Effects of Biomolecular Crowding on Protein Stability

The intracellular milieu is complex, heterogeneous and crowded-- an environment vastly different from dilute, buffered solutions where most biophysical studies are performed. The cytoplasm excludes about a third of the volume available to macromolecules in dilute solution. This exclusion arises from the sum of two phenomena: steric repulsions and chemical interactions, often called hard and soft interactions, respectively. Most efforts to understand crowding have focused on steric repulsions. Globular protein stability is the difference in free energy between the compact, biologically functional native state and the ensemble of less compact, nonfunctional denatured state. The hard-core repulsive component of crowding stabilizes globular proteins because the decrease in available volume favors compact species. The effect of soft interactions can be stabilizing or destabilizing. Soft repulsive interactions reinforce the stabilizing influence of hard-core repulsions. However, the equilibrium is shifted towards the denatured state in systems dominated by attractive non-specific interactions, because unfolding exposes more reactive surface. In Chapter 1, I introduce the concepts of hard and soft interactions in more depth and discuss how they are expected to affect the equilibrium thermodynamic stability of globular proteins. In Chapter 2, I describe experiments that test these concepts by using Escherichia coli cell lysates as the crowding agents, chymotrypsin inhibitor 2 (CI2) as the test protein and NMR-detected amide-proton exchange to measure stability. The lysate destabilizes CI2, and the destabilization increases with increasing lysate concentration. This observation shows that the cytoplasm interacts favorably, but non-specifically, with CI2, and these interactions overcome the stabilizing hard-core repulsions. In fact, the effects of the lysate are even stronger than those of homogeneous protein crowders, reinforcing the biological significance of weak, non-specific interactions. In Chapter 3, I test the idea that the net charge on the crowding proteins affects stability. To accomplish this goal, I isolated the anionic proteins from the lysate and used them as the crowding agent. CI2 is an anion under the chosen conditions, and, therefore, I expected the net repulsive interactions between CI2 and the crowders to increase the stability of CI2. Instead, the refined lysate also resulted in destabilization. Thus, even the anionic proteins, which have the same net charge as CI2, significantly interact with CI2's backbone non-specifically to overcome the stabilizing effect of steric repulsion. My in vitro studies show that weak chemical interactions play key roles in the cytosol. It will even be more difficult to identify soft interactions in living cells where reductionist approaches are difficult or impossible to apply. Nevertheless, endeavors aimed at quantifying soft interactions are essential for producing a physiologically relevant understanding of biophysics. Once the details of soft interactions are known, it should be possible to tune them so as to obtain bespoke behavior in test tubes and in cells. In summary, despite their weak and non-specific behavior, biologists of all types need to keep these interactions in mind when designing experiments to correlate in vitro studies with in vivo behavior.Doctor of Philosoph

An osmolyte mitigates the destabilizing effect of protein crowding: Crowding & Osmolytes

Most theories predict that macromolecular crowding stabilizes globular proteins, but recent studies show that weak attractive interactions can result in crowding-induced destabilization. Osmolytes are ubiquitous in biology and help protect cells against stress. Given that dehydration stress adds to the crowded nature of the cytoplasm, we speculated that cells might use osmolytes to overcome the destabilization caused by the increased weak interactions that accompany desiccation. We used NMR-detected amide proton exchange experiments to measure the stability of the test protein chymotrypsin inhibitor 2 under physiologically relevant crowded conditions in the presence and absence of the osmolyte glycine betaine. The osmolyte overcame the destabilizing effect of the cytosol. This result provides a physiologically relevant explanation for the accumulation of osmolytes by dehydration-stressed cells

Amide proton exchange of a dynamic loop in cell extracts

Intrinsic rates of exchange are essential parameters for obtaining protein stabilities from amide 1H exchange data. To understand the influence of the intracellular environment on stability, one must know the effect of the cytoplasm on these rates. We probed exchange rates in buffer and in Escherichia coli lysates for the dynamic loop in the small globular protein chymotrypsin inhibitor 2 using a modified form of the nuclear magnetic resonance experiment, SOLEXSY. No significant changes were observed, even in 100 g dry weight L−1 lysate. Our results suggest that intrinsic rates from studies conducted in buffers are applicable to studies conducted under cellular conditions

High monocytic MDSC signature predicts multi-drug resistance and cancer relapse in non-Hodgkin lymphoma patients treated with R-CHOP

