The mammalian Translocon-Associated Protein (TRAP) complex and co- translational protein transport

In eukaryotes, many proteins translocate into the endoplasmic reticulum (ER) and some insert themselves into the membrane. In 1971, G. Blobel proposed the “Signal Hypothesis”, explaining how proteins translocate from the cytosol into the ER. The signal peptides (SPs) have a tripartite structure but heterogeneous peptide sequence composition. SP complexity is essential for many processes, such as signal recognition particle binding, translocon gating, early folding prevention, and signal peptidase interaction/cleavage, as well as post-cleavage functions, such as antigen presentation. ER protein translocation takes place through the heterotrimeric Sec61 protein-conducting channel (PCC). Sec61α is a multi-spanning membrane protein that forms a complex with the single-spanning partners Sec61β and Sec61γ. During co-translational translocation, Sec61 is associated with the ribosome (via two cytosolic loops) and many accessory components (ribosome-channel complex), such as the translocon-associated protein (TRAP) complex. TRAP has four subunits—TRAP α (ssr1), TRAP β (ssr2), TRAP γ (ssr3), and TRAP δ (ssr4). The subunits α, β, and δ are single-spanning transmembrane (TM) proteins with luminal and cytosolic domains (type I), while the subunit γ has four TM domains and a prominent cytosolic domain. Recently, microscopic techniques, such as cryo-EM and cryo-ET, have enabled the determination of the translocation machinery structure. However, at present there is a lack of understanding regarding the roles of some of its components and domains. Protein function is determined by many different aspects, including localisation, sequence, structure, expression, post- translational modifications, and interactions. The present study aimed to contribute to the understanding of TRAP functions. Analyses of protein-protein interactions (PPIs), sequences (motifs), and expressions were carried out using experimental and computational methods. Importantly, we found that the TRAP complex interacts with the translocon Sec61 (peptide array). These PPIs may be essential for translocating substrates or stabilising translocon machinery. The PPIs occur in the ER luminal side between Sec61α1 loop 5 and TRAP α/β subunits. The latter also interact with one another, as is expected for elements of a complex (pull-down assays). The computational analysis identified a calcium-binding domain at the N-terminus of the TRAP α subunit, which may have a functional role. In Eukaryonten müssen viele Proteine in das ER transferiert werden, und einige von ihnen werden in die Membran eingebracht. Ein Meilenstein im Verständnis der Protein-Translokation ist die von G. Blobel 1971 vorgeschlagene "Signalhypothese", die erklärt, wie die Proteine aus dem Cytosol in das ER transloziert werden. Das Signalpeptid (SP) hat typischerweise eine dreiteilige Struktur, ist aber in der Aminosäuresequenz sehr heterogen, was eine schnelle Entwicklung im Verlauf der Evolution impliziert, die wahrscheinlich mit dem reifen Protein verbunden ist. Heutzutage wissen wir, dass die Komplexität des SP mit vielen biologischen Prozessen verbunden ist: Signalerkennungspartikel (SRP)-Bindung, translokale Interaktion (Gating), frühe Faltungsprävention, Signalpeptidase (SPase)-Interaktion und - Spaltung und sogar Post-Cleavage- Funktionen wie Antigenpräsentation. Die Translokation erfolgt über einen heterotrimeren proteinleitenden Kanal (PCC): Sec61α ist ein multi- spannendes Membranprotein und bildet mit den Single-Spanning-Partnern Sec61β und Sec61γ einen Komplex. Während der kotranslationalen Translokation ist Sec61 dem Ribosom (über zwei zytosolische Schleifen) und einer großen Anzahl von weiteren Komponenten zugeordnet (RCC, Ribosom-Kanal- Komplex). Zu diesen zusätzlichen Bestandteilen gehört der heterotetramere Translocon-Associated Protein (TRAP)-Komplex aus TRAP α (ssr1), TRAP β (ssr2), TRAP γ (ssr3) und TRAP δ (ssr4). Die Untereinheiten α, β und δ sind Single-Spanning- Transmembran (TM)-Proteine mit ER-luminalen und zytosolischen Domänen, während die γ Untereinheit vier TM-Domänen und eine prominente zytosolische Domäne aufweist. In letzter Zeit wurden große Fortschritte bei der Untersuchung der Struktur der Translokationsmaschinen erzielt, auch dank der verbesserten mikroskopischen Techniken wie