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The article is a summary of the report prepared by the Helsinki Foundation for Human Rights under the "Children Rights Behind Bars 2.0" project. The authors - based on the analysis of information taken on the basis of access to public information, the conclusions from the report of the Supreme Audit Office [pl. Najwyższa Izba Kontroli] and 8 interviews and focus groups with persons working in the system of juvenile social rehabilitation and foster care - indicate doubts related to the functioning of solutions supporting the reintegration of pupils of social rehabilitation institutions. Among the observed problems, they identify a multitude of entities responsible for the functioning of the system, lack of coordination of activities, lack of tools to verify the effectiveness of social rehabilitation work, as well as numerous formal criteria limiting the availability of reintegration services.

Over 20 mutations in β-cardiac myosin heavy chain (β-MHC), expressed in cardiac and slow muscle fibers, cause Laing early-onset distal myopathy (MPD-1), a skeletal muscle myopathy. Most of these mutations are in the coiled-coil tail and commonly involve a mutation to a proline or a single-residue deletion, both of which are predicted to strongly affect the secondary structure of the coiled coil. To test this, we characterized the effects of two MPD-1 causing mutations: A1603P and K1617del in vitro and in cells. Both mutations affected secondary structure, decreasing the helical content of 15 heptad and light meromyosin constructs. Both mutations also severely disrupted the ability of glutathione S-transferase–light meromyosin fusion proteins to form minifilaments in vitro, as demonstrated by negative stain electron microscopy. Mutant eGFP-tagged β-MHC accumulated abnormally into the M-line of sarcomeres in cultured skeletal muscle myotubes. Incorporation of eGFP-tagged β-MHC into sarcomeres in adult rat cardiomyocytes was reduced. Molecular dynamics simulations using a composite structure of part of the coiled coil demonstrated that both mutations affected the structure, with the mutation to proline (A1603P) having a smaller effect compared to K1617del. Taken together, it seems likely that the MPD-1 mutations destabilize the coiled coil, resulting in aberrant myosin packing in thick filaments in muscle sarcomeres, providing a potential mechanism for the disease., Graphical abstract Unlabelled Image, Highlights • It is unclear how mutations in the coiled coil of β-myosin heavy chain cause distal myopathy. • A1603P and K1617del mutations reduce helicity and affect filament formation in vitro. • eGFP-tagged β-myosin heavy chain abnormally accumulates at the M-line of sarcomeres in skeletal myotubes. • Molecular dynamics simulations provide a molecular understanding for these experiments. • Effects on structure and packing into the thick filament provide a molecular basis for the disease.

Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.

Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species.

Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species.

Background: INCENP is predicted to have a coiled coil domain. Results: The coil is actually a stable single α-helix (SAH) domain that is highly extendable and directly binds microtubules. Conclusion: This flexible dog leash may allow Aurora B to associate with dynamic targets in the outer kinetochore. Significance: The SAH domain allows CPC flexibility without requiring complex dimerization., The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible “dog leash,” allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.

We have discovered that two mutations at the actin binding domain (ABD) of α-actinin-2 (ACTN2), which cause hypertrophic cardiomyopathy (HCM), have minor effects on its structure and ability to bind actin and integrate into Z-discs, providing a potential disease mechanism., α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease.

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Histone deacetylase 3 removes acetyl groups from lysine residues, thereby modifying protein function. It is found in both the nucleus and the cytoplasm. We have discovered that it can indirectly deacetylate tubulin, a cytoplasmic protein that forms microtubules, thus modifying the microtubule., Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3–silencing mediator of retinoic and thyroid receptors (SMRT)–deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation.

The human genome contains 39 myosin genes, divided up into 12 different classes. The structure, cellular function and biochemical properties of many of these isoforms remain poorly characterized and there is still some controversy as to whether some myosin isoforms are monomers or dimers. Myosin isoforms 6 and 10 contain a stable single α-helical (SAH) domain, situated just after the canonical lever. The SAH domain is stiff enough to be able to lengthen the lever allowing the myosin to take a larger step. In addition, atomic force microscopy and atomistic simulations show that SAH domains unfold at relatively low forces and have a high propensity to refold. These properties are likely to be important for protein function, enabling motors to carry cargo in dense actin networks, and other proteins to remain attached to binding partners in the crowded cell.