IntroductionNon-Hodgkin Lymphoma (NHL) is a heterogeneous lymphoproliferative malignancy with B cell origin. Combinatorial treatment of rituximab, cyclophsphamide, hydroxydaunorubicin, oncovin, prednisone (R-CHOP) is the standard treatment regimen for NHL, yielding a complete remission (CR) rate of 40-50%. Unfortunately, considerable patients undergo relapse after CR or initial treatment, resulting in poor clinical implications. Patient’s response to chemotherapy varies widely from static disease to cancer recurrence and later is primarily associated with the development of multi-drug resistance (MDR). The immunosuppressive cells within the tumor microenvironment (TME) have become a crucial target for improving the therapy efficacy. However, a better understanding of their involvement is needed for distinctive response of NHL patients after receiving chemotherapy to design more effective front-line treatment algorithms based on reliable predictive biomarkers.MethodsPeripheral blood from 61 CD20+ NHL patients before and after chemotherapy was utilized for immunophenotyping by flow-cytometry at different phases of treatment. In-vivo and in-vitro doxorubicin (Dox) resistance models were developed with murine Dalton’s lymphoma and Jurkat/Raji cell-lines respectively and impact of responsible immune cells on generation of drug resistance was studied by RT-PCR, flow-cytometry and colorimetric assays. Gene silencing, ChIP and western blot were performed to explore the involved signaling pathways.ResultsWe observed a strong positive correlation between elevated level of CD33+CD11b+CD14+CD15- monocytic MDSCs (M-MDSC) and MDR in NHL relapse cohorts. We executed the role of M-MDSCs in fostering drug resistance phenomenon in doxorubicin-resistant cancer cells in both in-vitro, in-vivo models. Moreover, in-vitro supplementation of MDSCs in murine and human lymphoma culture augments early expression of MDR phenotypes than culture without MDSCs, correlated well with in-vitro drug efflux and tumor progression. We found that MDSC secreted cytokines IL-6, IL-10, IL-1β are the dominant factors elevating MDR expression in cancer cells, neutralization of MDSC secreted IL-6, IL-10, IL-1β reversed the MDR trait. Moreover, we identified MDSC secreted IL-6/IL-10/IL-1β induced STAT1/STAT3/NF-κβ signaling axis as a targeted cascade to promote early drug resistance in cancer cells.ConclusionOur data suggests that screening patients for high titre of M-MDSCs might be considered as a new potential biomarker and treatment modality in overcoming chemo-resistance in NHL patients

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 1

Protein Crowder Charge and Protein Stability

Macromolecular crowding effects arise from steric repulsions and weak, nonspecific, chemical interactions. Steric repulsions stabilize globular proteins, but the effect of chemical interactions depends on their nature. Repulsive interactions such as those between similarly charged species should reinforce the effect of steric repulsions, increasing the equilibrium thermodynamic stability of a test protein. Attractive chemical interactions, on the other hand, counteract the effect of hard-core repulsions, decreasing stability. We tested these ideas by using the anionic proteins from <i>Escherichia coli</i> as crowding agents and assessing the stability of the anionic test protein chymotrypsin inhibitor 2 at pH 7.0. The anionic protein crowders destabilize the test protein despite the similarity of their net charges. Thus, weak, nonspecific, attractive interactions between proteins can overcome the charge–charge repulsion and counterbalance the stabilizing effect of steric repulsion

Macromolecular Crowding and Protein Stability

An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells

Physicochemical Properties of Cells and Their Effects on Intrinsically Disordered Proteins (IDPs)

It has long been axiomatic that a protein’s structure determines its function. Intrinsically disordered proteins (IDPs) and disordered protein regions (IDRs) defy this structure–function paradigm. They do not exhibit stable secondary and/or tertiary structures and exist as dynamic ensembles of interconverting conformers with preferred, nonrandom orientations.(1-4) The concept of IDPs and IDRs as functional biological units was initially met with skepticism. For a long time, disorder, intuitively implying chaos, had no place in our perception of orchestrated molecular events controlling cell biology. Over the past years, however, this notion has changed. Aided by findings that structural disorder constitutes an ubiquitous and abundant biological phenomenon in organisms of all phyla,(5-7) and that it is often synonymous with function,(8-11) disorder has become an integral part of modern protein biochemistry. Disorder thrives in eukaryotic signaling pathways(12) and functions as a prominent player in many regulatory processes.(13-15) Disordered proteins and protein regions determine the underlying causes of many neurodegenerative disorders and constitute the main components of amyloid fibrils.(16) They further contribute to many forms of cancer, diabetes and to cardiovascular and metabolic diseases.(17, 18) Research into disordered proteins produced significant findings and established important new concepts. On the structural side, novel experimental and computational approaches identified and described disordered protein ensembles(3, 19, 20) and led to terms such as secondary structure propensities, residual structural features, and transient long-range contacts.(1, 21) The discovery of coupled folding-and-binding reactions defined the paradigm of disorder-to-order transitions(22) and high-resolution insights into the architectures of amyloid fibrils were obtained.(23, 24) On the biological side, we learned about the unexpected intracellular stability of disordered proteins, their roles in integrating post-translational protein modifications in cell signaling and about their functions in regulatory processes ranging from transcription to cell fate decisions.(15, 25, 26) One open question remaining to be addressed is how these in vitro structural insights relate to biological in vivo effects. How do complex intracellular environments modulate the in vivo properties of disordered proteins and what are the implications for their biological functions (Figure 1)?(27-29