Cryo-EM und Cryo-ET; die Forschung hat jedoch stets gezeigt, dass das Verständnis für die Rolle(n) einiger ihrer Komponenten und Domänen fehlt. Eine Proteinfunktion wird unter Berücksichtigung vieler Aspekte untersucht: intrazelluläre Lokalisation, Sequenz, Struktur, Expressionsprofil, post-translationale Modifikationen, Interaktionen. Der Hauptzweck dieser Forschungsarbeit ist es, zum Verständnis der TRAP- Funktion(en) beizutragen, indem Sequenzen (Motive), Expressionen und vor allem Protein- Protein-Interaktionen innerhalb des Komplexesund mit den umgebenden Strukturen durch computergestützte und experimentelle Methoden analysiert werden. Die relevanteste Erkenntnis ist, dass der TRAP-Komplex mit dem Translokon interagiert (Peptid- Array). Diese Wechselwirkungen könnten für die Translokation einiger Substrate oder auch nur für die Stabilisierung der Translokomaschinerie von wesentlicher Bedeutung sein. Die Interaktionen finden auf der ER-Lumenseite zwischen Sec61α1 loop 5 und den TRAP α /β Untereinheiten statt, letztere interagieren auch untereinander, wie es für Elemente eines Komplexes erwartet wird (Pulldown-Assay). Die Computeranalyse zeigt eine Calcium- Bindungsdomäne am N-Terminus der TRAP alpha Untereinheit auf, die eine funktionelle Rolle spielen könnte.In Eukaryonten müssen viele Proteine in das ER transferiert werden, und einige von ihnen werden in die Membran eingebracht. Ein Meilenstein im Verständnis der Protein-Translokation ist die von G. Blobel 1971 vorgeschlagene "Signalhypothese", die erklärt, wie die Proteine aus dem Cytosol in das ER transloziert werden. Das Signalpeptid (SP) hat typischerweise eine dreiteilige Struktur, ist aber in der Aminosäuresequenz sehr heterogen, was eine schnelle Entwicklung im Verlauf der Evolution impliziert, die wahrscheinlich mit dem reifen Protein verbunden ist. Heutzutage wissen wir, dass die Komplexität des SP mit vielen biologischen Prozessen verbunden ist: Signalerkennungspartikel (SRP)-Bindung, translokale Interaktion (Gating), frühe Faltungsprävention, Signalpeptidase (SPase)-Interaktion und - Spaltung und sogar Post-Cleavage- Funktionen wie Antigenpräsentation. Die Translokation erfolgt über einen heterotrimeren proteinleitenden Kanal (PCC): Sec61α ist ein multi- spannendes Membranprotein und bildet mit den Single-Spanning-Partnern Sec61β und Sec61γ einen Komplex. Während der kotranslationalen Translokation ist Sec61 dem Ribosom (über zwei zytosolische Schleifen) und einer großen Anzahl von weiteren Komponenten zugeordnet (RCC, Ribosom-Kanal- Komplex). Zu diesen zusätzlichen Bestandteilen gehört der heterotetramere Translocon-Associated Protein (TRAP)-Komplex aus TRAP α (ssr1), TRAP β (ssr2), TRAP γ (ssr3) und TRAP δ (ssr4). Die Untereinheiten α, β und δ sind Single-Spanning- Transmembran (TM)-Proteine mit ER-luminalen und zytosolischen Domänen, während die γ Untereinheit vier TM-Domänen und eine prominente zytosolische Domäne aufweist. In letzter Zeit wurden große Fortschritte bei der Untersuchung der Struktur der Translokationsmaschinen erzielt, auch dank der verbesserten mikroskopischen Techniken wie Cryo-EM und Cryo-ET; die Forschung hat jedoch stets gezeigt, dass das Verständnis für die Rolle(n) einiger ihrer Komponenten und Domänen fehlt. Eine Proteinfunktion wird unter Berücksichtigung vieler Aspekte untersucht: intrazelluläre Lokalisation, Sequenz, Struktur, Expressionsprofil, post-translationale Modifikationen, Interaktionen. Der Hauptzweck dieser Forschungsarbeit ist es, zum Verständnis der TRAP- Funktion(en) beizutragen, indem Sequenzen (Motive), Expressionen und vor allem Protein- Protein-Interaktionen innerhalb des Komplexesund mit den umgebenden Strukturen durch computergestützte und experimentelle Methoden analysiert werden. Die relevanteste Erkenntnis ist, dass der TRAP-Komplex mit dem Translokon interagiert (Peptid- Array). Diese Wechselwirkungen könnten für die Translokation einiger Substrate oder auch nur für die Stabilisierung der Translokomaschinerie von wesentlicher Bedeutung sein. Die Interaktionen finden auf der ER-Lumenseite zwischen Sec61α1 loop 5 und den TRAP α /β Untereinheiten statt, letztere interagieren auch untereinander, wie es für Elemente eines Komplexes erwartet wird (Pulldown-Assay). Die Computeranalyse zeigt eine Calcium- Bindungsdomäne am N-Terminus der TRAP alpha Untereinheit auf, die eine funktionelle Rolle spielen könnte

