Understanding the Role of Protein Kinases Kin1 and Kin2 in the Protein Folding Pathways in the Yeast Saccharomyces Cerevisiae

Eukaryotic protein kinases catalyze the transfer of the -phosphate of an ATP to a serine/threonine/tyrosine residue present in a protein substrate. The phosphorylation of proteins has profound effects on their activity and protein-protein interactions, thus regulating a plethora of cellular processes, including cell growth, differentiation and protein homeostasis (or proteostasis). Our lab is the first to demonstrate that protein kinases Kin1 and its paralog Kin2 in the budding yeast Saccharomyces cerevisiae, orthologs of human microtubule affinity-regulating kinase (MARK), contribute to protein-folding homeostasis inside the endoplasmic reticulum (ER), in addition to their canonical roles in cellular exocytosis. The main aim of my studies is to fully understand the Kin kinase signaling pathway and how it contributes to the ER protein-folding homeostasis in the yeast Saccharomyces cerevisiae. Specifically, I study how Kin kinases are activated and what their upstream and downstream effectors are. My studies have revealed that the N-terminal half of Kin1 or Kin2 protein containing the kinase domain (KD) with a short kinase extension region (KER) was sufficient to complement the function of full-length Kin1 or Kin2. I have also found that phosphorylation of a single residue in Kin1 (Thr-302) or Kin2 (Thr-281) was important for their kinase domain function. Furthermore, I have found that phosphorylation of Thr-302 or Thr-281 occurred in trans by an upstream kinase. These results are published in Molecular and Cellular Biology. Further studies are directed towards identifying the Thr-302 or Thr-281 upstream kinase. One third of total cellular proteins fold and mature inside the E¬R. Due to abiotic or biotic stresses, unfolded proteins may accumulate inside the ER lumen, causing ER stress. During ER stress, a dual kinase RNase Ire1 is activated and it restores the ER protein-folding homeostasis in Saccharomyces cerevisiae as follows. The active Ire1 initiates a signaling pathway by removing an intervening sequence from the HAC1 mRNA by an unconventional splicing mechanism. Matured HAC1 mRNA then translates an active transcription factor Hac1, which enhances the expression of protein folding enzymes and chaperones that help mitigate ER stress. We and others have shown that HAC1 splicing requires co-localization of the HAC1 mRNA with the Ire1 protein, which is mediated by a bipartite element (BE) present in the 3’-UTR of the HAC1 mRNA. I have shown that the Kin kinases and a BE-RNA-protein complex (RNP) significantly contribute to HAC1 mRNA splicing. Here I have characterized and determined the role of a component of the proposed RNP, an uncharacterized protein Pal2. Our collaborator Dr. Benjamin Turk at Yale University identified a list of putative substrates of Kin kinases, using a phospho-proteomics based approach1. We have shown that Kin2 specifically phosphorylates the Ser-222 residue of Pal2. Further, molecular genetic studies showed that the yeast strain lacking Pal2 and its paralog Pal1 was deficient in maintaining ER protein homeostasis, which could be restored by expressing a wild-type Pal2 protein, but not by its unphosphorylated form. These data suggest that both Kin kinases and its substrate Pal2 significantly contribute to ER protein homeostasis. Overall, my finding of Pal2 phosphorylation by Kin kinases provides a novel mechanistic insight into the physiological signaling pathways mediated by the Kin kinases

