[Hereditary cancer syndromes: a modern paradigm].

About 5-10% of malignant neoplasms (MN) are hereditary. Carriers of mutations associated with hereditary tumor syndromes (HTS) are at high risk of developing tumors in childhood and young age and synchronous and metachronous multiple tumors. At the same time, this group of diseases remains mainly an oncological problem, and clinical decisions are made only when MNs are detected in carriers of pathogenic mutations.Individual recommendations for cancer screening, treatment, and prevention should be developed for carriers of mutations associated with HTS to prevent an adverse outcome of the disease. It is essential to identify patients at risk by doctors of all specialties for further referral to medical and genetic counseling with molecular genetic testing (in case of indications). The problems of standardization of enrollment criteria for genetic tests, further tactics of prevention, screening, and treatment of many hereditary oncological diseases remain unsolved.This review was created to inform doctors of various specialties, including endocrinologists, about the HTS. This allows them to get acquainted with main clinical features of specific syndromes, helps to understand the difference between hereditary and non-hereditary cancer, recognize signs of hereditary cancer, and introduce the indications for genetic examination and genetic counseling of the patient. Also, significant differences between international and domestic recommendations on screening measures, diagnosis, and treatment of HTS underline the need to review the existing and develop new algorithms for medical support of patients with HTS.

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex.

Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated (Cas) immunity relies on adaptive acquisition of spacers-short fragments of foreign DNA. For the type I-E CRISPR-Cas system from Escherichia coli, efficient "primed" adaptation requires Cas effector proteins and a CRISPR RNA (crRNA) whose spacer partially matches a segment (protospacer) in target DNA. Primed adaptation leads to selective acquisition of additional spacers from DNA molecules recognized by the effector-crRNA complex. When the crRNA spacer fully matches a protospacer, CRISPR interference-that is, target destruction without acquisition of additional spacers-is observed. We show here that when the rate of degradation of DNA with fully and partially matching crRNA targets is made equal, fully matching protospacers stimulate primed adaptation much more efficiently than partially matching ones. The result indicates that different functional outcomes of CRISPR-Cas response to two kinds of protospacers are not caused by different structures formed by the effector-crRNA complex but are due to the more rapid destruction of targets with fully matching protospacers.

Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems.

Chronic suppression of inositol 1,4,5-triphosphate receptor-mediated calcium signaling in cerebellar purkinje cells alleviates pathological phenotype in spinocerebellar ataxia 2 mice.

Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder characterized by progressive ataxia. SCA2 results from a poly(Q) (polyglutamine) expansion in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are primarily affected in SCA2, but the cause of PC dysfunction and death in SCA2 is poorly understood. In previous studies, we reported that mutant but not wild-type Atx2 specifically binds the inositol 1,4,5-trisphosphate receptor (InsP(3)R) and increases its sensitivity to activation by InsP3. We further proposed that the resulting supranormal calcium (Ca2+) release from the PC endoplasmic reticulum plays a key role in the development of SCA2 pathology. To test this hypothesis, we achieved a chronic suppression of InsP(3)R-mediated Ca2+ signaling by adenoassociated virus-mediated expression of the inositol 1,4,5-phosphatase (Inpp5a) enzyme (5PP) in PCs of a SCA2 transgenic mouse model. We determined that recombinant 5PP overexpression alleviated age-dependent dysfunction in the firing pattern of SCA2 PCs. We further discovered that chronic 5PP overexpression also rescued age-dependent motor incoordination and PC death in SCA2 mice. Our findings further support the important role of supranormal Ca2+ signaling in SCA2 pathogenesis and suggest that partial inhibition of InsP3-mediated Ca2+ signaling could provide therapeutic benefit for the patients afflicted with SCA2 and possibly other SCAs.

Polyclonal antibodies against human proteasome subunits PSMA3, PSMA5, and PSMB5.

A proteasome is a multi-subunit protein complex, which plays a central role in ubiquitin-dependent protein degradation in all eukaryotic cells. The 26S proteasome is composed of a catalytic 20S core complex and one or two 19S regulatory complexes. The 20S core complex forms a cylinder consisting of four stacked rings of seven α (PSMA1-7) or ß (PSMB1-7) subunits. Target proteins are degraded in the cavity of the 20S complex due to proteolytic activities of three ß subunits having catalytic sites located on the inner surface of the cylinder. The aim of this study was the generation of polyclonal antibodies against human proteasome subunits PSMA3, PSMA5, and PSMB5 and characterization of their experimental applications. To construct GST-fusion proteins, DNA sequences encoding PSMA3, PSMA5, and PSMB5 were cloned into prokaryotic expression vectors pGEX-5X-1 or pGEX-4T-3. Recombinant proteins GST-PSMA3, GST-PSMA5, and GST-PSMB5 were highly expressed in E. coli BL21 (DE3) cells, purified by glutathione-affinity chromatography and further used for rabbit immunization. The activity and specificity of the obtained antibody-containing sera were evaluated using Western blot analysis and immunoprecipitation. We have shown by Western blot analysis that our anti-PSMA3, anti-PSMA5, and anti-PSMB5 antibodies recognized both recombinant and endogenous proteins from different human cell lines. We have also shown that anti-PSMA3 and anti-PSMA5 sera were able to recognize and immunoprecipitate native forms of both endogenous and overexpressed FLAG-tagged proteins PSMA3 and PSMA5, respectively. Thus, the antibodies generated against PSMA3, PSMA5, and PSMB5 can be used in various experimental applications, including the evaluation of cellular levels of proteasome subunits in cell extracts and affinity purification of the endogenous and/or overexpressed proteasome subunits, which facilitates subsequent analysis of their post-translational modifications as well as protein-protein interactions in vivo.