Naturally occurring single alpha helical (SAH) domains are unique structural elements displaying high stability across a range of pH and ionic strength conditions. Rich in charged residues (E, K and R), which are thought to form a network of stabilizing ionic interactions, SAH domains play a key role as flexible elements that bridge functional domains in proteins. The best-studied examples of SAH domains come from myosin motor proteins in which they can replace the canonical lever. We recently showed that inner centromere protein (INCENP) has a long (>200 residue) SAH domain.

Background: Single α-helix (SAH) domains bridge two functional domains in proteins. Their force response is poorly understood. Results: Modeling and experiments show that SAH domains unfold non-cooperatively at low forces and maintain an approximately constant force as they unfold. Conclusion: SAH domains act as constant force springs. Significance: SAH domains are important mechanical elements in proteins., Single α-helix (SAH) domains are rich in charged residues (Arg, Lys, and Glu) and stable in solution over a wide range of pH and salt concentrations. They are found in many different proteins where they bridge two functional domains. To test the idea that their high stability might enable these proteins to resist unfolding along their length, the properties and unfolding behavior of the predicted SAH domain from myosin-10 were characterized. The expressed and purified SAH domain was highly helical, melted non-cooperatively, and was monomeric as shown by circular dichroism and mass spectrometry as expected for a SAH domain. Single molecule force spectroscopy experiments showed that the SAH domain unfolded at very low forces (

Membrane skeleton is a structure that provides strength and elasticity to the erythrocyte membrane. This features stem from the main component of this structure, a multifunctional protein called spectrin. Spectrin forms a network underlying membrane bilayer containing integral membrane proteins which interact with multiple proteins and lipid partners. Although membrane skeleton and spectrin structure have been described before, the latest discoveries show their new details and properties. In this work we summarize recent findings concerning structure and function of spectrin together with its possible role in pathology. We focus our interest on lately published structural data and we make an attempt to combine these findings with possible physiological functions.

Palmitoylation is one of the most common posttranslational lipid modifications of proteins and we now know quite a lot about it. However, the state of knowledge about the enzymes that catalyze this process is clearly insufficient. This review is focused on 23 human DHHC genes and their products – protein palmitoyltransferases. Here we describe mainly the structure and function of these proteins, but also, to a lesser degree, what the substrates of the enzymes are and whether they are related to various diseases. The main aim of this review was to catalogue existing information concerning the human DHHC family of genes/proteins, making them and their functions easier to understand.

It was shown previously that an ankyrin-sensitive, phosphatidylethanolamine/phosphatidylcholine (PE/PC) binding site maps to the N-terminal part of the ankyrin-binding domain of β-spectrin (ankBDn). Here we have identified the amino acid residues within this domain which are responsible for recognizing monolayers and bilayers composed of PE/PC mixtures. In vitro binding studies revealed that a quadruple mutant with substituted hydrophobic residues W1771, L1775, M1778 and W1779 not only failed to effectively bind PE/PC, but its residual PE/PC-binding activity was insensitive to inhibition with ankyrin. Structure prediction and analysis, supported by in vitro experiments, suggests that “opening” of the coiled-coil structure underlies the mechanism of this interaction. Experiments on red blood cells and HeLa cells supported the conclusions derived from the model and in vitro lipid-protein interaction results, and showed the potential physiological role of this binding. We postulate that direct interactions between spectrin ankBDn and PE-rich domains play an important role in stabilizing the structure of the spectrin-based membrane skeleton.

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na+K+ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.

