Signal Transduction in tandem HAMP domains

The incidence of HAMP tandems in bacterial signaling proteins is low and presently it is unknown what physiological advantage may be gained by using a tandem. Presently a simple general mechanism of HAMP signaling which satisfactorily accounts for all experimental data cannot be presented. To study signal transduction via HAMP domains we used an in vitro biochemical system in which the signal output is affected exclusively by the HAMP domain that is inserted between the Tsr receptor as the input and the Rv3645 adenylyl cyclase as output domain. Initially neither the HAMP tandem nor its respective monomers operated as signal transducers in our system. The introduction of five targeted mutations in the first α-helix of NpHAMP1 which adapted this sequence somewhat to the equivalent Tsr sequence was required to obtain a functional, i.e. signal-transducing HAMP tandem. Replacement of the entire α-helix NpAS11-mut5 by the equivalent sequence of HAMPTsr the chimeric HAMP monomer (AS1Tsr/NpAS2) was fully operational in that serine strongly inhibited AC activity. Furthermore, in combination with NpHAMP2 in tandem the sign of the output signal was inverted as predicted. This left us with two HAMP tandem constructs with opposite outputs of the serine signal as initiated by serine-binding to the periplasmic domain of Tsr. The differences between both constructs were confined to the first α-helix of the first HAMP domain in the tandem as all other segments remained unchanged. Both constructs received the same conformational signal from Tsr. One might then reasonably speculate that the first α-helix (α-helix-1) is ultimately responsible for formation of different ground states of the output domain which leads to differences in signal output. In a series of experiments, AS1 of NpHAMP1 was extensively mutated to decipher which residues actually might determine such different states. In NpAS11-Tsr and NpAS11-mut5, five amino acid residues in α-helix-1 were responsible for defining opposite ground states. Just manipulating the α-helix-1 in a HAMP tandem was sufficient to produce opposite signaling outputs. The data do not permit making a similar claim for signal transduction through a HAMP monomer. This finding is hard to explain with rotation as a major HAMP signaling mechanism. In N. pharaonis sensory rhodopsin-I and its cognate transducer complex, SRI-HtrI has a dual function by mediating attractant and repellant responses whereas SRII-HtrII mediates only repellant responses. Both transducers have a HAMP tandem. In such a system signal rapid changes in signal input may require a fast track system for adaptation which has been reported for SRI-HtrI complex signaling. Our data allow the speculation that HAMP tandems by virtue of their intrinsic sequences prime a signal transduction system for a distinct organismal response to peculiar environmental cues such as light in N. pharaonis. The results complicate predictions of HAMP mediated signaling based on our current structural knowledge base. According to the gearbox model of HAMP signal transduction one might consider that HAMP1 may rotate in both directions. However, such an interpretation would clash with the fact that the signal emanating from the Tsr membrane receptor is the same irrespective of the type of HAMP domain attached to its C-terminal membrane exit. It is similarly questionable whether other proposals for signal transduction such as the piston or the dynamic bundle models alone could plausibly explain the above results. Rotation as one structural parameter for HAMP signal transduction is not excluded, rather it ought to be seen in conjunction with other molecular movements which might control four helix bundle stability in essence by regulated unfolding. Stability in this context is not restricted to the HAMP module alone but includes adjacent regions with which the HAMP domain is in a continuous structural balance. The possibility to switch the sign of the output signal by a single amino acid mutation in a HAMP tandem context may be an evolutionary advantage in the multiplicity of HAMP-mediated signaling systems and may expand the