Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

Involvement of CacyBP/SIP in differentiation and the immune response of HaCaT keratinocytes.

CacyBP/SIP is a multifunctional protein present in various cells and tissues. However, its expression and role in the epidermis has not been explored so far. In this work, using RT-qPCR, Western blot analysis and three-dimensional (3D) organotypic cultures of HaCaT keratinocytes we show that CacyBP/SIP is present in the epidermis. To investigate the possible role of CacyBP/SIP in keratinocytes we obtained CacyBP/SIP knockdown cells and studied the effect of CacyBP/SIP deficiency on their differentiation and response to viral infection. We found that CacyBP/SIP knockdown results in reduced expression of epidermal differentiation markers in both undifferentiated and differentiated HaCaT cells. Since epidermis is engaged in immune defense, the impact of CacyBP/SIP knockdown on this process was also analyzed. By applying RT-qPCR and Western blot it was found that poly(I:C), a synthetic analog of double-stranded RNA that mimics viral infection, stimulated the expression of genes involved in antiviral response, such as IFIT1, IFIT2 and OASL. Interestingly, following poly(I:C) stimulation, the level of expression of these genes was significantly lower in cells with CacyBP/SIP knockdown than control ones. Since the signaling pathway mediating cellular responses to viral infection involves, among others, the STAT1 transcription factor, we measured its activity using luciferase assay and found that it was lower in CacyBP/SIP knockdown HaCaT cells. Altogether, the presented results indicate that CacyBP/SIP promotes epidermal differentiation and might be involved in response of the skin cells to viral infection.

Sgt1 Regulates α-Synuclein Subcellular Localization and Expression of Parkinson's Disease Related Genes, PINK1 and PARK9.

The SGT1 protein is highly expressed in the mammalian brain, particularly in neurons of the hippocampus and cortex, and in Purkinje cells of the cerebellum. There are literature data indicating that the protein may be involved in pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). In the present work we have found that SGT1 protected cells from the toxicity of rotenone, an agent that evokes behavioral and histopathological symptoms of PD. To gain more insight into the possible mechanism underlying the protective action of SGT1 we looked at α-synuclein subcellular distribution in HEK293 cells with an altered SGT1 level. By immunofluorescent staining we have found that in HEK293 cells overexpressing SGT1 α-synuclein was mainly localized in the cytoplasm while in control cells it was present in the nucleus. Accordingly, when SGT1 expression was silenced, α-synuclein was predominantly present in the nucleus. These results were then confirmed by subcellular fractionation and Western blot analysis. Moreover, we have found that altered level of SGT1 in HEK293 cells influenced the expression of PD related genes, PINK1 and PARK9. Altogether, our results point to SGT1 as an important factor that might be involved in the pathogenesis of Parkinson's disease (PD).

Calmodulin and Its Binding Proteins in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca2+ as a universal carrier of extracellular and intracellular signals that can initiate and control various cellular processes. Disturbances in Ca2+ homeostasis and dysfunction of Ca2+ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca2+ level. The best known and studied Ca2+ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca2+ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.

HSP90 Co-Chaperone, CacyBP/SIP, Protects α-Synuclein from Aggregation.

Recently, it has been found that the CacyBP/SIP protein acts as HSP90 co-chaperone and exhibits chaperone properties itself. Namely, CacyBP/SIP has been shown to protect citrate synthase from aggregation and to recover the activity of thermally denatured luciferase in vitro. In the present work, we have analyzed the influence of CacyBP/SIP on aggregation of α-synuclein, a protein present in Lewy bodies of Parkinson's disease brain. By applying a thioflavin T (ThT) fluorescence assay, we have found that CacyBP/SIP protects α-synuclein from aggregation and that the fragment overlapping the N-terminal part and the CS domain of CacyBP/SIP is crucial for this activity. This protective effect of CacyBP/SIP has been confirmed by results obtained using high-speed ultracentrifugation followed by dot-blot and by transmission electron microscopy (TEM). Interestingly, CacyBP/SIP exhibits the protective effect only at the initial phase of α-synuclein aggregation. In addition, we have found that, in HEK293 cells overexpressing CacyBP/SIP, there are less α-synuclein inclusions than in control ones. Moreover, these cells are more viable when treated with rotenone, an agent that mimics PD pathology. By applying proximity ligation assay (PLA) on HEK293 cells and in vitro assays with the use of purified recombinant proteins, we have found that CacyBP/SIP directly interacts with α-synuclein. Altogether, in this work, we show for the first time that CacyBP/SIP is able to protect α-synuclein from aggregation in in vitro assays. Thus, our results point to an important role of CacyBP/SIP in the pathology of Parkinson's disease and other synucleinopathies.