Almost covering all the layers of hypercube with multiplicities

Given a hypercube $\mathcal{Q}^{n} := \{0,1\}^{n}$ in $\mathbb{R}^{n}$ and $k \in \{0, \dots, n\}$, the $k$-th layer $\mathcal{Q}^{n}_{k}$ of $\mathcal{Q}^{n}$ denotes the set of all points in $\mathcal{Q}^{n}$ whose coordinates contain exactly $k$ many ones. For a fixed $t \in \mathbb{N}$ and $k \in \{0, \dots, n\}$, let $P \in \mathbb{R}\left[x_{1}, \dots, x_{n}\right]$ be a polynomial that has zeroes of multiplicity at least $t$ at all points of $\mathcal{Q}^{n} \setminus \mathcal{Q}^{n}_{k}$, and $P$ has zeros of multiplicity exactly $t-1$ at all points of $\mathcal{Q}^{n}_{k}$. In this short note, we show that $$deg(P) \geq \max\left\{ k, n-k\right\}+2t-2.$$Matching the above lower bound we give an explicit construction of a family of hyperplanes $H_{1}, \dots, H_{m}$ in $\mathbb{R}^{n}$, where $m = \max\left\{ k, n-k\right\}+2t-2$, such that every point of $\mathcal{Q}^{n}_{k}$ will be covered exactly $t-1$ times, and every other point of $\mathcal{Q}^{n}$ will be covered at least $t$ times. Note that putting $k = 0$ and $t=1$, we recover the much celebrated covering result of Alon and F\"uredi (European Journal of Combinatorics, 1993). Using the above family of hyperplanes we disprove a conjecture of Venkitesh (The Electronic Journal of Combinatorics, 2022) on exactly covering symmetric subsets of hypercube $\mathcal{Q}^{n}$ with hyperplanes. To prove the above results we have introduced a new measure of complexity of a subset of the hypercube called index complexity which we believe will be of independent interest. We also study a new interesting variant of the restricted sumset problem motivated by the ideas behind the proof of the above result.Comment: 16 pages, substantial changes from previous version, title and abstract changed to better reflect the content of the pape

On higher multiplicity hyperplane and polynomial covers for symmetry preserving subsets of the hypercube

Alon and F\"uredi (European J. Combin. 1993) gave a tight bound for the following hyperplane covering problem: find the minimum number of hyperplanes required to cover all points of the n-dimensional hypercube {0,1}^n except the origin. Their proof is among the early instances of the polynomial method, which considers a natural polynomial (a product of linear factors) associated to the hyperplane arrangement, and gives a lower bound on its degree, whilst being oblivious to the (product) structure of the polynomial. Thus, their proof gives a lower bound for a weaker polynomial covering problem, and it turns out that this bound is tight for the stronger hyperplane covering problem. In a similar vein, solutions to some other hyperplane covering problems were obtained, via solutions of corresponding weaker polynomial covering problems, in some special cases in the works of the fourth author (Electron. J. Combin. 2022), and the first three authors (Discrete Math. 2023). In this work, we build on these and solve a hyperplane covering problem for general symmetric sets of the hypercube, where we consider hyperplane covers with higher multiplicities. We see that even in this generality, it is enough to solve the corresponding polynomial covering problem. Further, this seems to be the limit of this approach as far as covering symmetry preserving subsets of the hypercube is concerned. We gather evidence for this by considering the class of blockwise symmetric sets of the hypercube (which is a strictly larger class than symmetric sets), and note that the same proof technique seems to only solve the polynomial covering problem

Studies on synthesis and characterization of magnesia based refractory aggregates developed from Indian magnesite

The present work intends to study the properties of magnesia based refractory aggregates developed from Indian magnesite by changing lime/silica ratio. The material has been sintered in the temperature range of 1550C - 1700C. The sintered samples are characterized in terms of bulk density, apparent porosity, true density, percentage densification, mechanical, thermo-mechanical properties like cold modulus of rupture, hot modulus of rupture and thermal shock resistance and structural properties by XRD. The developed microstructures at different temperatures are studied through FESEM study and compositional analysis of the developed phases is done by EDX study

Effect of citric acid and polymer blend on characteristics of ofloxacin floating matrix tablets by factorial design

The present investigation deals with the development of floating matrix tablet containing Ofloxacin, to prolong the gastric residence time, thereby effective in eradication of Helicobacter pylori from the gastric mucosa. A 32 factorial design was employed to formulate floating matrix tablet selecting polymer blend ratio [hydroxypropyl methylcellulose (HPMC) / sodium carboxymethylcellulose (SCMC)] and content of citric acid as independent variables. Time required for 50 % of drug release (t50 %), percentage drug release at 8 h (Q8), floating duration (h) and diffusion exponent (n) were selected as dependent variables. Multiple regression analysis with two way ANOVA revealed statistically significant effect of the two independent variables on the responses studied (P 8 varied from ~ 76 % to ~100 % whereas t50 % ranged from 1.7 h to 3.7 h. The kinetics of drug release fitted best to Higuchi diffusion controlled model.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Studies on densification, mechanical, micro-structural and structure-properties relationship of magnesium aluminate spinel refractory aggregates prepared from Indian magnesite