versatility of such units.HAMP* vermittelte Signaltransduktion ist allgegenwärtig (> 28.000 HAMP Datenbank Einträge). In den meisten Fällen ist zwischen einem Sensor und einem Ausgabemodul eine HAMP Domäne (HAMP Monomer) eingesetzt. Sie dient wahrscheinlich als Adapter zwischen der Sensor- und Effektordomäne. Der vorgeschlagene Mechanismus der HAMP Signaltransduktion durch Drehung wurde durch eine Kristallstruktur einer seriellen dreifach-HAMP aus Pseudomonas aeruginosa gestärkt. Das Rotationsmodell würde vorhersagen, dass sich mit jeder zusätzlichen HAMP Domäne das Vorzeichen des Ausgangssignals umkehrt. Diese Vorhersage wurde durch biochemische Experimente überprüft, indem eine HAMP-Tandem Domäne des HtrII Photorezeptors aus N. pharaonis verwendet wurde. Das grundlegende Design unserer getesteten Konstrukte mit Tsr als Sensor und Rv3645 AC als Effektor wurde beibehalten. Das grundlegende Design unserer getesten Konstrukte war jeweils Tsr als Sensor und Rv3645 AC als Effecktor mit der zu untersuchenden HAMP Domäne dazwischen. Es war nicht verwunderlich, dass zunächst weder das HAMP-Tandem noch seine jeweiligen Monomere in den getesteten Konstrukten als Signalgeber fungierten, weil in NpHtrII das Lichtsignal zwischen sensorischem Rhodopsin II und der Chemotaxiseinheit HtrII innerhalb der Membran weiter gegeben wird. Die Einführung von fünf gezielten Mutationen in der ersten α-Helix von NpHAMP1, die deren Sequenz stärker an die von Tsr angleicht, war erforderlich, um eine funktionale, d.h. signaltransduzierende HAMP-Tandem Domäne zu erhalten. Beide HAMP Monomere allein waren inaktiv als Signalgeber. Der überraschende Befund war, dass das Vorzeichen des Ausgangssignals nicht wie vorhergesagt umgedreht wurde.Wenn die gesamte erste α-Helix (AS1) im NpHAMP Monomer durch die äquivalente α-Helix von HAMPTsr ersetzt wurde (AS11-Tsr/NpH1), wurde das Konstrukt gehemmt. Mit NpHAMP2 als HAMP-Tandem allerdings wurde das Vorzeichen des Ausgangssignals invertiert, d.h. das Tandem-Konstrukt wurde aktiviert. Die Unterschiede im Ausgangssignal zwischen beiden Konstrukten können der ersten α-Helix der ersten HAMP Domäne im Tandem zugerechnet werden, da alle anderen Segmente unverändert blieben. Man kann annehmen, dass die erste α-Helix letztlich verantwortlich ist für die Bildung von unterschiedlichen Grundzuständen der Effektordomäne.In einer Serie von Experimenten wurde durch zahlreiche Mutationen in AS1 von NpHAMP1 untersucht, welche Aminosäuren die verschiedenen Zustände bestimmen. Das Ergebnis von 48 Mutationen in NpAS11-mut5 und AS11-Tsr/NpH1 Tandem Konstrukten war, dass fünf Aminosäuren für die gegensätzlichen Grundzustände verantwortlich waren. Dies würde dafür sprechen, dass das Signal, das vom Tsr Membranrezeptor ausgeht, immer das gleiche ist, unabhängig von der Art der angeschlossenen HAMP Domäne. So ist es fraglich, ob andere Modelle der Signaltransduktion wie z.B. das Kolben-Modell oder das ''dynamic bundle model'' die obigen Ergebnisse plausibel erklären. Rotation als alleiniger struktureller Parameter für die HAMP Signaltransduktion wird kaum ausreichen, sondern sollte in Verbindung mit anderen molekularen Bewegungen gesehen werden, welche Einfluss auf die Stabilität des Vierhelixbündels der HAMP Domäne nehmen können. Hierzu lässt sich z.B. das „regulated unfolding’’ nennen. Bei dieser These ist die Stabilität nicht auf das HAMP Modul allein beschränkt, sondern umfasst auch benachbarte Bereiche, mit denen sich die HAMP Domäne in einem kontinuierlichen Strukturgleichgewicht befindet. Eine plausible Interpretation wäre, dass HAMP Domänen verschiedene Grundzustände eines sensorischen Systems definieren und entsprechend gegensätzliche physiologische Reaktionen auslösen können. In dem einen Grundzustand lagert sich das katalytische AC Homodimer richtig zusammen, sodass bei Stimulation des Tsr Rezeptors sich die Untereinheiten voneinander distanzieren, wodurch es zu einer Hemmung der Enzymaktivität kommt. Im Gegensatz dazu kommen im anderen Grundzustand die Untereinheiten durch ein Serinsignal zusammen, was zu einer erhöhten Enzymaktivität führt. *- Histidine kinases, Adenylyl cyclases, Methyl-accepting chemotaxis proteins and Phosphatases

