distinctive structural features and different specificity pockets (S1, S2, S3, etc.) where the substrate side chains (P1, P2, P3, etc.) are bound (5). Consequently, the correlation of structural features of the S1 pockets with the distinctive cleavage products has led to the association of β1, β2, and β5 with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively (6).The catalytically active subunits are substituted in immune cells of vertebrate organisms by the immune β-subunits β1i, β2i, and β5i as part of an adaptive immune response. These substitutions cause substantial functional differences between the constitutive (cCP) and immuno (iCP) species, reflected in higher yield of peptides that are recognized by the major histocompatibility complex (MHC) class I generated by iCP (7). Additionally, it has been observed that iCP achieves higher degradation rates than cCP, in both in vitro and cellular assays (8–13).Some sequence variations between the constitutive and immune subunits provide explanations to the observed catalytic differences. Most conspicuously, and first seen in the eukaryotic proteasome crystal structure from yeast (yCP) (3) and confirmed by the murine constitutive and immune CP structures (mcCP and miCP) (14), Arg45 of the β1 subunit, located at the base of the S1 pocket, is replaced by leucine in β1i, thereby causing a specific change of the electrostatic milieu, in line with the observed low postacidic activity of the iCP (15).Despite the high sequence similarity between β5 subunits of mcCP and miCP including identical active sites, a peptidic α-β-epoxyketone inhibitor, PR-957, showed higher affinity to iCP by one order of magnitude. The structural comparison of cCP and iCP in their apo and PR-957 liganded states suggested an explanation. On binding of PR-957, the cCP β5 backbone displays significant deformations, whereas the iCP β5 backbone remains unchanged. This observation, together with our experience in constructing β5 models for virtual screening purposes, prompted us to reinvestigate the vast amount of structural data for yCP by a procedure that facilitates discovery of global changes: principal component analysis (PCA).We focus our study on the β5 subunit, because β5 inactivation in yeast renders a lethal phenotype (16) and therefore β5 harbors an essential enzymatic activity, and because almost all crystallographically defined complexes are liganded at their β5 active site.Here we present a detailed investigation of the wealth of yeast and mouse proteasome ligand complex structures that led us to embark on structural comparisons beyond the immediate vicinity of the ligands to obtain a view of the global response of the core particle of yeast and mouse proteasome to complex formation. This study (i) is evidence of the structural plasticity of the β, specifically β5, subunits; (ii) offers perspectives for the analysis of the structure-function relationship of the CP; and (iii) provides an aid for the design and development of ligands as drugs for this intensively studied target for cancer and autoimmune diseases.     

Treatment of AL Amyloidosis: Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART) Consensus Statement 2020 Update

Immunoglobulin light chain (AL) amyloidosis is a clonal plasma cell disorder leading to progressive and life-threatening organ failure. The heart and the kidneys are the most commonly involved organs, but almost any organ can be involved. Because of the nonspecific presentation, diagnosis delay is common, and many patients are diagnosed with advanced organ failure. In the era of effective therapies and improved outcomes for patients with AL amyloidosis, the importance of early recognition is further enhanced as the ability to reverse organ dysfunction is limited in those with a profound organ failure. As AL amyloidosis is an uncommon disorder and given patients’ frailty and high early death rate, management of this complex condition is challenging. The treatment of AL amyloidosis is based on various anti–plasma cell therapies. These therapies are borrowed and customized from the treatment of multiple myeloma, a more common disorder. However, a growing number of phase 2/3 studies dedicated to the AL amyloidosis population are being performed, making treatment decisions more evidence-based. Supportive care is an integral part of management of AL amyloidosis because of the inherent organ dysfunction, limiting the delivery of effective therapy. This extensive review brings an updated summary on the management of AL amyloidosis, sectioned into the 3 pillars for survival improvement: early disease recognition, anti–plasma cell therapy, and supportive care.     

PS-341对小鼠重症急性胰腺炎核因子-κB活化的影响

目的观察PS-341对重症急性胰腺炎(SAP)小鼠胰腺组织核因子-κB(NF-κB)活化的影响,阐明PS-341对SAP的作用机制。方法采用连续7次腹内注射雨蛙素(每次间隔1h)及脂多糖制作小鼠SAP模型,将60只ICR雌性小鼠随机分为PS-341治疗组(注射脂多糖前0.5h腹内注射0.5mg/kgPS-341),模型对照组(注射脂多糖前0.5h腹内注射50%DMSO),空白对照组(生理盐水制模)。最后一次注射雨蛙素后2h麻醉小鼠,取血检测血淀粉酶,光镜下观察小鼠胰腺和肺脏的病理形态,测定胰腺组织中髓过氧化物酶水平,实时荧光定量PCR测定胰腺组织中黏附分子(ICAM-1)的含量,免疫印迹法(Westernblotting)检测胰腺组织中IκBα的表达,电泳迁移率实验(EMSA)测定NF-κB活性。结果 PS-341治疗组和模型对照组比较,胰腺组织中I-κB降解减少,NF-κB活性显著下降,血清淀粉酶、MPO、胰腺组织中细胞黏附分子ICAM-1的表达都明显降低(P<0.05)。胰腺和肺脏组织病理学有明显的改善(P<0.05)。结论 PS-341通过抑制胰腺内NF-κB活化而抑制炎症反应。PS-341可能是治疗SAP的一个新措施。     

20S蛋白酶体β5亚单位基因沉默对人骨髓间充质干细胞增殖能力的影响

目的:应用基因沉默技术,观察下调20S蛋白酶体β5亚单位(PSMB5)对人骨髓间充质干细胞(hBMSCs)增殖能力的影响及其潜在机制,寻求调节干细胞活力的关键靶点。方法:构建PSMB5-shRNA慢病毒载体感染早期hBMSCs,根据感染条件不同将细胞分为绿色荧光蛋白(GFP)对照组和shRNA组。倒置荧光显微镜观察转染效率,RT-PCR和免疫印迹检测PSMB5沉默效率,荧光分光光度法检测蛋白酶体活性;BrdU掺入实验观察PSMB5基因沉默对早期hBMSCs增殖潜能的影响,流式细胞术检测细胞周期分布,免疫印迹检测细胞周期相关蛋白1(cyclin D1)及其激酶CDK4的表达变化。结果:慢病毒感染早期hBMSCs 72 h可见约95%以上细胞表达绿色荧光蛋白。shRNA组PSMB5 mRNA和蛋白表达水平较GFP对照组分别降低93.31%±0.59%和56.83%±13.31%,且蛋白酶体活性较对照组降低37.47%±0.41%。此外,shRNA组BrdU阳性率26.14%±8.13%较对照组49.53%±11.18%显著降低,G1期细胞较GFP对照组增多,而S期和G2/M期细胞却较对照组减少,Cyclin D1和CDK4的表达水平分别下降54.55%±7.76%和63.26%±15.76%。结论:PSMB5基因沉默能够下调Cyclin D1和CDK4的表达,导致细胞G1/S转换停滞,影响hBMSCs增殖潜能。