Disrupted structure and aberrant function of CHIP mediates the loss of motor and cognitive function in preclinical models of SCAR16.

CHIP (carboxyl terminus of heat shock 70-interacting protein) has long been recognized as an active member of the cellular protein quality control system given the ability of CHIP to function as both a co-chaperone and ubiquitin ligase. We discovered a genetic disease, now known as spinocerebellar autosomal recessive 16 (SCAR16), resulting from a coding mutation that caused a loss of CHIP ubiquitin ligase function. The initial mutation describing SCAR16 was a missense mutation in the ubiquitin ligase domain of CHIP (p.T246M). Using multiple biophysical and cellular approaches, we demonstrated that T246M mutation results in structural disorganization and misfolding of the CHIP U-box domain, promoting oligomerization, and increased proteasome-dependent turnover. CHIP-T246M has no ligase activity, but maintains interactions with chaperones and chaperone-related functions. To establish preclinical models of SCAR16, we engineered T246M at the endogenous locus in both mice and rats. Animals homozygous for T246M had both cognitive and motor cerebellar dysfunction distinct from those observed in the CHIP null animal model, as well as deficits in learning and memory, reflective of the cognitive deficits reported in SCAR16 patients. We conclude that the T246M mutation is not equivalent to the total loss of CHIP, supporting the concept that disease-causing CHIP mutations have different biophysical and functional repercussions on CHIP function that may directly correlate to the spectrum of clinical phenotypes observed in SCAR16 patients. Our findings both further expand our basic understanding of CHIP biology and provide meaningful mechanistic insight underlying the molecular drivers of SCAR16 disease pathology, which may be used to inform the development of novel therapeutics for this devastating disease.

Changes in protein function underlie the disease spectrum in patients with CHIP mutations.

Monogenetic disorders that cause cerebellar ataxia are characterized by defects in gait and atrophy of the cerebellum; however, patients often suffer from a spectrum of disease, complicating treatment options. Spinocerebellar ataxia autosomal recessive 16 (SCAR16) is caused by coding mutations in STUB1, a gene that encodes the multifunctional enzyme CHIP (C terminus of HSC70-interacting protein). The disease spectrum of SCAR16 includes a varying age of disease onset, cognitive dysfunction, increased tendon reflex, and hypogonadism. Although SCAR16 mutations span the multiple functional domains of CHIP, it is unclear whether the location of the mutation and the change in the biochemical properties of CHIP contributes to the clinical spectrum of SCAR16. In this study, we examined relationships between the clinical phenotypes of SCAR16 patients and the changes in biophysical, biochemical, and functional properties of the corresponding mutated protein. We found that the severity of ataxia did not correlate with age of onset; however, cognitive dysfunction, increased tendon reflex, and ancestry were able to predict 54% of the variation in ataxia severity. We further identified domain-specific relationships between biochemical changes in CHIP and clinical phenotypes and specific biochemical activities that associate selectively with either increased tendon reflex or cognitive dysfunction, suggesting that specific changes to CHIP-HSC70 dynamics contribute to the clinical spectrum of SCAR16. Finally, linear models of SCAR16 as a function of the biochemical properties of CHIP support the concept that further inhibiting mutant CHIP activity lessens disease severity and may be useful in the design of patient-specific targeted approaches to treat SCAR16.

CHIP Regulates Aquaporin-2 Quality Control and Body Water Homeostasis.

The importance of the kidney distal convoluted tubule (DCT) and cortical collecting duct (CCD) is highlighted by various water and electrolyte disorders that arise when the unique transport properties of these segments are disturbed. Despite this critical role, little is known about which proteins have a regulatory role in these cells and how these cells can be regulated by individual physiologic stimuli. By combining proteomics, bioinformatics, and cell biology approaches, we found that the E3 ubiquitin ligase CHIP is highly expressed throughout the collecting duct; is modulated in abundance by vasopressin; interacts with aquaporin-2 (AQP2), Hsp70, and Hsc70; and can directly ubiquitylate the water channel AQP2 in vitro shRNA knockdown of CHIP in CCD cells increased AQP2 protein t1/2 and reduced AQP2 ubiquitylation, resulting in greater levels of AQP2 and phosphorylated AQP2. CHIP knockdown increased the plasma membrane abundance of AQP2 in these cells. Compared with wild-type controls, CHIP knockout mice or novel CRISPR/Cas9 mice without CHIP E3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. We did not observe significant changes in other water- or sodium-transporting proteins in the gene-modified mice. In summary, these results suggest that CHIP regulates AQP2 and subsequently, renal water handling.