Long-term outcomes of direct acting antivirals in post-transplant advanced hepatitis C virus recurrence and fibrosing cholestatic hepatitis.

Long-term functional outcomes of sofosbuvir-based antiviral treatment were evaluated in a cohort study involving 16 Italian centres within the international compassionate use programme for post-transplant hepatitis C virus (HCV) recurrence. Seventy-three patients with cirrhosis (n=52) or fibrosing cholestatic hepatitis (FCH, n=21) received 24-week sofosbuvir with ribavirin\ub1pegylated interferon or interferon-free sofosbuvir-based regimen with daclatasvir/simeprevir+ribavirin. The patients were observed for a median time of 103 (82-112) weeks. Twelve of 73 (16.4%) died (10 non-FCH, 2 FCH) and two underwent re-LT. Sustained virological response was achieved in 46 of 66 (69.7%): 31 of 47 (66%) non-FCH and 15 of 19 (79%) FCH patients. All relapsers were successfully retreated. Comparing the data of baseline with last follow-up, MELD and Child-Turcotte-Pugh scores improved both in non-FCH (15.3\ub16.5 vs 10.5\ub13.8, P<.0001 and 8.4\ub12.1 vs 5.7\ub11.3, P<.0001, respectively) and FCH (17.3\ub15.9 vs 10.1\ub12.8, P=.001 and 8.2\ub11.6 vs 5.5\ub11, P=.001, respectively). Short-treatment mortality was higher in patients with baseline MELD 6525 than in those with MELD<25 (42.9% vs 4.8%, P=.011). Long-term mortality was 53.3% among patients with baseline MELD 6520 and 7.5% among those with MELD<20 (P<.0001). Among deceased patients 75% were Child-Turcotte-Pugh class C at baseline, while among survivors 83.9% were class A or B (P<.0001). Direct acting antivirals-based treatments for severe post-transplant hepatitis C recurrence, comprising fibrosing cholestatic hepatitis, significantly improve liver function, even without viral clearance and permit an excellent long-term survival. The setting of severe HCV recurrence may require the identification of "too-sick-to-treat patients" to avoid futile treatments

Bibliografia degli scritti di Illuminato Peri

Bibliografia degli scritti di Illuminato Peri (1925-1996), professore ordinario di Storia Medievale presso la Facoltà di Magistero dell'Università di Palerm

I testamenti di Matteo Sclafani (1333-1354)