An Analyzation of UI/UX of Tuudo

Presently, the Tuudo company is diligently engaged in enhancing and advancing its career paths feature and the overall Tuudo application. In pursuit of this objective, the company seeks valuable insights and recommendations derived from real user experiences. This thesis project aims to provide an in-depth analysis of Tuudo, focusing on its project structure, key features, and a comprehensive comparison with alternative applications in the market. The project begins by introducing essential terminologies to establish a foundation of understanding for the reader. It then delves into Tuudo's background as both an app and a company, providing relevant information to contextualize the subsequent analysis. A detailed examination of Tuudo's key features follows, with particular emphasis on the career paths feature, offering valuable insights into its functionality and purpose. To support the research, user surveys and user research are conducted, generating valuable data and feedback. The results of these efforts are presented, providing insights into user perceptions, experiences, and preferences regarding Tuudo and its features. Additionally, the comparative analysis provides information on the positioning of Tuudo in the market and enables a comprehensive evaluation of its strengths and weaknesses in comparison to its alternatives. Overall, this thesis study provides insightful information about Tuudo, its features, and its competitive landscape. It provides a more in-depth understanding of the app's functionality and possibilities for improvement, making it a significant resource for the Tuudo company to further proceed

CAPRIN1(P512L) causes aberrant protein aggregation and associates with early-onset ataxia

CAPRIN1 is a ubiquitously expressed protein, abundant in the brain, where it regulates the transport and translation of mRNAs of genes involved in synaptic plasticity. Here we describe two unrelated children, who developed early-onset ataxia, dysarthria, cognitive decline and muscle weakness. Trio exome sequencing unraveled the identical de novo c.1535C > T (p.Pro512Leu) missense variant in CAPRIN1, affecting a highly conserved residue. In silico analyses predict an increased aggregation propensity of the mutated protein. Indeed, overexpressed CAPRIN1(P512L) forms insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders (ATXN2, GEMIN5, SNRNP200 and SNCA). Moreover, the CAPRIN1(P512L) mutation in isogenic iPSC-derived cortical neurons causes reduced neuronal activity and altered stress granule dynamics. Furthermore, nano-differential scanning fluorimetry reveals that CAPRIN1(P512L) aggregation is strongly enhanced by RNA in vitro. These findings associate the gain-of-function Pro512Leu mutation to early-onset ataxia and neurodegeneration, unveiling a critical residue of CAPRIN1 and a key role of RNA-protein interactions

Estimating the costs of blindness and moderate to severe visual impairment among people with diabetes in India

Objectives This study provides an estimate of the annual cost of blindness and moderate to severe visual impairment (MSVI) among people with diabetes aged 40 years and above in India in the year 2019.Design A cost of illness study.Setting India.Participants People with diabetes aged 40 years and above in India in the year 2019.Primary and secondary outcome measures Estimates are provided for the total costs of screening for most common vision-threatening eye conditions, treatment of these conditions, economic activity lost by these people and their family carers whose ability to work is affected, and loss of quality of life experienced by people with diabetes and blindness or MSVI.Results It is estimated that for people with diabetes aged 40 years or above, annual screening followed by eye examination where required would cost around 42.3 billion Indian rupees (INR) (4230 crores) per year; treating sight problems around 2.87 billion INR (287 crores) per year if 20% of those needing treatment receive it; and lost economic activity around 472 billion INR (47 200 crores). Moreover, 2.86 million (0.286 crores) quality-adjusted life years (QALYs) are lost annually due to blindness and MSVI. The estimate of lost production is highly sensitive to the proportion of people with MSVI able to work and how their output compares with that of a person with no visual impairment.Conclusions This is the first study to estimate the cost of blindness and MSVI for people aged 40 years and over with diabetes in India. The annual cost to the Indian economy is substantial. This cost will be expected to fall if a successful screening and treatment plan is introduced in India. Further work is suggested using more robust data, when available, to estimate the loss of productivity and loss of QALYs, as this would be worthwhile

Genome-wide SWAp-Tag yeast libraries for proteome exploration

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.</p