The present work intends to study the development of magnesium aluminate spinel aggregates from Indian magnesite in a single firing stage. The raw magnesite has been evaluated in terms of chemical analysis, differential thermal analysis, thermogravimetric analysis, infrared spectroscopy, and X-ray diffraction. The experimental batch containing Indian magnesite and calcined alumina has been sintered in the temperature range of 1550 degrees C-1700 degrees C. The sintered material has been characterized in terms of physico-chemical properties like bulk density, apparent porosity, true density, relative density and thermo-mechanical/mechanical properties like hot modulus of rupture, thermal shock resistance, cold modulus of rupture and structural properties by X-ray diffraction in terms of phase identification and evaluation of crystal structure parameters of corresponding phases by Rietveld analysis. The microstructures developed at different temperatures have been analyzed by field emission scanning electron microscope study and compositional analysis of the developed phase has been carried out by energy dispersive X-ray study. (C) 2014 Elsevier Inc. All rights reserved

Effect of ZrO2 on the densification behavior and properties of Indian magnesite

Natural magnesite is the primary source for magnesia-based refractory materials. India has vast deposits of magnesite in Salem and Almora regions. However, due to the presence of large amount of impurities which forms low-melting compounds at elevated temperature, its high-temperature application is restricted. Raw magnesite was evaluated in terms of chemical analysis, differential thermal analysis, thermogravimetric analysis, and phase assemblage. Zirconia (1-5 wt %) was added to Indian natural magnesite of Salem region to minimize the low-melting phase formation at high temperatures. Samples were sintered in the temperature range of 1550-1700 degrees C. Sintered samples were characterized in terms of densification, mechanical and thermo-mechanical properties, phase assemblage, and microstructure. It was found that the addition of zirconia reduced the formation of detrimental phases like monticellite and thereby improved the high-temperature mechanical properties

Studies on densification, mechanical, microstructural and structure-properties relationship of refractory aggregates prepared from Indian magnesite by changing lime-silica ratio

The present work intends to study the development of refractory aggregates from Indian magnesite by modifying the lime silica ratio. The raw magnesite has been evaluated in terms of chemical analysis, DTA-TG, IR Spectroscopy, XRD analysis. The material has been sintered at temperatures ranging from 1550 degrees C to 1700 degrees C along with modification of the lime silica ratio. The sintered material has been characterized in terms of bulk density, apparent porosity, true density, relative density, cold modulus of rupture, hot modulus of rupture, thermal shock resistance, structural properties by XRD in terms of phase identification and evaluation of crystal structure parameters of corresponding phases by Rietveld analysis. The microstructures developed at different temperatures have been analyzed by FESEM study and compositional analysis of the developed phases has been carried out by EDAX study. (C) 2014 Elsevier Ltd and Techna Group S.r.l. All rights reserved

Effect of Yb2O3 and TiO2 on reaction sintering and properties of magnesium aluminate spinel

Magnesium aluminate spinel with an initial MgO: Al2O3 molar ratio of 2:1 was prepared from its constituent oxides through a solid-state sintering process at temperatures ranging from 1550 to 1700 degrees C in a normal air atmosphere. The effect of varying amount (0.25-1.0 wt%) of TiO2 and Yb2O3 on densification, phase assemblage, mechanical, thermo-mechanical properties and microstructure of magnesia-rich spinel were investigated under static heating condition. The addition of TiO2 and Yb2O3 favours the densification of magnesia-rich spinel, which is discernible up to 1650 degrees C. This beneficial effect may be attributed to the development of the secondary phase and formation of solid solution due to the dissolution of the additive ions in the spinel structure. A marginal increase in the average grain size of the samples along with a narrower grain size distribution occurred with the incorporation of both the additives. Both the additives improved the mechanical properties of the magnesia-rich spinel; however, better room temperature flexural strength was achieved with Yb2O3 as compared to TiO2 addition. For the samples sintered at 1550 degrees C, 1.0 wt% Yb2O3 addition resulted in 30% increase in flexural strength; however, same amount of TiO2 addition increased the strength by 20%. In case of thermal shock resistance, 1.0 wt% TiO2 and 0.25 wt% Yb2O3 addition demonstrated promising result among all the samples