Ubiquitin ligase STUB1 destabilizes IFNγ-receptor complex to suppress tumor IFNγ signaling.

The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identify STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through IFNγ-R1K285 and JAK1K249. Conversely, STUB1 inactivation amplifies IFNγ signaling, sensitizing tumor cells to cytotoxic T cells in vitro. This is corroborated by an anticorrelation between STUB1 expression and IFNγ response in ICB-treated patients. Consistent with the context-dependent effects of IFNγ in vivo, anti-PD-1 response is increased in heterogenous tumors comprising both wildtype and STUB1-deficient cells, but not full STUB1 knockout tumors. These results uncover STUB1 as a critical regulator of IFNγ-R1, and highlight the context-dependency of STUB1-regulated IFNγ signaling for ICB outcome.

With or without You: Co-Chaperones Mediate Health and Disease by Modifying Chaperone Function and Protein Triage.

Heat shock proteins (HSPs) are a family of molecular chaperones that regulate essential protein refolding and triage decisions to maintain protein homeostasis. Numerous co-chaperone proteins directly interact and modify the function of HSPs, and these interactions impact the outcome of protein triage, impacting everything from structural proteins to cell signaling mediators. The chaperone/co-chaperone machinery protects against various stressors to ensure cellular function in the face of stress. However, coding mutations, expression changes, and post-translational modifications of the chaperone/co-chaperone machinery can alter the cellular stress response. Importantly, these dysfunctions appear to contribute to numerous human diseases. Therapeutic targeting of chaperones is an attractive but challenging approach due to the vast functions of HSPs, likely contributing to the off-target effects of these therapies. Current efforts focus on targeting co-chaperones to develop precise treatments for numerous diseases caused by defects in protein quality control. This review focuses on the recent developments regarding selected HSP70/HSP90 co-chaperones, with a concentration on cardioprotection, neuroprotection, cancer, and autoimmune diseases. We also discuss therapeutic approaches that highlight both the utility and challenges of targeting co-chaperones.

Evaluating the efficacy of enzalutamide and the development of resistance in a preclinical mouse model of type-I endometrial carcinoma.

Androgen Receptor (AR) signaling is a critical driver of hormone-dependent prostate cancer and has also been proposed to have biological activity in female hormone-dependent cancers, including type I endometrial carcinoma (EMC). In this study, we evaluated the preclinical efficacy of a third-generation AR antagonist, enzalutamide, in a genetic mouse model of EMC, Sprr2f-Cre;Ptenfl/fl. In this model, ablation of Pten in the uterine epithelium leads to localized and distant malignant disease as observed in human EMC. We hypothesized that administering enzalutamide through the diet would temporarily decrease the incidence of invasive and metastatic carcinoma, while prolonged administration would result in development of resistance and loss of efficacy. Short-term treatment with enzalutamide reduced overall tumor burden through increased apoptosis but failed to prevent progression of invasive and metastatic disease. These results suggest that AR signaling may have biphasic, oncogenic and tumor suppressive roles in EMC that are dependent on disease stage. Enzalutamide treatment increased Progesterone Receptor (PR) expression within both stromal and tumor cell compartments. Prolonged administration of enzalutamide decreased apoptosis, increased tumor burden and resulted in the clonal expansion of tumor cells expressing high levels of p53 protein, suggestive of acquired Trp53 mutations. In conclusion, we show that enzalutamide induces apoptosis in EMC but has limited efficacy overall as a single agent. Induction of PR, a negative regulator of endometrial proliferation, suggests that adding progestin therapy to enzalutamide administration may further decrease tumor burden and result in a prolonged response.

CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury.

Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

Performance of a gravitational marrow separator, multidirectional bone marrow aspiration needle, and repeated bone marrow collections on the production of concentrated bone marrow and separation of mesenchymal stem cells in horses.

Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, MSCs, and cell viability. Results-Concentrated bone marrow samples obtained with the marrow separator had 5- to 19-fold bone marrow-derived MSC, WBC, and platelet counts, compared with original bone marrow samples. Use of a multidirectional needle increased the frequency of obtaining MSC-richer concentrated bone marrow. Repeating bone marrow aspiration at 1 month yielded greater MSC numbers but slightly lower cell viability after processing. Conclusions and Clinical Relevance-The gravitational bone marrow separator and multidirectional needle were used to effectively harvest bone marrow and improve the quality of concentrated bone marrow. Comparable, or even greater, numbers of bone marrow-derived MSCs were collected by repeated bone marrow aspiration after a 1-month interval from the same aspiration sites. Use of the marrow separator and multidirectional bone marrow aspiration needle can facilitate a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of bone marrow-derived MSCs and growth factors from platelets and plasma.