The functionality of a protein depends on its correct folding, but newly synthesized proteins are susceptible to aberrant folding and aggregation. Heat shock proteins (HSPs) function as molecular ...chaperones that aid in protein folding and the degradation of misfolded proteins. Trinucleotide (CAG) repeat expansion in the Huntingtin gene (HTT) results in the expression of misfolded Huntingtin protein (Htt), which contributes to the development of Huntington's disease. We previously found that the degradation of mutated Htt with polyQ expansion (Htt103QP) depends on both ubiquitin proteasome system and autophagy. However, the role of heat shock proteins in the clearance of mutated Htt remains poorly understood. Here, we report that cytosolic Hsp70 (Ssa family), its nucleotide exchange factors (Sse1 and Fes1), and a Hsp40 co-chaperone (Ydj1) are required for inclusion body formation of Htt103QP proteins and their clearance via autophagy. Extended induction of Htt103QP-GFP leads to the formation of a single inclusion body in wild-type yeast cells, but mutant cells lacking these HSPs exhibit increased number of Htt103QP aggregates. Most notably, we detected more aggregated forms of Htt103QP in sse1Δ mutant cells using an agarose gel assay. Increased protein aggregates are also observed in these HSP mutants even in the absence Htt103QP overexpression. Importantly, these HSPs are required for autophagy-mediated Htt103QP clearance, but are less critical for proteasome-dependent degradation. These findings suggest a chaperone network that facilitates inclusion body formation of misfolded proteins and the subsequent autophagic clearance.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the ...Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function.
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•Cdc48 segregase is required for the degradation of misfolded proteins in yeast•Cdc48 deficiency leads to a decreased pool of free ubiquitin that compromises the UPS•San1 and Ubr1 ubiquitinate misfolded proteins, reducing the free ubiquitin pool•Restoring free ubiquitin suppresses the toxicity associated with Cdc48 deficiency
Misfolded protein accumulation causes cytotoxicity, but the mechanism remains poorly understood. Using budding yeast as a model organism, Higgins et al. show that ubiquitination of misfolded proteins depletes free ubiquitin, which compromises ubiquitin-dependent cellular functions and causes cytotoxicity. The Cdc48/p97 segregase antagonizes this cytotoxicity by promoting ubiquitin recycling from misfolded proteins.
Background: Activating mutations in MYD88 are prevalent in many B-cell malignancies, including Waldenström Macroglobulinemia (95-97%), primary CNS lymphoma (70-80%), ABC DLBCL (40%), marginal zone ...lymphoma (5-10%), and CLL (5-15%). Mutated MYD88 transcriptionally upregulates the SRC family member HCK, which in turn serves as a master signal for triggering multiple pro-survival cascades in mutated MYD88 lymphoma cells including BTK/NFKB, SYK, and ERK (Blood 127:3237-52; Blood Adv 4:141-153; Blood Adv 6:3332-38). In previous work, we developed and characterized a novel HCK kinase inhibitor, KIN-8194 (Blood 138:1966-79). KIN-8194 blocked both HCK and BTK and was active both in vitro and in vivo in MYD88 mutated xenograft lymphoma models, including BTK Cys481Ser ibrutinib resistant models. Proteolysis targeting chimeras (PROTACs) represent a novel approach for blocking kinase based signaling and may provide an advantage over kinase inhibitors with greater selectivity and sustained target inhibition. We therefore initiated a medicinal chemistry campaign to develop highly potent, selective, and bio-available HCK/BTK targeting PROTACS using KIN-8194 scaffold for the treatment of MYD88 driven lymphomas. Methods: We developed and characterized a large series of PROTACs using KIN-8194 as a scaffold. We rationally designed linkers to facilitate E3 ubiquitin ligase binding and improve physical properties leading to degraders with ideal oral pharmacokinetic properties and bioavailability. We characterized their kinome profiling by performing a KINOMEscan assay against a panel of 468 kinases at 1 uM ( Fig. 1). Subsequently, we conducted rigorous biological efficacy screenings using the ATP-based Cell Titer-Glo Luminescent Cell Viability Assay to identify the most potent compounds. Compounds with IC 50 scores lower than KIN-8194 were selected for further validation through flow cytometry based quantitative assays that verified apoptotic and cytotoxic effects on WM and MYD88-mutated lymphoma cell lines, as well as primary human peripheral blood mononuclear cells (PBMCs), utilizing Annexin V and Propidium iodide (PI) staining. Results: We identified and characterized several novel PROTACs from the KIN-8194 scaffold that exhibited potent dual inhibition of HCK and BTK kinase activity at 1 nM, as well as marked degradation of HCK and BTK by western blot analysis at dose levels as low as 100 nM in MYD88 mutated WM and ABC DLBCL cell lines. KINOMEscan profiling showed greater selectivity for novel PROTACs as exemplified in Fig. 1. Enhanced cell killing due to degradation of HCK and BTK versus inhibition alone was confirmed using respective PROTAC analogues with modified IMID warheads that do not bind cereblon. We observed suppression of downstream pro-survival NFKB and ERK signaling in response to the dual HCK/BTK PROTACs. Moreover, several HCK/BTK PROTACs (DFCI-004, -005, -006) demonstrated remarkable low IC 50 values (1-100 nM) in proliferation assays of MYD88 mutated BCWM.1 WM and TMD-8 ABC DLBCL cells. Importantly, the dual HCK/BTK PROTACs elicited higher levels of apoptosis in BCWM.1 WM and TMD-8 ABC DLBCL cells in comparison to the native KIN-8194 kinase inhibitor and showed sparing of healthy donor B- and T-cells akin to KIN-8194. Finally, murine PK studies showed that the HCK/BTK PROTACs DFCI-005 and -006 were highly bioavailable with F=40-42%, and C Max of 700-2700 nM at single oral dose ranges of 10-60 mg/kg. Conclusions: In these studies, we demonstrated the development and characterization of novel, dual HCK/BTK PROTACs that demonstrate potent and selective kinase inhibition and protein degradation of HCK and BTK. Importantly, the novel PROTACs showed enhanced apoptosis of MYD88 mutated WM and ABC DLBCL cells over the native kinase inhibitor KIN-8194 and sparing of healthy donor B- and T-cells. Lastly, novel HCK/BTK PROTACs exhibited high levels of bioavailability. Our studies therefore provide a framework for the advancement of dual HCK/BTK PROTACs for the treatment of MYD88 mutated lymphomas.