Stable single alpha helical (SAH) domains are found in ∼0.2% of proteins and are rich in charged amino acids; E,R and K. Ionic interactions between oppositely charged amino acids (either i, i+3 and/or i, i+4) are responsible for their high stability. We recently showed that SAH domains can act as ‘constant force springs (Wolny et al., J. Biol. Chem. 2014). Thus they do not simply act as a ‘spacer’ between two protein domains, but can actively respond to force.It is still unclear what the minimal requirements are for a peptide to form a SAH domain. To test this, we used modelling and have characterised 96 residue constructs that vary in the number of charged interactions (E-R or E-K) from 1 every 7 residues (repeating 7mer pattern of (AAEAAKA)n or (AAEAARA)n to 3 every 7 residues ((AEEEKKK)n or (AEEERRR)n) to determine how many is ‘enough’ to stabilize a long (96 residue) alpha helix. 96 residue (AAEAAKA)n or (AAEAARA)n peptides tended to aggregate, and were only soluble in very low salt, when they were found to be highly helical, but unstable. Thus, sparsely placed charged residues (1 pair per heptad) are not sufficient to keep polyalanine stable in higher salt concentrations. 96 residue (AEEEKKK)n peptides were highly stable and helical (>90%) and melted non-cooperatively as expected for SAH domains. Surprisingly, 96 residue (AEEERRR)n peptides tended to aggregate, were only soluble at pH

It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301–1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy. Background: It is unclear how mutations in the coiled-coil tail of β-cardiac myosin cause heart disease. Results: Effects of disease-causing mutations in the myosin tail were studied in vivo and in vitro. Conclusion: Mutations that reduce helical content in vitro reduce sarcomere incorporation of myosin in vivo. Significance: A change in myosin tail structure can lead to heart disease.

Spectrin and ankyrin are the key components of the erythrocyte cytoskeleton. The recently published crystal structure of the spectrin–ankyrin complex has indicated that their binding involves complementary charge interactions as well as hydrophobic interactions. However, only the former is supported by biochemical evidence. We now show that nonpolar interactions are important for high affinity complex formation, excluding the possibility that the binding is exclusively mediated by association of distinctly charged surfaces. Along these lines we report that substitution of a single hydrophobic residue, F917S in ankyrin, disrupts the structure of the binding site and leads to complete loss of spectrin affinity. Finally, we present data showing that minimal ankyrin binding site in spectrin is formed by helix 14C together with the loop between helices 15 B/C.

While the last decade has seen a growth of support for harm reduction around the world, the availability and accessibility of quality harm reduction services in prison settings is uneven and continues to be inadequate compared to the progress achieved in the broader community. This article provides a brief overview of harm reduction in prisons in Catalonia (Spain), Greece, Ireland, Italy, Latvia, Poland, and Portugal. While each country provides a wide range of harm reduction services in the broader community, the majority fail to provide these same services or the same quality of these services, in prison settings, in clear violation of international human rights law and minimum standards on the treatment of prisoners. Where harm reduction services have been available and easily accessible in prison settings for some time, better health outcomes have been observed, including significantly reduced rates of HIV and HCV incidence. While the provision of harm reduction in each of these countries' prisons varies considerably, certain key themes and lessons can be distilled, including around features of an enabling environment for harm reduction, resource allocation, collection of disaggregated data, and accessibility of services.

In most proteins, α-helices are stabilised by interactions with neighbouring secondary structure elements (e.g. coiled-coils). However, we recently showed that many proteins contain single α-helical (SAH) domains, which are stable in isolation and commonly inserted between two different functional domains. SAH domains are rich in arginine, lysine and glutamic acid residues. Their stability arises from the many potential (i, i ± 4) and (i, i ± 3) intrahelical interactions between either R and E, or K and E.To date, the best-studied SAH domains are those from myosins 6 and 10, and the Dictyostelium myosin myoM, where they are likely to form part of the functional lever. A SAH domain is also predicted for myosin 7a. We have shown that the SAH domain can functionally substitute for the canonical lever in myosin 5a in vitro. In cells, myosin 10 missing its SAH domain still moves to the tips of filopodia but with a reduced velocity.We have now determined that the SAH domains unfold at forces of less than ∼50 pN using single molecule force microscopy. Moreover, SAH domains unfold non-co-operatively during thermal melting, and have a high propensity to refold completely when the temperature is returned back to 10 °C, measured by circular dichroism. Molecular simulations agree with all the experimental data available, confirming the surprising stability of SAH domains and revealing unique properties that set the SAH domains apart. Among these is the ability to recover helical conformation rapidly after being extended through application of force.Thus SAH domains appear to function as ‘stretchable’ elements, unfolding and rapidly refolding at relatively low forces. In myosins, this may enable these motors to hold on to and traffic their cargoes in the crowded environment of the cell.