Hsp90 and Its Co-Chaperones in Neurodegenerative Diseases.

Proper folding is crucial for proteins to achieve functional activity in the cell. However, it often occurs that proteins are improperly folded (misfolded) and form aggregates, which are the main hallmark of many diseases including cancers, neurodegenerative diseases and many others. Proteins that assist other proteins in proper folding into three-dimensional structures are chaperones and co-chaperones. The key role of chaperones/co-chaperones is to prevent protein aggregation, especially under stress. An imbalance between chaperone/co-chaperone levels has been documented in neurons, and suggested to contribute to protein misfolding. An essential protein and a major regulator of protein folding in all eukaryotic cells is the heat shock protein 90 (Hsp90). The function of Hsp90 is tightly regulated by many factors, including co-chaperones. In this review we summarize results regarding the role of Hsp90 and its co-chaperones in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and prionopathies.

HSP90 and Its Novel Co-Chaperones, SGT1 and CHP-1, in Brain of Patients with Parkinson's Disease and Dementia with Lewy Bodies.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of inclusions known as Lewy bodies in some brain regions. Lewy bodies consist of α-synuclein and many other proteins including chaperones. OBJECTIVE: To learn more about the role of chaperone complexes in PD and a related disorder, i.e., dementia with Lewy bodies (DLB), in this work we analyzed the expression of HSP90 and its two quite recently identified co-chaperones, SGT1 and CHP-1, in selected brain regions from patients suffering from these diseases. METHODS: To fulfill the aim of our study we used human material and applied immunohistochemistry, Western blot analysis and real time/quantitative PCR (RT-qPCR). RESULTS: We have found that HSP90 mRNA level is higher in the temporal cortex of PD and in frontal cortex of DLB brains, even though level of protein does not change significantly. The mRNA level of SGT1 is higher in the frontal and temporal cortex of PD and in substantia nigra of DLB brains while no significant changes in the level of protein were noticed. Similarly, the mRNA level of CHP-1 was found to be higher in the frontal and temporal cortex of PD and in all examined regions i.e. substantia nigra, frontal and temporal cortex of DLB brains. In the case of CHP-1 the protein level was found to be higher in frontal cortex of PD and in all examined areas of DLB patients. CONCLUSIONS: Our data indicate that the level of HSP90, SGT1 and CHP-1 is upregulated in the majority of cases of PD and DLB, which suggests that the examined proteins might be involved in these pathologies.

Role of Mitogen Activated Protein Kinase Signaling in Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder caused by insufficient dopamine production due to the loss of 50% to 70% of dopaminergic neurons. A shortage of dopamine, which is predominantly produced by the dopaminergic neurons within the substantia nigra, causes clinical symptoms such as reduction of muscle mass, impaired body balance, akinesia, bradykinesia, tremors, postural instability, etc. Lastly, this can lead to a total loss of physical movement and death. Since no cure for PD has been developed up to now, researchers using cell cultures and animal models focus their work on searching for potential therapeutic targets in order to develop effective treatments. In recent years, genetic studies have prominently advocated for the role of improper protein phosphorylation caused by a dysfunction in kinases and/or phosphatases as an important player in progression and pathogenesis of PD. Thus, in this review, we focus on the role of selected MAP kinases such as JNKs, ERK1/2, and p38 MAP kinases in PD pathology.