The identification of acute injury of the kidney relies on serum creatinine (SCr), a functional marker with poor temporal resolution as well as limited sensitivity and specificity for cellular ...injury. In contrast, urinary biomarkers of kidney injury have the potential to detect cellular stress and damage in real time.
To detect the response of the kidney to injury, we have tested a lateral flow dipstick that measures a urinary protein called neutrophil gelatinase-associated lipocalin (NGAL). Analysis of urine was performed in a prospective cohort of 479 patients (final cohort N = 426) entering an emergency department in New York City and subsequently admitted for inpatient care.
Colorimetric development had high interrater reliability (88% concordance rate) and correlated with traditional enzyme-linked immunosorbent assay (ELISA) measurements (ρ = 0.732, P < .0001). Of the 14% of the cohort who met Acute Kidney Injury Network (AKIN) SCr criteria for acute kidney injury (AKI), 67% demonstrated transient (<2 days) and 33% demonstrated sustained (>2 days) elevation of SCr. Comparing the outcomes of patients with sustained versus transient or undetectable changes in SCr revealed that the urinary NGAL (uNGAL) dipstick had high specificity and negative predictive value (NPV) (high- vs. low-intermediate readings, sensitivity = 0.55, specificity = 0.91, positive predictive value = 0.24, NPV = 0.97, χ2 = 20.39, P < 0.001).
We show that the introduction of a bedside uNGAL dipstick permits accurate triage by identifying individuals who do not have tubular injury. In an era of shortening length of stay and rapid decisions based on isolated SCr measurements, real-time exclusion of kidney injury by a dipstick will be particularly useful to overcome the retrospective, insensitive, and nonspecific attributes of SCr.
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The purpose of this study was to evaluate condom use and the incidence of breakage and slippage during vaginal intercourse among female prostitutes in legal Nevada brothels, where use of condoms is ...required by law.
Forty-one licensed prostitutes in three brothels were enrolled in a prospective trial in August 1993. Used condoms were collected to verify reported breaks visually. Retrospective breakage and slippage rates were obtained in a standardized interview.
Condoms were used for every act of vaginal intercourse with a brothel client during the study period, as well as in the previous year. In the prospective study phase, condoms were used in 353 acts of vaginal intercourse with clients. No condoms broke, and none fell off the penis during intercourse. Only twice (0.6%) did condoms completely fall off during withdrawal. Twelve times (3.4%) during intercourse and 15 times (4.3%) during withdrawal, condoms slipped down the penis but did not fall off.
These findings, among the lowest breakage and slippage rates published, suggest that regular condom use may lead to condom mastery and the development of techniques to reduce the likelihood of breakage and slippage.
This study examined condom use in legal Nevada brothels.
Forty female prostitutes in two brothels were interviewed about client resistance to condoms and techniques for facilitating condom use.
Of ...3290 clients in the previous month, 2.7% (95% confidence interval CI = 2.2%,3.4%) were reluctant to use condoms. Of these individuals, 72% ultimately used condoms, while 12% chose nonpenetrative sex without condoms. The remaining 16% left the brothels without services. Condom use rates were markedly lower with nonpaying sex partners (lowers) than with clients.
Brothel prostitutes may be at greater risk for acquiring HIV and other sexually transmitted diseases from lovers than from clients.
For those who choose to be sexually active, condoms are the best available means of protection against sexually transmitted diseases including the human immunodeficiency virus (HIV), which causes ...acquired immunodeficiency syndrome (AIDS). Condoms are also an effective method for preventing pregnancy. Unfortunately, condoms are not 100% effective at preventing pregnancy or the spread of infection, in part because condoms do break. In order to gain insight into condom breakage, a questionnaire was administered to women attending a municipal hospital family planning clinic. Thirty-six percent of the 106 subjects had experienced at least one condom breakage. Condom breakage occurred in approximately 1 out of 100 acts of intercourse using condoms, with a lifetime breakage rate of 10 per 1000 condom uses and a past year breakage rate of 8 per 1000 condom uses. Breakage rates did not differ substantially by age. Five percent of the women's unplanned pregnancies were attributed to broken condoms. The results of this study corroborate previously reported rates. Factors associated with these women's most recent breakage experiences included: vaginal intercourse, minimal foreplay, and breakage prior to ejaculation. Controlled studies will be needed to determine how the condom can be used to reduce the likelihood of breakage.
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