Edizione dei quattro testamenti di Matteo Sclafani (1333, 1345, 1348, 1354). La lettura dei quattro testamenti permette di delineare la parabola della vita del conte di Adernò, dall'ascesa ai vertici della società siciliana del Trecento, all'espulsione da Palermo e al sequestro dei beni. La comparazione dei testamenti permette, inoltre, di ricostruire il ruolo politico- economico e militare dello Sclafani e i suoi rapporti familiari

Beatrice Rosso Spatafora e i Luna (XV secolo)

Beatrice Rosso Spatafora, contessa di Sclafani e signora di Caltavuturo, sposa in prime nozze Carlo Luna conte di Caltabellotta e, dopo aver ottenuto la sentenza di nullità del matrimonio per impotentia coeundi del marito e la dispensa per la consanguineità, sposa l’ex cognato Sigismondo Luna. Attraverso le vicende legate al duplice matrimonio, il saggio dà un contributo alla ricostruzione della storia di una famiglia, i Luna, che, per il ramo insediatosi in Sicilia alla fine del XIV secolo, è ancora poco nota.Beatrice Rosso Spatafora, countess of Sclafani and Lady of Caltavuturo, first married Carlo Luna, earl of Caltabellotta. After obtaining the annulment of this marriage on the grounds of the impotentia coeundi of the husband and receiving dispensation from the impediment of consanguinity, she then married her former brother-in-law Sigismondo Luna. Through the events related to the double wedding, this essay helps reconstruct the history of the Luna family, focussing specifically on the branch which settled in Sicily at the end of the fourteenth century, about which little is known

Bibliografia ragionata degli scritti di Laura Sciascia

Bibliografia ragionata degli scritti di Laura Sciasci

Matteo Sclafani: paura della morte e desiderio di eternità

Nel saggio si delinea la parabola della vita di Matteo Sclafani, conte di Adernò, seguendo il mutare delle sue volontà testamentarie, dall'ascesa ai vertici della società siciliana del Trecento all'espulsione dall'amata Palermo, dalla formazione di un vasto patrimonio al sequestro dei beni, dall'ambizione di vedere perpetuati nome e insegne familiari alla vanificazione del suo desiderio

THE INHIBITORY EFFECT OF PROPOLIS AND CAFFEIC ACID PHENETHYLESTER ON CYCLOOXYGENASE ACTIVITY IN J774 MACROPHAGES.

The effect of an ethanolic extract of propolis, with and without CAPE, and some of its components on cyclooxygenase (COX-1 and COX-2) activity in J774 macrophages has been investigated. COX-1 and COX-2 activity, measaured as prostaglandin E-2 (PGE(2)) production, were concentration-dependently inhibited by propolis (C x 10(-3)-3 x 10(2) mugml(-1)) with an IC50 of 2.7 mugml(-1) and 4.8 x 10(-2) mugml(-1), respectively. Among the compounds tested pinocembrin and caffeic, ferulic, cinnamic and chlorogenic acids did not affect the activity of COX isoforms. Conversely, CAPE (2.8 x 10(-4)-28 mugml(-1); 10(-9)-10(-4) M) and galangin (2.7 x 10(-4)-27 mugml(-1); 10(-9)-10(-4) M) were effective, the last being about ten-twenty times less potent. In fact the IC50 of CAPE for COX-1 and COX-2 were 4.4 x 10(-1) mugml(-1) (1.5 x 10(-6) M) and 2 x 10(-3) mugml(-1) (6.3 x 10(-9) M), respectively. The IC50 of galangin were 3.7 mugml(-1) (15 x 10(-6) M) and 3 x 10(-2) mugml(-1) (120 x 10(-1) M), for COX-1 and COX-2 respectively. To better investigate the role of CAPE, we tested the action of the ethanolic extract of propolis deprived of CAPE, which resulted about ten times less potent than the extract with CAPE in the inhibition of both COX-1 and COX-2, with an IC50 of 30 mugml(-1) and 5.3 x 10(-1) mugml(-1), respectively. Moreover the comparison of the inhibition curves showed a significant difference (p < 0.001). These results suggest that both CAPE and galangin contribute to the overall activity of propolis, CAPE being more effective

Spontaneous intracranial hypotension : two steroid-responsive cases

Purpose: Spontaneous intracranial hypotension (SIH) is characterised by orthostatic headache, low cerebrospinal fluid pressure and diffuse pachymeningeal enhancement after intravenous gadolinium contrast administration. Magnetic resonance imaging (MRI) often plays a crucial role for correct diagnosis. Case description: We described two similar cases of SIH, whose clinical and imaging features are typical for this pathology. At MRI brain scan, both patients showed diffuse and intense pachymeningeal enhancement and moderate venous distension and epidural vein engorgement. The two patients were treated with bed rest and oral steroid therapy, with complete and long-lasting symptomatic relief. Conclusions: Orthostatic nature of headache is the most indicative clinical feature suggesting SIH; contrast-enhanced MRI provides definite imaging diagnostic findings. Conservative treatment coupled to steroid therapy is often sufficient to obtain complete disappearance of symptoms

Effects of hyperglycaemia on small intestinal and anorectal motor function in humans

This thesis presents studies which relate to the effects of changes in the blood glucose concentration on the organisation and control of motility in the small intestine and anorectum. The motor mechanisms by which acute hyperglycaemia slows small intestinal transit in healthy humans were investigated. Hyperglycaemia (blood glucose concentration -15mmol/l) stimulated phase III activity, but there was overall suppression of small intestinal pressure waves' when compared to euglycaemia- These results indicate that marked hyperglycaemia has major effects on small intestinal motility in normal subjects. The observed suppression of small intestinal motility provides a possible mechanism for the slowing of small intestinal transit during hyperglycaemia. Among the candidate mechanisms mediating the effects of hyperglycaemia on small intestinal motility is modification of tonic inhibition mediated by enteric nerves containing nitric oxide, The effects of a specific inhibitor of nitric oxide (NO) synthase, NG-monomethyl-L-arginine (L-NMMA), on small intestinal motor activity, were evaluated in healthy human volunteers. Administration of L-NMMA was associated with stimulation of small intestinal phase III activity and a reduction in the duration of phase I activity. These results indicate that NO mechanisms are involved in the initiation of small intestinal phase III activity- Disordered defaecation occurs commonly in patients with diabetes mellitus and is associated with heterogenous anorectal motor and sensory dysfunctions' These abnormalities may potentially be due to hyperglycaemia, rather than irreversible neural dysfunction. In healthy humans, measurements of anorectal motility and sensation were performed, during euglycaemia (4mmol/l) and hyperglycaemia (8mmol/l and 12mmol/l). At a blood glucose concentration of 12mmol/l the number of spontaneous internal anal sphincter relaxations was greater and the strength of the external anal sphincter less when compared to euglycaemia. The threshold for perception of rectal balloon distension was lower at a blood glucose of 12mmol/l when compared to 4mmol/l. These observations demonstrate that acute changes in the blood glucose concentration affect both the smooth and striated muscle of the anal sphincter, as well as rectal sensation, and suggest that hyperglycaemia may contribute to incontinence in patients with diabetes mellitus. Autonomic nervous system dysfunction occurs frequently in patients with diabetes mellitus and is associated with disordered gastrointestinal motor function. The possibility that the blood glucose concentration may affect cardiovascular autonomic (parasympathetic and sympathetic) function was evaluated in healthy human volunteers by performing paired studies during euglycaemia and hyperglycaemia (∼15mmol/l). Hyperglycaemia was associated with changes in cardiovascular parasympathetic function. This observation indicates that acute changes in the blood glucose concentration affect cardiovascular autonomic function, which may be important in mediating the effects of hyperglycaemia on gastrointestinal motor function. The studies reported in this thesis provide new insights into the control of gastrointestinal motility in healthy humans and effects of changes in the blood glucose concentration. The latter observations are likely to be relevant to an understanding of gastrointestinal motor function in patients with diabetes mellitus.Thesis (M.Med.Sc.)-- University of Adelaide, Dept. of Medicine, 199